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.

• 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
    2. Add 5 diploid oysters, 5 triploid oysters to each tub. Make sure the oysters are labeled as to which ploidy they are.
    3. 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

• 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.
     1. Shuck, dissect, and split tissues into separate vials for storage - clearly label each tissue sample
  3. Take tissue samples and do an RNA, protein extraction for all 30 oysters.‍

Data

pH

Day	pH
	High CO2	Control
0	5.24	7.24
1	5.31	7.60
2
3		7.14
4
5		7.24
6

Survival (# found dead)

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