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

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700

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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

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Hinge Length by Treatment and Day

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Shell Height by Treatment and Day

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Oyster Morphometrics: Results

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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

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Heat Shock Protein 70

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Oxidative Stress Genes

STRESS Stress Response

•  Increase metabolism •  Kill pathogens

ROS Prx6

GPx SOD

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Superoxide Dismutase

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Glutathione Peroxidase

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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