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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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0 20 40 60 80 100 120 140 160
Hours post spawn
Bod
y le
ngth
(µm
)
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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270
Bod
y Le
ngth
Tip
to O
ral h
ood
B G O
Treatment
Body Length: 48 h
380 540 970CO2 (ppm)
Total Skeleton: 48 h
500
600
700
800
900
Tota
l ske
leto
n
B G OTreatment380 540 970
CO2 (ppm)
Mean Body Length and Total Skeleton vs. Time
Mean Body Length Over Time
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330
8/2/2007 8/3/2007 8/4/2007 8/5/2007 8/6/2007
Time (d)
len
gth
(µ
m)
380ppm540ppm970ppm
Mean Total Skeleton Over Time
500
600
700
800
900
1000
1100
8/2/2007 8/3/2007 8/4/2007 8/5/2007 8/6/2007
Time (d)le
ng
th µ
m
380ppm540ppm970ppm
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
Turned off
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
-238
1318232833
0:00 12:0 24:0 36:0 48:0 60:0 72:0
DAY 1 DAY 2 DAY 3
Bod
y te
mpe
ratu
re o
f m
usse
ls (o
C)
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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0.5
1.0
1.5
2.0
2.5
3.0
15 19 21 23 25 27 29 31
Rel
ativ
e ex
pres
sion
Temp ºC
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
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
COLP-3_ SM30-_
mR
NA
Exp
ress
ion
Rel
ativ
e to
actin
Air
CO2
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)