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Gene Expression as an Indicator of Environmental Stress in the Pacific Oyster, Crassostrea gigas Emma Timmins-Schiffman University of Washington
Steven Roberts SAFS
Mackenzie Gavery
AFS, Conservation Genetics and Genomics in Fisheries
September 8, 2011
Outline
¤ Climate change in the Pacific Northwest
¤ Oyster mortalities in hatcheries
¤ Transcriptomic tools for monitoring environmental change ¤ Targeted gene approach
¤ Whole transcriptome (NGS) approach
¤ Applications of transcriptomics
Climate Change in the PNW
¤ Ocean acidification ¤ As CO2 dissolves in
seawater, pH goes down.
1750 1800 1850 1900 1950 2000 2050 2100
300
400
500
600
700
800
IPCC (2007) Projected Estimates of Atmospheric CO2
Year
Estim
ate
of
atm
osp
he
ric C
O2
(p
pm
)
northesouth gradients in salinity (27.5e30.5), pH (7.50e7.85), andUarg values (0.50e0.85). Oxygen concentrations throughout theMain Basin and South Sound were undersaturated (oxygen satu-ration at observed water densities of Puget Sound waters isw280e321 mmol kg!1; calculated as in Garcia and Gordon, 1992),but remained substantially above the hypoxic levels (w62 mmol kg!1
or about 2 mg O2 L!1; the common de!nition of hypoxia). Oxygenconcentrations in Hood Canal were strongly strati!ed, ranged from119 to 279 mmol kg!1 (3.8e8.9 mg L!1), and grew increasinglyundersaturated with depth and proximity to the southern endof Hood Canal, where the lowest oxygen concentrations wereobserved. The conditions observed in winter, when the watercolumn should mix most deeply in all areas, re"ect the watercolumn baseline for the seasonal evolution of chemical conditionsfrom spring to fall as rates of biological processes in surface watersincrease with the warming and increased strati!cation of the watercolumn.
The summertime (August 2008) distributions of salinity,oxygen, pH, and aragonite saturation in the Strait of Juan de Fuca,Main Basin, South Sound, and Hood Canal are shown in Fig. 4. LowpH (<7.75), low aragonite saturation state (Uarg " w0.9), highsalinity (>31.0) water undersaturated in oxygen enters the Strait ofJuan de Fuca in the deeper waters and "ows eastward towardAdmiralty Inlet where it mixes upward due to strong tidal mixing(Fig. 2). A portion of this water returns to the mouth of the strait inthe out"owing surface water, lowering pH and Uarg values to nearsaturation. The remaining fraction "ows over the sill at AdmiraltyInlet and spills into the deeper basins. The pH values of the deepwaters "owing into Puget Sound over the sill range from 7.64 to
7.78, with Uarg values ranging from 0.77 to 1.05, and oxygenconcentrations of 105e184 mmol kg!1. Out"owing surface watersat the sill have higher pH values that range from 7.75 to 7.81,with Uarg values from 0.99 to 1.12, and oxygen concentrations of168e192 mmol kg!1. In the shallow surface waters (depth < 8 m)of Main Basin, South Sound, and Hood Canal, the pH rangedfrom 7.77 to 8.25, Uarg was saturated to supersaturated every-where (1.01e2.79), and oxygen concentrations ranged from192 mmol kg!1 to 385 mmol kg!1. Below 50 m in the Main Basin andSouth Sound, the pH values were lower (7.71e7.91), the watersranged from slightly supersaturated to undersaturated withrespect to aragonite (Uarg " 0.86e1.35), and oxygen concentrationsranged from 142 to 217 mmol kg!1. In contrast, the deep waters ofHood Canal had markedly lower pH and Uarg values and oxygenconcentrations (7.32e7.75, 0.34e0.97, and 57e175 mmol kg!1,respectively) than the deep waters of the Main Basin. These highlycorrosivewaterswithpHvalues<7.4 andUarg<0.6 reach as shallowa depth as 50m in the southernpart of the Hood Canal Basin.WithinPuget Sound, only the deepest sample from the southernmoststation in Hood Canal was hypoxic (57 mmol kg!1 "1.9 mg L!1), andsurface (<2 m depth) nitrate concentrations in this area werebetween 0.07 and 1.36 mmol kg!1, indicating strong strati!cationand nutrient-limited phytoplankton growth. However, we werenot able to sample in Lynch Cove, the arm of Hood Canal wherethe most severe hypoxia has previously been observed (Fig. 1),because the ship was too large to navigate there. Thus, it is possiblethat more extensive hypoxic conditions were present at thetime that may have affected the aragonite saturation values inthat arm.
Fig. 4. Distribution of (A) salinity, (B) oxygen, (C) pH, and (D) Uarg along a transect from the coast through Puget Sound’s Main Basin and into South Sound; and (E) salinity,(F) oxygen, (G) pH, and (H) Uarg on a transect from the coast to the southern end of Hood Canal during August 2008. Note that color scales for summer cross-sections span largerranges for some parameters than winter cross-sections. Black dots represent sampling depths.
R.A. Feely et al. / Estuarine, Coastal and Shelf Science 88 (2010) 442e449 445
Kilometers along transect
Feely et al. 2010
5
WA Dept. of Ecology 2007
Commercial/Industry
High Population Density
Hatchery mortalities ¤ Larval mortalities have been linked
to acidic, upwelled waters and disease
Elston et al. 2008
Transcriptomic Tools for Conservation
¤ What are transcriptomics?
¤ Measurement of physiological response
¤ Range/plasticity of response
¤ Plasticity of population response linked to underlying genetic diversity
Population Environmental Change
Acclimatization?
Bottleneck?
Adaptation?
Mortality?
Transcriptomics and Conservation ¤ How do these host of environmental changes affect all
life stages of Pacific oyster?
¤ How can we learn about population conservation and response to environmental change through use of transcriptomic tools?
Transcriptomic Tools for Conservation
¤ 2 methods ¤ Targeted gene approach (bottom up)
¤ Global transcriptome/next-generation sequencing (top down)
Which genes/pathways might change? What response do we see?
Targeted Gene Approach
¤ Oyster larvae exposed to 3 levels of pCO2 for 4 days – ambient (400 µatm), and 2 elevated (700 and 1000 µatm)
¤ Variables measured: water chemistry, size, calcification, developmental stage, and gene expression
¤ Genes chosen: ¤ Molecular chaperone – hsp70
¤ Oxidative stress – SOD, GPx, Prx6
D1 400 D1 700 D1 1000 D3 400 D3 700 D3 1000
3040
5060
70
Hinge Length by Treatment and Day
Day and pCO2 (!atm)
Hin
ge L
engt
h (!
m)
D1 400 D1 700 D1 1000 D3 400 D3 700 D3 1000
4050
6070
80
Shell Height by Treatment and Day
Day and pCO2 (!atm)
She
ll H
eigh
t (!m
)
Oyster Morphometrics: Results
400 700 1000 400 700 1000 400 700 1000 400 700 1000
Treatment (µatm) Treatment (µatm)
Day 1 Day 3 Day 1 Day 3
Hsp70
STRESS Stress Response
Protein damage/unfolding
Chaperones bind to proteins to either repair or remove
Hsp70
400 700 1000
510
1520
25
Heat Shock Protein 70
Treatment (!atm)
Fold
Ove
r Min
imum
Exp
ress
ion
Oxidative Stress Genes
STRESS Stress Response
• Increase metabolism • Kill pathogens
ROS Prx6
GPx SOD
400 700 1000
0.00
0.05
0.10
0.15
0.20
Superoxide Dismutase
Treatment (!atm)
Fold
Ove
r Min
imum
Exp
ress
ion
400 700 1000
0.0e+00
5.0e+24
1.0e+25
1.5e+25
Glutathione Peroxidase
Treatment (!atm)
Fold
Ove
r Min
imum
Exp
ress
ion
400 700 1000
0e+00
1e+22
2e+22
3e+22
4e+22
5e+22
6e+22
Peroxiredoxin 6
Treatment (!atm)
Fold
Ove
r Min
imum
Exp
ress
ion
Targeted Gene Summary
¤ Changes in pCO2 affect larval size – what underlying physiological mechanisms are affected?
¤ Oxidative stress is an important response to ocean acidification
¤ Acidification may cause larval oysters to increase production of ROS (metabolism?), necessitating up-regulation of oxidative stress genes
Whole Transcriptome Approach
¤ 2 sites: “pristine” and high human impact
¤ Oysters collected cDNA pooled transcriptomes sequenced using SOLiD
v 32 million reads
17 million matched Publicly available contigs
29 thousand contigs
Upregulated contigs| min 10 unique hits & 2 fold increase
1329 22 specific
1316 25 specific
cell recognition
regulation of multicellular organismal processes
cell projection organization
cellular component movement
cell adhesion
cell communication
immune response
regulation of biological quality hormone metabolism
maintenance of location
maintenance of location in cell
cellular developmental process
developmental maturation localization of
cell
negative regulation of cellular component organization
semantic space X
sem
an
tic s
pa
ce
Y
p-value 0.04
8E-15
Whole Transcriptome: Results
-1 0 1 2
-2-1
01
PCA of Oyster Gene Expression at 15 Genes
PC1 (31%)
PC
2 (3
0%)
SPI
HMGP
CP17AGNRR2CALLGSTA
DPGN
MDR49
CIQT4
TIMP3.DH
CATLGSTO1
DEF1
RETSTTIMP3.BB
Big Beef CreekDrayton Harbor
Whole Transcriptome Summary
¤ A variety of novel processes involved in environmental response were discovered (cell adhesion)
¤ Global picture of how the environment at the 2 sites is affecting the oyster physiological response
Conclusions
¤ Both the targeted gene and the whole transcriptome approaches provide valuable tools for understanding oysters’ plastic response to environmental change.
¤ Gene expression analyses should be considered important management tools for populations that may be experiencing environmental stress.
Acknowledgements
Emily Carrington•Matt George•Jeff Hard•Michelle Herko•Laura Newcomb•Ken Sebens•Jim & Lisa Seeb•Richard Strathmann•Adam Summers•Billie Swalla•Sam White
NOAA Saltonstall-Kennedy grant