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



                          David Metzger

                     University of Washington
               School of Aquatic and Fishery Sciences

                            Committee:
                        Dr. Steven Roberts
                       Dr. Carolyn Friedman
                         Dr. 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 Acidification




                             -
 CO2 + H2O        H2CO3   HCO3 + H+




CO2          pH
                                      Photo: David Mack
Ocean Acidification




                                         -
 CO2 + H2O           H2CO3            HCO3 + H+


                                                      -
             CaCO3           Ca2+   + CO32- + H+   HCO3


CO2           pH             Calcium Carbonate
                                                    Photo: David Mack
Ocean Acidification




• Calcium carbonate molecules are less available

• Calcification become a more energetically demanding process



CO2           pH           Calcium Carbonate
                                                      Photo: David Mack
Ocean Acidification




CO2   pH     Calcium Carbonate
                                 Photo: David Mack
Ocean Acidification
                  Ocean Surface
Primary Production              Respiration



           Sinking Particles



           Decomposition

    CO2         pH             Calcium Carbonate
                  Ocean Floor
Thermohaline Circulation




                  Photo: Bureau of Meteorology
Upwelling
                     N



 Surface H2O
 Ambient pCO2
                            S


High pCO2
Impact of Ocean Acidification on
       Marine Calcifiers
Pteropods
Orr et al, 2005 & Lischka et al., 2010




                                   Photo: National Geographic Images
Impact of Ocean Acidification on
              Marine Calcifier Larvae
Kurihara et al, 2008
Mytilus galloprovincialis
(Blue mussel)
Impact of Ocean Acidification on
              Marine Calcifier Larvae
Kurihara et al, 2008
Mytilus galloprovincialis
(Blue mussel)




                                    Kurihara et al, 2007
                                    Crassostrea gigas
                                    (Pacific oyster)
Impact of Ocean Acidification on
              Marine Calcifier Larvae
Kurihara et al, 2008
Mytilus galloprovincialis
(Blue mussel)




O’Donnell et al, 2010               Kurihara et al, 2007
Lytechinus pictus                   Crassostrea gigas
(Purple sea urchin)                 (Pacific oyster)
Impact of Ocean Acidification on
    Marine Calcifier Larvae




                            Photo: Somkey Bay
Manila Clam Aquaculture



                      4000      Global Production
                                3.6 million tons in 2010
                      3000
       Tons (x1000)



                      2000

                      1000
                         1950


                                    1960


                                           1970


                                                  1980


                                                           1990


                                                                  2000


                                                                         2010
      http://www.fao.org/
Meet the Manila Clam
• Culturally important
                           • Important food source




• Environmentally
  important
  - Accumulate heavy metals and toxins
Manila Clam Life Cycle
Manila Clam Habitat
• Intertidal and coastal environments

                 Hypoxia       Salinity

   Temperature                            Disease
Manila Clam Habitat
Hypothesis:
   – 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 NWFSC
Ocean Acidification Facility
Methods

• Exposed for 2 weeks at 2
  pCO2 Levels                                      CO2 CO2
                                                       FREE
• 6 Replicates chambers/
  pCO2 treatment

• ~30,000 veliger
  larvae/chamber


     Temperature (°C)        Ambient pCO2   Elevated pCO2
           18                355 ± 17μatm   898 ± 48μatm
                               (pH 8.07)       (pH 7.71)
Larval Growth and Survival
                    100%                                        Ambient
                     80%                                        Elevated

 Survival
                     60%
                     40%
                     20%
                      0%
                            1   4         7         11     14

                    20000
 Shell Area (μm2)




                    18000
                    16000
                    14000
                    12000
                    10000
                                                   Mean + SE
                     8000
                            1   4         7        11     14

                                    Sampling Day
Physiology and Transcriptomics



Ocean acidification



   Environment
High Throughput Sequencing

                    Generated 244,082,559
                         Total Reads




Illumina HiSeq
Reference Assembly
Manila clam transcriptome database
     http://compgen.bio.unipd.it/ruphibase/
     (Milan et al., 2011)


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
Reference Assembly
Manila clam transcriptome database
     http://compgen.bio.unipd.it/ruphibase/
     (Milan et al., 2011)
                                              Sequence “X”


Map reads to
reference sequences



   Total number of reads from elevated and       243,416,187
   ambient libraries combined
   Total number of contigs                       27,390
Reference Assembly
Manila clam transcriptome database
     http://compgen.bio.unipd.it/ruphibase/
     (Milan et al., 2011)
                                              Sequence “X”

Map reads to
reference sequences



Characterize by gene ontology
Reference Assembly            cell cycle and
          cell adhesion                                   cell organization
                                           proliferation
                                                           and biogenesis
                                                           cell-cell
                                                          signaling
                                                                     death
                  transport
                                                    developmental
                                                      processes
                                                                     DNA
                                                                  metabolism

                                                   protein
                                                  metabolism

stress response

          signal                 RNA
      transduction            metabolism
RNA-seq
• Expression analysis
                                  Sequence “X”




     Ambient CO2   Elevated CO2
RNA-seq
                  1000              Fold > 1.5
                                    P value < 0.1
        RPKM (Elevated pCO2)


                          100




                               10




                               1

                                0

                                    0   1           10      100     1000
                                              RPKM (Ambient pCO2)

RPKM = Reads Per Kilobase of exon model per Million mapped reads
RNA-seq
              1000              Fold > 1.5
                                P value < 0.1
    RPKM (Elevated pCO2)


                      100




                           10

                                                Number of Contigs
Differentially Expressed Contigs                3,954
                           1
Contigs with higher expression in               3,792 (96%)
        0
elevated pCO2
             0 1         10         100                   1000
Contigs with lower expression in     162 (4%)
                      RPKM (Ambient CO2)
elevated pCO2
OA and Gene Expression
    Manila clam                   4%                          10%   Sea Urchin
Metzger et al., in review                                           Todgham & Hofmann 2009




                                   96%                  90%
                                             Higher
                                             Lower
                                                                    Coral
       Sea Urchin                                                   Moya et al., 2012
   O’Donnell et al., 2010              21%
                                                      39%

                                                                61%
                            79%
Enrichment Analysis
Every gene in library VS. Differentially expressed
 (Reference assembly)                                         (RNAseq)
                                 1000




                        RPKM (Elevated pCO2)
                                         100




                                               10




                                               1

                                               0

                                                    0   1     10         100      1000
                                                            RPKM (Ambient pCO2)
Enrichment Analysis
Every gene in library VS. Differentially expressed
 (Reference assembly)             (RNAseq)

       27,390                      3,954
Enrichment Analysis
Gene Expression
ATP Synthesis
H+ Transport
Oxidative Stress
 Environment     Cell Signaling    Metabolism



                     ROS

               Hydrogen Peroxide



           DNA and Protein Damage
Development and Growth
                   20000
Shell Area (μm2)



                   18000
                   16000
                   14000
                   12000
                   10000
                    8000
                           1   4     7    11   14
                               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
• 2 pCO2 Levels
   • Elevated                              CO2      CO2
                                                   FREE
   • Ambient
• 8 Replicates/treatment
• 10 Juvenile clams/Replicate




      Temperature ( C)      Ambient pCO2     Elevated pCO2
            13              424 45μatm      1146 312μatm
                              (pH 8.01)         (pH 7.63)
Experimental Design



      1 Week
                             Gene
                           Expression
         2 Weeks


       3 Weeks
                           Quantitative PCR
     Sampled gill tissue
Calcification and Ion Transport
                 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.
   Ambient CO2
   Elevated CO2
Mean + SE                   Perlucin-6                          Calmodulin
              6.0                                 1.5
              5.0
Fold Change




              4.0                                  1
              3.0
              2.0                                 0.5
              1.0
              0.0                                  0
                    Week1    Week2       Week3          Week1     Week2      Week3
Protein Translation and Stability
               Organisms respond to stress by changing gene expression and protein
                synthesis.
               Therefore genes involved with protein translation would also increase.
   Ambient CO2
   Elevated CO2
Mean + SE                    Cathepsin-L                               Elongation Factor 2
   4.0                                                      1.2
                                                              1
               3.0
Fold Change




                                                            0.8
               2.0                                          0.6
                                                            0.4
               1.0
                                                            0.2
               0.0                                            0
                      Week1       Week2       Week3                 Week1       Week2    Week3

               Cathepsin-L consistently lower though differences are not significant.
Stress Response
      Transcription molecular chaperones and genes involved in cell
       stress response increase in response to environmental stress

   Ambient CO2
   Elevated CO2                                HSP90
Mean + SE                       1.4
                                1.2
                  Fold Change




                                  1
                                0.8
                                0.6
                                0.4
                                0.2
                                  0

                                       Week1   Week2   Week3
Oxidative Stress
 Environmental stress, increases in metabolism, and cell signaling can
  increase production of ROS.
 Genes that catabolize ROS would therefore increase .
   Ambient CO2
   Elevated CO2                         Glutathione Peroxidase 3
Mean + SE
                                1.5
                  Fold Change




                                 1

                                0.5

                                 0
                                       Week1     Week2      Week3
    Glutathione peroxidase 3 is lower in elevated pCO2 exposed animals at
     weeks one and two but difference is not significant
Does this affect the response
          to 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
              Hypoxia          Salinity

Temperature                               Disease




               Ocean Acidification
Experimental Design
       1 Week
                          Gene
                        Expression
          2 Weeks


        3 Weeks




    1 hour heat shock
    1 week recovery
Thermal tolerance and OA
                                     38°C                                        39°C
                   100%                                        100%
                   80%                                         80%
Percent Survival




                   60%                                         60%
                   40%                                         40%
                   20%                                         20%
                    0%                                          0%
                           1    2    3    4    5    6    7
                          Day1 Day2 Day3 Day4 Day5 Day6 Day7           1    2    3    4    5    6    7
                                                                      Day1 Day2 Day3 Day4 Day5 Day6 Day7
                             Days Post Heat Shock                         Days Post Heat Shock

                                  Ambient pCO2
Thermal tolerance and OA
                                     38°C                                        39°C
                   100%                                        100%
                   80%                                         80%
Percent Survival




                   60%                                         60%
                   40%                                         40%
                   20%                                         20%
                    0%                                          0%
                           1    2    3    4    5    6    7
                          Day1 Day2 Day3 Day4 Day5 Day6 Day7           1    2    3    4    5    6    7
                                                                      Day1 Day2 Day3 Day4 Day5 Day6 Day7
                             Days Post Heat Shock                         Days Post Heat Shock

                                  Ambient pCO2
Thermal tolerance and OA
                                     38°C                                        39°C
                   100%                                        100%
                   80%                                         80%
Percent Survival




                   60%                                         60%
                   40%                                         40%
                   20%                                         20%
                    0%                                          0%
                           1    2    3    4    5    6    7
                          Day1 Day2 Day3 Day4 Day5 Day6 Day7          1     2    3    4    5    6    7
                                                                      Day1 Day2 Day3 Day4 Day5 Day6 Day7
                             Days Post Heat Shock                         Days Post Heat Shock

                                  Ambient pCO2

                                  Elevated pCO2
Summary

Hypothesis:
   Ocean acidification will negatively impact manila clams.
Summary
1. No affect on larval growth and                                             2. Larvae increase expression of
mortality.                                                                    genes involved in essential biological
                                                                              processes.
 150%                                    23000

 100%                                    18000

  50%                                    13000

  0%                                         8000
        1   4       7     11    14                     1    4   7   11   14




3. Juvenile clams to not respond to                                           4. Elevated CO2 does not impact
elevated pCO2 by increasing gene                                              thermal tolerance.
expression.
            1.5
                                                                              100%                                  100%
                1                                                              80%                                   80%
                                                                               60%                                   60%
            0.5                                                                40%                                   40%
                                                                               20%                                   20%
                0                                                               0%                                    0%
                        Week1        Week2          Week3                            Day1Day2Day3Day4Day5Day6Day7          Day1Day2Day3Day4Day5Day6Day7
Impact of Multiple Stressors
              Hypoxia          Salinity

Temperature                               Disease




               Ocean Acidification
Summary
Hypothesis:
   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 Washington   Friday Harbor Laboratories   NOAA NWFSC
Roberts Lab:               Carrington Lab:              Shallin Busch
Sam White                  Emily Carrington             Paul McElhany
Steven Roberts             Moose O’Donnell              Mike Maher
Emma Timmins-Schiffman                                  Jason Miller
Caroline Storer                                         Sarah Norberg
Mackenzie Gavery

Friedman Lab:                                           Taylor Shellfish
Carolyn Friedman                                        Greg Jacob
Brent Vadopalas                                         Joth Davis
Lisa Crosson
Elene Dorfmeier                                         Funding
Sammi Brombacker                                        Washington Sea Grant
Robyn Strenge                                           Saltonstall-Kennedy
                                                        University of Washington


                                Georgia O’Keeffe’s 1926 pastel “Slightly Open Clam Shell”
Acknowledgements
 FRIENDS AND FAMILY!!




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

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Metzger MS defense

  • 1. Characterizing the effects of ocean acidification in larval and juvenile Manila clam, Ruditapes philippinarum, using a transcriptomic approach David Metzger University of Washington School of Aquatic and Fishery Sciences Committee: Dr. Steven Roberts Dr. Carolyn Friedman Dr. Linda Rhodes
  • 2. 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
  • 3. Ocean Acidification - CO2 + H2O H2CO3 HCO3 + H+ CO2 pH Photo: David Mack
  • 4. Ocean Acidification - CO2 + H2O H2CO3 HCO3 + H+ - CaCO3 Ca2+ + CO32- + H+ HCO3 CO2 pH Calcium Carbonate Photo: David Mack
  • 5. Ocean Acidification • Calcium carbonate molecules are less available • Calcification become a more energetically demanding process CO2 pH Calcium Carbonate Photo: David Mack
  • 6. Ocean Acidification CO2 pH Calcium Carbonate Photo: David Mack
  • 7. Ocean Acidification Ocean Surface Primary Production Respiration Sinking Particles Decomposition CO2 pH Calcium Carbonate Ocean Floor
  • 8. Thermohaline Circulation Photo: Bureau of Meteorology
  • 9. Upwelling N Surface H2O Ambient pCO2 S High pCO2
  • 10. Impact of Ocean Acidification on Marine Calcifiers Pteropods Orr et al, 2005 & Lischka et al., 2010 Photo: National Geographic Images
  • 11. Impact of Ocean Acidification on Marine Calcifier Larvae Kurihara et al, 2008 Mytilus galloprovincialis (Blue mussel)
  • 12. Impact of Ocean Acidification on Marine Calcifier Larvae Kurihara et al, 2008 Mytilus galloprovincialis (Blue mussel) Kurihara et al, 2007 Crassostrea gigas (Pacific oyster)
  • 13. Impact of Ocean Acidification on Marine Calcifier Larvae Kurihara et al, 2008 Mytilus galloprovincialis (Blue mussel) O’Donnell et al, 2010 Kurihara et al, 2007 Lytechinus pictus Crassostrea gigas (Purple sea urchin) (Pacific oyster)
  • 14. Impact of Ocean Acidification on Marine Calcifier Larvae Photo: Somkey Bay
  • 15. Manila Clam Aquaculture 4000 Global Production 3.6 million tons in 2010 3000 Tons (x1000) 2000 1000 1950 1960 1970 1980 1990 2000 2010 http://www.fao.org/
  • 16. Meet the Manila Clam • Culturally important • Important food source • Environmentally important - Accumulate heavy metals and toxins
  • 18. Manila Clam Habitat • Intertidal and coastal environments Hypoxia Salinity Temperature Disease
  • 19. Manila Clam Habitat Hypothesis: – 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
  • 22. Methods • Exposed for 2 weeks at 2 pCO2 Levels CO2 CO2 FREE • 6 Replicates chambers/ pCO2 treatment • ~30,000 veliger larvae/chamber Temperature (°C) Ambient pCO2 Elevated pCO2 18 355 ± 17μatm 898 ± 48μatm (pH 8.07) (pH 7.71)
  • 23. Larval Growth and Survival 100% Ambient 80% Elevated Survival 60% 40% 20% 0% 1 4 7 11 14 20000 Shell Area (μm2) 18000 16000 14000 12000 10000 Mean + SE 8000 1 4 7 11 14 Sampling Day
  • 24. Physiology and Transcriptomics Ocean acidification Environment
  • 25. High Throughput Sequencing Generated 244,082,559 Total Reads Illumina HiSeq
  • 26. Reference Assembly Manila clam transcriptome database http://compgen.bio.unipd.it/ruphibase/ (Milan et al., 2011) 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
  • 27. Reference Assembly Manila clam transcriptome database http://compgen.bio.unipd.it/ruphibase/ (Milan et al., 2011) Sequence “X” Map reads to reference sequences Total number of reads from elevated and 243,416,187 ambient libraries combined Total number of contigs 27,390
  • 28. Reference Assembly Manila clam transcriptome database http://compgen.bio.unipd.it/ruphibase/ (Milan et al., 2011) Sequence “X” Map reads to reference sequences Characterize by gene ontology
  • 29. Reference Assembly cell cycle and cell adhesion cell organization proliferation and biogenesis cell-cell signaling death transport developmental processes DNA metabolism protein metabolism stress response signal RNA transduction metabolism
  • 30. RNA-seq • Expression analysis Sequence “X” Ambient CO2 Elevated CO2
  • 31. RNA-seq 1000 Fold > 1.5 P value < 0.1 RPKM (Elevated pCO2) 100 10 1 0 0 1 10 100 1000 RPKM (Ambient pCO2) RPKM = Reads Per Kilobase of exon model per Million mapped reads
  • 32. RNA-seq 1000 Fold > 1.5 P value < 0.1 RPKM (Elevated pCO2) 100 10 Number of Contigs Differentially Expressed Contigs 3,954 1 Contigs with higher expression in 3,792 (96%) 0 elevated pCO2 0 1 10 100 1000 Contigs with lower expression in 162 (4%) RPKM (Ambient CO2) elevated pCO2
  • 33. OA and Gene Expression Manila clam 4% 10% Sea Urchin Metzger et al., in review Todgham & Hofmann 2009 96% 90% Higher Lower Coral Sea Urchin Moya et al., 2012 O’Donnell et al., 2010 21% 39% 61% 79%
  • 34. Enrichment Analysis Every gene in library VS. Differentially expressed (Reference assembly) (RNAseq) 1000 RPKM (Elevated pCO2) 100 10 1 0 0 1 10 100 1000 RPKM (Ambient pCO2)
  • 35. Enrichment Analysis Every gene in library VS. Differentially expressed (Reference assembly) (RNAseq) 27,390 3,954
  • 40. Oxidative Stress Environment Cell Signaling Metabolism ROS Hydrogen Peroxide DNA and Protein Damage
  • 41. Development and Growth 20000 Shell Area (μm2) 18000 16000 14000 12000 10000 8000 1 4 7 11 14 Sampling Day
  • 42. 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
  • 43. 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
  • 44. Question 2: Juvenile Manila Clams
  • 46. FHL CO2 System • 2 pCO2 Levels • Elevated CO2 CO2 FREE • Ambient • 8 Replicates/treatment • 10 Juvenile clams/Replicate Temperature ( C) Ambient pCO2 Elevated pCO2 13 424 45μatm 1146 312μatm (pH 8.01) (pH 7.63)
  • 47. Experimental Design 1 Week Gene Expression 2 Weeks 3 Weeks Quantitative PCR Sampled gill tissue
  • 48. Calcification and Ion Transport  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. Ambient CO2 Elevated CO2 Mean + SE Perlucin-6 Calmodulin 6.0 1.5 5.0 Fold Change 4.0 1 3.0 2.0 0.5 1.0 0.0 0 Week1 Week2 Week3 Week1 Week2 Week3
  • 49. Protein Translation and Stability  Organisms respond to stress by changing gene expression and protein synthesis.  Therefore genes involved with protein translation would also increase. Ambient CO2 Elevated CO2 Mean + SE Cathepsin-L Elongation Factor 2 4.0 1.2 1 3.0 Fold Change 0.8 2.0 0.6 0.4 1.0 0.2 0.0 0 Week1 Week2 Week3 Week1 Week2 Week3  Cathepsin-L consistently lower though differences are not significant.
  • 50. Stress Response  Transcription molecular chaperones and genes involved in cell stress response increase in response to environmental stress Ambient CO2 Elevated CO2 HSP90 Mean + SE 1.4 1.2 Fold Change 1 0.8 0.6 0.4 0.2 0 Week1 Week2 Week3
  • 51. Oxidative Stress  Environmental stress, increases in metabolism, and cell signaling can increase production of ROS.  Genes that catabolize ROS would therefore increase . Ambient CO2 Elevated CO2 Glutathione Peroxidase 3 Mean + SE 1.5 Fold Change 1 0.5 0 Week1 Week2 Week3  Glutathione peroxidase 3 is lower in elevated pCO2 exposed animals at weeks one and two but difference is not significant
  • 52. Does this affect the response to 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?
  • 53. Impact of Multiple Stressors Hypoxia Salinity Temperature Disease Ocean Acidification
  • 54. Experimental Design 1 Week Gene Expression 2 Weeks 3 Weeks 1 hour heat shock 1 week recovery
  • 55. Thermal tolerance and OA 38°C 39°C 100% 100% 80% 80% Percent Survival 60% 60% 40% 40% 20% 20% 0% 0% 1 2 3 4 5 6 7 Day1 Day2 Day3 Day4 Day5 Day6 Day7 1 2 3 4 5 6 7 Day1 Day2 Day3 Day4 Day5 Day6 Day7 Days Post Heat Shock Days Post Heat Shock Ambient pCO2
  • 56. Thermal tolerance and OA 38°C 39°C 100% 100% 80% 80% Percent Survival 60% 60% 40% 40% 20% 20% 0% 0% 1 2 3 4 5 6 7 Day1 Day2 Day3 Day4 Day5 Day6 Day7 1 2 3 4 5 6 7 Day1 Day2 Day3 Day4 Day5 Day6 Day7 Days Post Heat Shock Days Post Heat Shock Ambient pCO2
  • 57. Thermal tolerance and OA 38°C 39°C 100% 100% 80% 80% Percent Survival 60% 60% 40% 40% 20% 20% 0% 0% 1 2 3 4 5 6 7 Day1 Day2 Day3 Day4 Day5 Day6 Day7 1 2 3 4 5 6 7 Day1 Day2 Day3 Day4 Day5 Day6 Day7 Days Post Heat Shock Days Post Heat Shock Ambient pCO2 Elevated pCO2
  • 58. Summary Hypothesis: Ocean acidification will negatively impact manila clams.
  • 59. Summary 1. No affect on larval growth and 2. Larvae increase expression of mortality. genes involved in essential biological processes. 150% 23000 100% 18000 50% 13000 0% 8000 1 4 7 11 14 1 4 7 11 14 3. Juvenile clams to not respond to 4. Elevated CO2 does not impact elevated pCO2 by increasing gene thermal tolerance. expression. 1.5 100% 100% 1 80% 80% 60% 60% 0.5 40% 40% 20% 20% 0 0% 0% Week1 Week2 Week3 Day1Day2Day3Day4Day5Day6Day7 Day1Day2Day3Day4Day5Day6Day7
  • 60. Impact of Multiple Stressors Hypoxia Salinity Temperature Disease Ocean Acidification
  • 61. Summary Hypothesis: 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
  • 62. 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?
  • 63. Acknowledgements University of Washington Friday Harbor Laboratories NOAA NWFSC Roberts Lab: Carrington Lab: Shallin Busch Sam White Emily Carrington Paul McElhany Steven Roberts Moose O’Donnell Mike Maher Emma Timmins-Schiffman Jason Miller Caroline Storer Sarah Norberg Mackenzie Gavery Friedman Lab: Taylor Shellfish Carolyn Friedman Greg Jacob Brent Vadopalas Joth Davis Lisa Crosson Elene Dorfmeier Funding Sammi Brombacker Washington Sea Grant Robyn Strenge Saltonstall-Kennedy University of Washington Georgia O’Keeffe’s 1926 pastel “Slightly Open Clam Shell”
  • 64. Acknowledgements FRIENDS AND FAMILY!! Georgia O’Keeffe’s 1926 pastel “Slightly Open Clam Shell”
  • 65. Georgia O’Keeffe’s 1926 pastel “Slightly Open Clam Shell”

Editor's Notes

  1. Upwelling of corrosive water has been shown to be detrimental to early developmental stages of calcifying organisms. Conseuqently, several studies have been conducted to assess the suseptability of important aquaculture species such as mussels and oysters. Results from these studies indicated that high pCO2 levels will reduce production as a result of incrased mortality and slower growth. No studies, however, have been conducted that assess the impact of ocean acidification manila clams.
  2. Upwelling of corrosive water has been shown to be detrimental to early developmental stages of calcifying organisms. Conseuqently, several studies have been conducted to assess the suseptability of important aquaculture species such as mussels and oysters. Results from these studies indicated that high pCO2 levels will reduce production as a result of incrased mortality and slower growth. No studies, however, have been conducted that assess the impact of ocean acidification manila clams.
  3. Upwelling of corrosive water has been shown to be detrimental to early developmental stages of calcifying organisms. Conseuqently, several studies have been conducted to assess the suseptability of important aquaculture species such as mussels and oysters. Results from these studies indicated that high pCO2 levels will reduce production as a result of incrased mortality and slower growth. No studies, however, have been conducted that assess the impact of ocean acidification manila clams.
  4. Upwelling of corrosive water has been shown to be detrimental to early developmental stages of calcifying organisms. Conseuqently, several studies have been conducted to assess the suseptability of important aquaculture species such as mussels and oysters. Results from these studies indicated that high pCO2 levels will reduce production as a result of incrased mortality and slower growth. No studies, however, have been conducted that assess the impact of ocean acidification manila clams.
  5. Upwelling of corrosive water has been shown to be detrimental to early developmental stages of calcifying organisms. Conseuqently, several studies have been conducted to assess the suseptability of important aquaculture species such as mussels and oysters. Results from these studies indicated that high pCO2 levels will reduce production as a result of incrased mortality and slower growth. No studies, however, have been conducted that assess the impact of ocean acidification manila clams.
  6. Manila clam production has expanded rapidly since 1990
  7. Temp is within range for optimal spawning and larval development
  8. Temp is representative of optimal growing conditions
  9. Whygil tissue? Constantly exposed to water and site of ion and gas exchange with the environment. Also rich with hemocytes which are the cells primarily involved in stress response
  10. Increase CO2 exposure and see if that elicits more of a responseNGS on juvenilesDig deeper into larval NGS data2. Can learn a lot from these experiments but multi generation and long term monitoring are the direction research needs to be going