Metzger MS defense

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  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Manila clam production has expanded rapidly since 1990
  • Temp is within range for optimal spawning and larval development
  • Temp is representative of optimal growing conditions
  • 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
  • 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
  • Metzger MS defense

    1. 1. Characterizing the effects of ocean acidification in larvaland 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. 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. 3. Ocean Acidification - CO2 + H2O H2CO3 HCO3 + H+CO2 pH Photo: David Mack
    4. 4. Ocean Acidification - CO2 + H2O H2CO3 HCO3 + H+ - CaCO3 Ca2+ + CO32- + H+ HCO3CO2 pH Calcium Carbonate Photo: David Mack
    5. 5. Ocean Acidification• Calcium carbonate molecules are less available• Calcification become a more energetically demanding processCO2 pH Calcium Carbonate Photo: David Mack
    6. 6. Ocean AcidificationCO2 pH Calcium Carbonate Photo: David Mack
    7. 7. Ocean Acidification Ocean SurfacePrimary Production Respiration Sinking Particles Decomposition CO2 pH Calcium Carbonate Ocean Floor
    8. 8. Thermohaline Circulation Photo: Bureau of Meteorology
    9. 9. Upwelling N Surface H2O Ambient pCO2 SHigh pCO2
    10. 10. Impact of Ocean Acidification on Marine CalcifiersPteropodsOrr et al, 2005 & Lischka et al., 2010 Photo: National Geographic Images
    11. 11. Impact of Ocean Acidification on Marine Calcifier LarvaeKurihara et al, 2008Mytilus galloprovincialis(Blue mussel)
    12. 12. Impact of Ocean Acidification on Marine Calcifier LarvaeKurihara et al, 2008Mytilus galloprovincialis(Blue mussel) Kurihara et al, 2007 Crassostrea gigas (Pacific oyster)
    13. 13. Impact of Ocean Acidification on Marine Calcifier LarvaeKurihara et al, 2008Mytilus galloprovincialis(Blue mussel)O’Donnell et al, 2010 Kurihara et al, 2007Lytechinus pictus Crassostrea gigas(Purple sea urchin) (Pacific oyster)
    14. 14. Impact of Ocean Acidification on Marine Calcifier Larvae Photo: Somkey Bay
    15. 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. 16. Meet the Manila Clam• Culturally important • Important food source• Environmentally important - Accumulate heavy metals and toxins
    17. 17. Manila Clam Life Cycle
    18. 18. Manila Clam Habitat• Intertidal and coastal environments Hypoxia Salinity Temperature Disease
    19. 19. 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
    20. 20. Question 1
    21. 21. NOAA NWFSCOcean Acidification Facility
    22. 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. 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. 24. Physiology and TranscriptomicsOcean acidification Environment
    25. 25. High Throughput Sequencing Generated 244,082,559 Total ReadsIllumina HiSeq
    26. 26. Reference AssemblyManila 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. 27. Reference AssemblyManila clam transcriptome database http://compgen.bio.unipd.it/ruphibase/ (Milan et al., 2011) Sequence “X”Map reads toreference sequences Total number of reads from elevated and 243,416,187 ambient libraries combined Total number of contigs 27,390
    28. 28. Reference AssemblyManila clam transcriptome database http://compgen.bio.unipd.it/ruphibase/ (Milan et al., 2011) Sequence “X”Map reads toreference sequencesCharacterize by gene ontology
    29. 29. Reference Assembly cell cycle and cell adhesion cell organization proliferation and biogenesis cell-cell signaling death transport developmental processes DNA metabolism protein metabolismstress response signal RNA transduction metabolism
    30. 30. RNA-seq• Expression analysis Sequence “X” Ambient CO2 Elevated CO2
    31. 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. 32. RNA-seq 1000 Fold > 1.5 P value < 0.1 RPKM (Elevated pCO2) 100 10 Number of ContigsDifferentially Expressed Contigs 3,954 1Contigs with higher expression in 3,792 (96%) 0elevated pCO2 0 1 10 100 1000Contigs with lower expression in 162 (4%) RPKM (Ambient CO2)elevated pCO2
    33. 33. OA and Gene Expression Manila clam 4% 10% Sea UrchinMetzger 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. 34. Enrichment AnalysisEvery 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. 35. Enrichment AnalysisEvery gene in library VS. Differentially expressed (Reference assembly) (RNAseq) 27,390 3,954
    36. 36. Enrichment Analysis
    37. 37. Gene Expression
    38. 38. ATP Synthesis
    39. 39. H+ Transport
    40. 40. Oxidative Stress Environment Cell Signaling Metabolism ROS Hydrogen Peroxide DNA and Protein Damage
    41. 41. Development and Growth 20000Shell Area (μm2) 18000 16000 14000 12000 10000 8000 1 4 7 11 14 Sampling Day
    42. 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 TranslationElongation 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. 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. 44. Question 2: Juvenile Manila Clams
    45. 45. FHL Ocean Acidification Facility
    46. 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. 47. Experimental Design 1 Week Gene Expression 2 Weeks 3 Weeks Quantitative PCR Sampled gill tissue
    48. 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 CO2Mean + SE Perlucin-6 Calmodulin 6.0 1.5 5.0Fold Change 4.0 1 3.0 2.0 0.5 1.0 0.0 0 Week1 Week2 Week3 Week1 Week2 Week3
    49. 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 CO2Mean + SE Cathepsin-L Elongation Factor 2 4.0 1.2 1 3.0Fold 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. 50. Stress Response  Transcription molecular chaperones and genes involved in cell stress response increase in response to environmental stress Ambient CO2 Elevated CO2 HSP90Mean + SE 1.4 1.2 Fold Change 1 0.8 0.6 0.4 0.2 0 Week1 Week2 Week3
    51. 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 3Mean + 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. 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. 53. Impact of Multiple Stressors Hypoxia SalinityTemperature Disease Ocean Acidification
    54. 54. Experimental Design 1 Week Gene Expression 2 Weeks 3 Weeks 1 hour heat shock 1 week recovery
    55. 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. 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. 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. 58. SummaryHypothesis: Ocean acidification will negatively impact manila clams.
    59. 59. Summary1. No affect on larval growth and 2. Larvae increase expression ofmortality. genes involved in essential biological processes. 150% 23000 100% 18000 50% 13000 0% 8000 1 4 7 11 14 1 4 7 11 143. Juvenile clams to not respond to 4. Elevated CO2 does not impactelevated 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. 60. Impact of Multiple Stressors Hypoxia SalinityTemperature Disease Ocean Acidification
    61. 61. 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
    62. 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. 63. AcknowledgementsUniversity of Washington Friday Harbor Laboratories NOAA NWFSCRoberts Lab: Carrington Lab: Shallin BuschSam White Emily Carrington Paul McElhanySteven Roberts Moose O’Donnell Mike MaherEmma Timmins-Schiffman Jason MillerCaroline Storer Sarah NorbergMackenzie GaveryFriedman Lab: Taylor ShellfishCarolyn Friedman Greg JacobBrent Vadopalas Joth DavisLisa CrossonElene Dorfmeier FundingSammi Brombacker Washington Sea GrantRobyn Strenge Saltonstall-Kennedy University of Washington Georgia O’Keeffe’s 1926 pastel “Slightly Open Clam Shell”
    64. 64. Acknowledgements FRIENDS AND FAMILY!! Georgia O’Keeffe’s 1926 pastel “Slightly Open Clam Shell”
    65. 65. Georgia O’Keeffe’s 1926 pastel “Slightly Open Clam Shell”

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