OAoysters


 * The Effect of Ocean Acidification on Pacific Oysters**

[|Google Docs Final Paper Draft]


 * __Group members__
 * Andy D.
 * Ashley T.
 * Thurs Oct 20th 3-5pm (measure pH, check survival)
 * Len T
 * Ying-Ying
 * Fri Oct 21st afternoon (measure pH, check survival)
 * Len T
 * Selina
 * Morgan- do we want to do the afternoon to keep it consistent?
 * We need to do it in the morning on the weekend because that is when there will be somebody to less us in- Andy
 * Saturday morning (measure pH, check survival)
 * Morgan- someone who knows how to shuck an oyster should probably be there...I've only had experienced with ones that have gone "pop"
 * Sunday morning (measure pH, check survival)
 * Monday 1:30pm (measure pH, check survival)
 * Morgan- I can come between 1230 and 115 if that helps
 * Ashley T.
 * Morgan- I can come between 1230 and 115 if that helps
 * Ashley T.

>>
 * __Purpose__
 * To determine and measure the gene regulation of Pacific oysters (//Crassostrea gigas//) in varying pH acidification.
 * __Hypothesis__
 * Triploid oysters will express higher levels of gene _ than diploid oysters under acidic conditions.


 * Materials
 * 18 Diploid oysters
 * 15 Triploid oysters
 * Seawater (Trashcan full)
 * CO2 treated water
 * 2 garbage cans
 * pH Meter
 * Oyster food


 * Basic Set-up:
 * 2 Treatment Groups with varying pHs (7.24, 5.24)
 * 7.24 (Seawater) - 12 Diploid Oysters,10 Triploid Oysters
 * 24 hour drying- 6 diploid, 5 triploid
 * 5.24 (C02 water) - 6 Diploid Oysters, 5 Triploid Oysters
 * Method
 * DAY 1 (Wednesday 10/19/11)
 * 1) Set up 3 tubs for three separate pH groups - 6.8, 6.7, 6.5
 * For 6.8 pH group: Add 8000 mL seawater
 * For 6.7 pH group: Add 80 mL CO2 water + 7920 mL seawater
 * For 6.5 pH group: Add 240 mL CO2 water + 7760 mL seawater
 * 1) Add 5 diploid oysters, 5 triploid oysters to each tub. Make sure the oysters are labeled as to which ploidy they are.
 * 2) Feed oysters and leave in same environment until next day. Record observations about oysters - pictures, notes, etc.


 * DAY 2 (Thursday 10/20/11)
 * 1) Record observations about oysters - pictures, notes, etc.
 * 2) Replace water in each group - make sure they are the same pH as before (see DAY 1 Step 2)
 * 3) sample all dead oysters from high CO2 due to high mortality

=Data= pH Survival (# found dead)
 * DAY 3 (Friday 10/21/11)
 * Record observations about oysters - pictures, notes, etc.
 * Replace water in each group- make sure they are the same pH as before (see DAY 1 Step 2)
 * DAY 4 (Saturday 10/22/11)
 * 1) Record observations about oysters - pictures, notes, etc.
 * 2) Replace water in each group- make sure they are the same pH as before (see DAY 1 Step 2)
 * DAY 5 (Sunday 10/23/11)
 * 1) Record observations about oysters - pictures, notes, etc.
 * 2) Replace water in each group- make sure they are the same pH as before (see DAY 1 Step 2)
 * DAY 6 (Monday 10/24/11)
 * 1) Record observations about oysters - pictures, notes, etc.
 * 2) Replace water in each group- make sure they are the same pH as before (see DAY 1 Step 2)
 * DAY 7 (Tuesday 10/25/11)
 * 1) Record observations about oysters - pictures, notes, etc.
 * 2) Extract mantle tissue samples from oysters.
 * 3) Shuck, dissect, and split tissues into separate vials for storage - clearly label each tissue sample
 * 4) Take tissue samples and do an RNA, protein extraction for all 30 oysters.‍
 * Day |||| pH ||
 * || High CO2 || Control ||
 * 0 || 5.24 || 7.24 ||
 * 1 || 5.31 || 7.60 ||
 * 2 ||  ||   ||
 * 3 ||  || 7.14 ||
 * 4 ||  ||   ||
 * 5 ||  || 7.24 ||
 * 6 ||  ||   ||
 * Day |||| High CO2 |||| Control ||
 * || Triploid || Diploid || Triploid || Diploid ||
 * 0 || 0 || 0 || 0 || 0 ||
 * 1 || 2 || 5 || 0 || 0 ||
 * 3 || 0 || 0 || 0 || 0 ||
 * 4 || 0 || 0 || 0 || 0 ||
 * 5 || 0 || 0 || 0 || 0 ||
 * 6 || 0 || 0 || 0 || 0 ||

=**Background Information/Research**= = =

[|Identification of Genes Directly Involved in Shell Formation and Their Functions in Pearl Oyster,]//[|Pinctada fucata]//
 * Summary: article focuses on shell matrix proteins control the CaCO3 crystallization. Related proteins include: "chitin-binding protein, collagen, sialic acid-binding lectin, and glycine-rich shell matrix protein". RT-PCR analysis was used most successfully on mantle tissue. We can also test other tissues, like the foot or gills though.

[|Shematrin: a family of glycine-rich structural proteins in the shell of the pearl oyster Pinctada fucata.]
 * Summary: cDNA constructed from mantle tissue mRNA reveals shematrin proteins that seem to be important in calcification... I wonder if it should be the same protein for Pacific oysters?

[|Impact of elevated CO2 on shellfish calcification]
 * Highlights a linear relationship between calcification and increasing pCO2 levels in Pacific oysters and mussels.

[|Hsp70 genes]
 * Can't get access to the paper, but it has the primers necessary to look at Hsp70 expression if we want to measure stress that way

@http://www.sciencedirect.com/science/article/pii/S037811190400575X
 * This article has a lot of useful information regarding specific genes of the Pacific oyster and methods.

[|Using functional genomics to explore the effects of ocean acidification on calcifying marine organism]
 * This paper describes methods used to study ocean acidification via quantitative PCR. It includes which genomic sequences were used for research in different organisms, as well as accession number for [|GenBank].

[|Impact on energy metabolism on oysters]
 * this article looks at bicarbonate ion concentrations in hemolymph and alanine+ATP levels in mantle tissue of oysters in response to a lower pH environment

[|changes in gene expression of Crassostrea virginica to pH and temperature changes]\
 * this article looks at changes in gene expression by looking at heat shock proteins and DNA histones in response to environmental changes including pH and temperature