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- 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
- 17. Manila Clam Life Cycle
- 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
- 20. Question 1
- 21. NOAA NWFSC Ocean Acidification Facility
- 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
- 37. Gene Expression
- 38. ATP Synthesis
- 39. H+ Transport
- 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
- 45. FHL Ocean Acidification Facility
- 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”
Notas del editor
- 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