Metzger MS defense

Characterizing the effects of ocean acidification in larval and juvenile Manila clam, Ruditapes philippinarum, using

a transcriptomic approach

David Metzger

University of WashingtonSchool of Aquatic and Fishery Sciences

Committee:Dr. Steven Roberts

Dr. Carolyn FriedmanDr. Linda Rhodes

Outline• Introduction and Background

• Ocean acidification• Manila clam Ruditapes philippinarum

• Question 1:How does elevated pCO2 affect larval Manila clam physiology?

• Question 2: Does elevated pCO2 affect the susceptibility of juvenile clams to other environmental stressors?

• Conclusions and future directions

Ocean AcidificationCO2

CO2 H2O H2CO3+

Photo: David Mack

CO2 pH

HCO3

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

+ H+-


CO2 H2O H2CO3+

Photo: David Mack

CO2 pH Calcium Carbonate

HCO3

H++ CO32-Ca2+ +CaCO3

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

+ H+

HCO3

-

-


Photo: David Mack


CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

• Calcium carbonate molecules are less available

• Calcification become a more energetically demanding process


Photo: David Mack


CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

Ocean Acidification

Sinking Particles

Decomposition

Ocean Surface

Ocean Floor

Primary Production Respiration

pH Calcium CarbonateCO2

Thermohaline Circulation

Photo: Bureau of Meteorology

High pCO2

Surface H2O

Ambient pCO2

Upwelling

S

~~~WIND~~~

N

Impact of Ocean Acidification on Marine Calcifiers

Pteropods

Photo: National Geographic Images

Orr et al, 2005 & Lischka et al., 2010

Impact of Ocean Acidification on Marine Calcifier Larvae

Kurihara et al, 2008Mytilus galloprovincialis(Blue mussel)



Kurihara et al, 2007Crassostrea gigas(Pacific oyster)



Kurihara et al, 2007Crassostrea gigas(Pacific oyster)

O’Donnell et al, 2010Lytechinus pictus(Purple sea urchin)


Photo: Somkey Bay

http://www.fao.org/

Manila Clam Aquaculture

4000

2000

Tons

(x10

00) 3000

1000

2010

2000

1990

1980

1970

1960

1950

Global Production3.6 million tons in 2010

8.5 million pounds$17 million dollars

Meet the Manila Clam

• Culturally important

• Environmentally important

- Accumulate heavy metals and toxins

• Important food source

Manila Clam Life Cycle

Manila Clam Habitat

Temperature

Hypoxia Salinity

Disease

• Intertidal and coastal environments

Manila Clam HabitatHypothesis:

– Ocean acidification will negatively impact manila clams.

Why?– Calcification, growth, and maintaining ion homeostasis will become more

energetically demanding.

– Less resources to cope with additional stressors of inhabiting intertidal communities

Question 1

NOAA NWFSCOcean Acidification Facility

Methods

• Exposed for 2 weeks at 2 pCO2 Levels

• 6 Replicates chambers/ pCO2 treatment

• ~30,000 veliger larvae/chamber

Temperature (°C) Ambient pCO2 Elevated pCO2

18 355 ± 17μatm(pH 8.07)

898 ± 48μatm(pH 7.71)

CO2 CO2

FREE

Larval Growth and Survival

1 4 7 11 1480009000

1000011000120001300014000150001600017000

Shel

l Are

a (μ

m2)

1 4 7 11 140%

20%

40%

60%

80%

100%

Sampling Day

Surv

ival

Mean + SE

Ambient

Elevated

Physiology and Transcriptomics

Environment

Ocean acidification

High Throughput Sequencing

Illumina HiSeq

Generated 244,082,559 Total Reads

Manila clam transcriptome database

What is RuphiBase?• 32,606 contiguous sequences from 454 Roche pyrosequencing• Average length 546bps

• 5,656 Sanger expressed sequence tags

• 51 mRNA sequences from NCBI

http://compgen.bio.unipd.it/ruphibase/(Milan et al., 2011)

Reference Assembly

http://compgen.bio.unipd.it/ruphibase/


Manila clam transcriptome databasehttp://compgen.bio.unipd.it/ruphibase/(Milan et al., 2011)

Reference Assembly

Sequence “X”

Map reads to reference sequences

Total number of reads from elevated and ambient libraries combined

243,416,187

Total number of contigs 27,390



Manila clam transcriptome databasehttp://compgen.bio.unipd.it/ruphibase/(Milan et al., 2011)

Reference Assembly

Sequence “X”

Map reads to reference sequences

Characterize by gene ontology



cell adhesion cell cycle and proliferationcell organiza-tion and bio-

genesis

cell-cell signal-ing

deathdevelop-mental pro-

cessesDNA metabo-

lism

protein metabo-

lismRNA metabolismsignal transduc-

tion

stress response

transport

Reference Assembly

• Expression analysisSequence “X”

Ambient CO2 Elevated CO2

RNA-seq

10 10000 100

RPKM (Ambient pCO2)1

RPKM

(Ele

vate

d pC

O2)

10

1000

0

100

1

RNA-seqFold > 1.5P value < 0.1

RPKM = Reads Per Kilobase of exon model per Million mapped reads

10 10000 100

RPKM (Ambient CO2)1

RPKM

(Ele

vate

d pC

O2)

10

1000

0

100

1

Number of Contigs

Differentially Expressed Contigs 3,954

Contigs with higher expression in elevated pCO2

3,792 (96%)

Contigs with lower expression in elevated pCO2

162 (4%)

RNA-seqFold > 1.5P value < 0.1

OA and Gene Expression

21%

79%

10%

90%96%

4%

61%

39%

Manila clam

Sea Urchin

Sea Urchin

CoralMoya et al., 2012

O’Donnell et al., 2010

Todgham & Hofmann 2009Metzger et al., in review

Higher

Lower

Every gene in library VS. Differentially expressed (Reference assembly) (RNAseq)

Enrichment Analysis

10 10000 100RPKM (Ambient pCO2)

1

RPKM

(Ele

vate

d pC

O2)

10

1000

0

100

1

Every gene in library VS. Differentially expressed (Reference assembly) (RNAseq)

Enrichment Analysis

3,95427,390

Enrichment Analysis

Gene Expression

ATP Synthesis

H+ Transport

Oxidative Stress

Hydrogen Peroxide

DNA and Protein Damage

ROS

Environment MetabolismCell Signaling

Development and Growth

1 4 7 11 1480009000

1000011000120001300014000150001600017000

Shel

l Are

a (μ

m2)

Sampling Day

Candidate Gene Identification

Gene Name Ruphibase ID Fold Change Gene Function

Perlucin 6 ruditapes_lrc29501 133 Calcification

Calmodulin ruditapes_c670 4.4 Calcium binding

Cathepsin L ruditapes_c11131 3 Protein Translation

Elongation factor 2 ruditapes2_c46 1.7 Protein Translation

Hsp90 ruditapes_c1528 2.5 Stress response

Glutathione Peroxidase 3 ruditapes2_c3709 3.4 Oxidative Stress

Summary

• No effect of elevated pCO2 on growth or survival

• Manila clam larvae increase transcription in response to an elevated pCO2 environment

• Characterized biological processes impacted by elevated pCO2

• Identified candidate genes for further study

Question 2: Juvenile Manila Clams

FHL Ocean Acidification Facility

FHL CO2 System

CO2CO2

FREE

• 2 pCO2 Levels

• Elevated• Ambient

• 8 Replicates/treatment• 10 Juvenile clams/Replicate

Temperature (°C) Ambient pCO2 Elevated pCO2

13 424 ± 45μatm(pH 8.01)

1146 ± 312μatm(pH 7.63)

Experimental Design

1 Week

2 Weeks

3 Weeks

GeneExpression

Sampled gill tissue

Quantitative PCR

Calcification and Ion Transport

Week1 Week2 Week30.00.51.01.52.02.53.03.5

Week1 Week2 Week30

0.20.40.60.8

11.2

Perlucin-6 Calmodulin

Fold

Cha

nge

Ambient CO2

Elevated CO2

Calcium carbonate abundance decreases making calcification more difficult.

Genes associated with calcification and calcium ion transport would increase to increase scavenging efforts of calcium ions.

Mean + SE

Week1 Week2 Week30

0.20.40.60.8

11.2

Week1 Week2 Week30.0

0.5

1.0

1.5

2.0

2.5

Protein Translation and Stability

Cathepsin-L Elongation Factor 2

Fold

Cha

nge

Ambient CO2

Elevated CO2

Cathepsin-L consistently lower though differences are not significant.

Organisms respond to stress by changing gene expression and protein synthesis.

Therefore genes involved with protein translation would also increase.

Mean + SE

Stress Response

HSP90

Week1 Week2 Week30

0.2

0.4

0.6

0.8

1

1.2

Fold

Cha

nge

Ambient CO2

Elevated CO2

Transcription molecular chaperones and genes involved in cell stress response increase in response to environmental stress

Mean + SE

Oxidative Stress

Week1 Week2 Week30

0.20.40.60.8

11.2

Glutathione Peroxidase 3

Glutathione peroxidase 3 is lower in elevated pCO2 exposed animals at weeks one and two but difference is not significant

Ambient CO2

Elevated CO2

Fold

Cha

nge

Environmental stress, increases in metabolism, and cell signaling can increase production of ROS.

Genes that catabolize ROS would therefore increase .

Mean + SE

Does this affect the responseto other stressors?

• Juvenile Manila clams do not change transcription levels of candidate genes when exposed to elevated pCO2.

• Do juvenile clams still possess the

physiological potential to cope with multiple stressors when exposed to a high pCO2 environment?

Impact of Multiple Stressors

Temperature

Hypoxia Salinity

Disease

Ocean Acidification

Experimental Design

1 Week

2 Weeks

3 Weeks

GeneExpression

1 hour heat shock1 week recovery

Day1 Day2 Day3 Day4 Day5 Day6 Day70%

20%

40%

60%

80%

100%


20%

40%

60%

80%

100%39°C38°C

Perc

ent S

urvi

val

Thermal tolerance and OA

1 2 3 4 5 6 7 1 2 3 4 5 6 7Days Post Heat Shock Days Post Heat Shock

Ambient pCO2

Thermal tolerance and OA


20%

40%

60%

80%

100%


20%

40%

60%

80%

100%39°C38°C

Perc

ent S

urvi

val

1 2 3 4 5 6 7 1 2 3 4 5 6 7

Ambient pCO2

Elevated pCO2

Days Post Heat Shock Days Post Heat Shock

Summary

Hypothesis:Ocean acidification will negatively impact manila clams.

REJECT

Summary1. No affect on larval growth and mortality.

2. Larvae increase expression of genes involved in essential biological processes.

3. Juvenile clams to not respond to elevated pCO2 by increasing gene expression.

4. Elevated CO2 does not impact thermal tolerance.

1 4 7 11 148000

10000

12000

14000

16000

18000

1 4 7 11 140%

20%40%60%80%

100%120%

Week1 Week2 Week30

0.2

0.4

0.6

0.8

1

1.2


20%

40%

60%

80%

100%


20%

40%

60%

80%

100%

Impact of Multiple Stressors

Temperature

Hypoxia Salinity

Disease

Ocean Acidification

SummaryHypothesis:

Ocean acidification will negatively impact manila clams.

Constant exposure to changing environmental conditions has conditioned Manila clams to effectively cope with increasing pCO2 levels as a result of ocean acidification

Future Studies• Are other biological processes in juvenile

Manila clams impacted by OA?

• Does ocean acidification impact reproduction and fertilization?

• Is there an effect on calcification?

Acknowledgements

University of WashingtonRoberts Lab:Sam WhiteSteven RobertsEmma Timmins-SchiffmanCaroline StorerMackenzie Gavery

Friedman Lab: Carolyn FriedmanBrent VadopalasLisa CrossonElene DorfmeierSammi BrombackerRobyn Strenge

NOAA NWFSCShallin BuschPaul McElhanyMike MaherJason MillerSarah Norberg

Taylor ShellfishGreg JacobJoth Davis

FundingWashington Sea GrantSaltonstall-KennedyUniversity of Washington

Friday Harbor LaboratoriesCarrington Lab:Emily CarringtonMoose O’Donnell

Georgia O’Keeffe’s 1926 pastel “Slightly Open Clam Shell”

Acknowledgements


FRIENDS AND FAMILY!!

QUESTIONS?


Metzger MS defense

Technology

Transcript of Metzger MS defense