Using functional genomicsto explore the impacts ofocean acidification oncalcifying marine organisms

Gretchen HofmannUC, Santa Barbara USA

Thanks to: Workshop Steering CommitteeOCB &NSF’s Office of Polar Programs

Outline of the presentation

• Highlight gene expression approaches

• Sample data from sea urchin studies– Organismal response

larval skeleton development

– Compensation/plasticity/adaptation

– Synergistic effect of temperature and CO2

• Strategies for future research

From: Ungerer et al. (2007) Heredity, 1-6

#1 Organismal response: skeleton formation in sea urchin embryos and larvae

Juvenile purple sea urchin© Gerardo Amador

4-arm echinopluteus larvae

Strongylocentrotus purpuratus

1) Utility of early life history stages2) Sea urchin: Developmental model

Embryoswith fertilization membrane

Our multidisciplinary approach

• Morphometrics– Measure the elements of

the larval skeleton

• Gene expression analysis– Ask the embryo to tell you

the answer.– Generating “physiological

fingerprints”4-arm stageLytechinus pictus

First-cut experimental approach

• IPCC gives predictions

• We use B1 and A1F1

Gas Mix

Experimental approach? Use IPCC scenarios for future CO2 levels

• Used IPCC Scenarios (by 2100)– Present 380 ppm CO2– B1 540 ppm CO2– A1F1 970 ppm CO2

• Raise larval cultures at 15 °C

Postdoctoral FellowDr. Michael ‘Moose’ O’Donnell

Body Length vs. Development in Lythechinus pictus larvae

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Larval Measurements1. Body length to tip of oral hood

2. BL to ventral arch

3. Left body rod

4. Left post-oral arm

5. Right body rod

6. Right post-oral arm

7. Left transverse rod

8. Right transverse rod

9. Body width

10. Width at tip

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Treatment

Body Length: 48 h

380 540 970CO2 (ppm)

Total Skeleton: 48 h

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Mean Body Length and Total Skeleton vs. Time

Mean Body Length Over Time

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Mean Total Skeleton Over Time

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Making Hard PartsWhat genes to choose?

From: Hofmann and O’Donnell (in prep) TS section on OA

#2: Linking plasticity & physiology with gene expression

Spicule Matrix Proteins Gene ID Exp. Notes

SMP30-A SPU_000825 High in prismSMP30-D SPU_000828 Low in prism

1. Biomineralization chip in prep.2. E.g., lots of spicule matrix proteins3. Oligo microarray from Agilent

~1,500 genes60-mers$170/array

Red = up-regulated

= down-regulatedGreen

Yellow = no difference

The “raw” data

Zoom in

Turned on

Zoom in

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Another way to look at the data on the chip

• Spots are genes we know• For example, gene chip for

mussels – ~3,000 genes of interest– Classified by function

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1318232833

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DAY 1 DAY 2 DAY 3

Bod

y te

mpe

ratu

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TimeHelmuth et al. 2002 Science 298: 1015-1017

An ecological example:Thermal stress in Intertidal mussels

Physiological fingerprints says: Oregon is hot!

• Mussels at Oregon site have strong up-regulation of “stress”genes in summer

Place, Hofmann (2007) MEPS (in review)

#3: Synergistic effects

The red sea urchin: S. franciscanus

• All cultures raised at 15 ºC• Three levels of CO2

• What happens to physiological plasticity?

(Not just biomineralization)

Changes in thermal phenotype

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HSP70 normalized by Ef1_a

380ppm 542ppm 970ppm

Strategies or what to do while we wait for the genome…

• Study highly conserved genes

• Work on targetedspecies with good genomic resources

• Vegas baby! Heterologoushybridizations

Jackson et al. (2007) Science

• Genes responsible for biomineralization in the pearl oyster

• Nacrein RT-primers from the pearl oyster used with other molluscs(Takeuchi and Endo 2006)

• PCR product size ~150-250bp

• Some success with other genes (msi60 and aspein)

• In process of sequencing the product

500 bp

300 bp

200 bp

Veliger:

N. ostrina

Mantle:

M. californianus

Nacrein

NacreousPrismatic

Periostracum

Modified from Takeuchi and Endo 2006

Veliger stage Nucella ostrina

Cross-species primers

Antarctic sea urchin

Sterechinus neumayeriblastula stage

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Cross hybridization to purp cDNA chipqPCR data

Sterechinus neumayeri

Suggested strategies & priorities for OA studies

• These approaches can be applied to many organisms

• Significant areas– Compensation?– Synergistic effects– Species interactions– Metagenomics– Genome project

proposals to JGI– Training gap and

collaborations

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Metazoan examples

AcknowledgementsData from: • Tim Crombie• LaTisha Hammond• Dr. Moose O’Donnell • Dr. Sean Place• Dr. Anne Todgham• Mackenzie Zippay

Supported by:U.S. National Science Foundation

&David and Lucile Packard Foundation

and the Moore Foundation (PISCO)