Timmins Schiffman PCSGA 2011

The Effects of Ocean Acidification onPacific Oyster Larval Developmentand PhysiologyEmma Timmins-SchiffmanSteven RobertsCarolyn FriedmanMichael O’DonnellUniversity of Washington PCSGA Salem, OR, September, 2011

How does OA affect larvae?Effect of OA Organism ReferenceDecreased shell Oyster, mussel, 1, 2, 3, 4, 9, 12size, strength, barnacle, crabcalcificationTranscriptome/ Urchin 5, 6, 10physiologyProtein Barnacle 7Developmental Urchin, shrimp, 8, 9, 13delay and change brittle starin energy budgetIncreased growth Sea star 11rateAbnormal Brittle star, urchin, 12, 2morphology oysterResponse to other Urchin, barnacle, 14, 3stressors crab

Which physiological mechanisms arechanging?¤ Calcification¤ Hydrogen ion balance across membranes¤ Energy metabolism¤ Timing of developmental processes¤ Stress response

How does ocean acidification affectdevelopment and physiology of Pacific oysterlarvae (Crassostrea gigas)

CO2-free air CO2 (canister)Honeywell Controller Treatment- equilibrated waterDuraFET pH probe Venturi injector

Experimental DesignEquilibrate treatment water Fertilization 1 hpf 6 hpf 24 hpf 72 hpf 96 hpfFix samples for Sample for transcriptomicsdevelopmental stage, size,and calcification

pH 8.5 8.0pH 7.5 400 !atm 700 !atm 1000 !atm 7.0 Time

Relationship between TA and Salinity Total Alkalinity 2100 2030 400 !atm 700 !atm 2020 1000 !atm 2050 2010 TA (!mol/kg)TA (!mol/kg) 2000 2000 1990 1950 1980 1970 1900 0 1 2 3 28.0 28.5 29.0 Day Salinity (ppt)

Dissolved Inorganic Carbon 2000 1900DIC (!mol/kg) 1800 1700 400 !atm 700 !atm 1000 !atm 1600 0 1 2 3 Day

Calcium Carbonate Saturation State 4 3Omega 2 Dissolution threshold 1 400 !atm Calcite 700 !atm Aragonite 1000 !atm 0 0 1 2 3 Day

Results: Larval Development, Growth,and Calcification¤ Larvae were fixed for later microscopy¤ Developmental stage was assessed¤ Growth was measured: hinge length, shell height¤ Calcification: double polarization of light

Proportion Larvae at Pre-Hatching Stage at 6hpf 1.0 400 !atm 700 !atm 1000 !atm 0.8Proportion at Pre-Hatching Significantly 0.6 fewer larvae are in an advanced 0.4 developmental stage at higher pCO2 (lower pH) 0.2 0.0 400 700 1000 Treatment

Larval Calcification: Methods¤ Double polarization of light¤ Qualify larval calcification

Larval Calcification at 24h More larvae 1.0 400 !atm 700 !atm have started 1000 !atm calcification 0.8 at higherProportion Calcified pCO2 0.6 0.4 0.2 0.0 400 700 1000 Treatment

Larval Calcification at 72h Fewer larvae 1.0 are fully calcified in the highest 0.8 pCO2Proportion Calcified (lowest pH) 0.6 treatment 0.4 400 !atm 0.2 700 !atm 1000 !atm 0.0 400 700 1000 Treatment

Larval Size: Methods¤ Size measured in 2 parameters – hinge length and shell height¤ Measurements are from 24 and 72 hours post fertilization

Hinge Length by Treatment and Day Shell Height by Treatment and Day 80 70 70 60Hinge Length (!m) Shell Height (!m) 50 60 40 50 30 40 D1 400 D1 700 D1 1000 D3 400 D3 700 D3 1000 D1 400 D1 700 D1 1000 D3 400 D3 700 D3 1000 Day and pCO2 (!atm) Day and pCO2 (!atm) Larvae are smaller at higher pCO2 at 3 days post-fertilization

Growth Rate by Treatment 15 Hinge Height Shell heightGrowth Rate/Day (!m) 10 growth rate is slower at the highest pCO2 5 0 400 700 1000 Treatment (!atm)

Gene Expression¤ 2 microcosms from each treatment at 96 hpf¤ Oxidative stress genes (SOD,Prx6) and molecular chaperone (Hsp70)

Hsp70 Stress ResponseSTRESS Protein damage/ unfolding Hsp70Chaperones bind to proteins to either repair or remove

Heat Shock Protein 70 25Fold Over Minimum Expression Increased 20 expression of hsp70 with 15 increased pCO2 could indicate 10 cellular stress 5 400 700 1000 Treatment (!atm)

Oxidative Stress Genes Stress ResponseSTRESS •  Increase metabolism •  Kill pathogens ROS Prx6 SOD

Superoxide Dismutase 0.20 0.15Expression 0.10 0.05 0.00 400 700 1000 Treatment (!atm)

Fold Over Minimum Expression 0e+00 1e+22 2e+22 3e+22 4e+22 5e+22 6e+22 400 700Treatment (!atm) Peroxiredoxin 6 1000

Oxidative Stress Genes Greater expression of SOD and Prx6 may indicate increased oxidative stress during exposure to ocean acidification

Conclusions¤  pCO2 of 700 and 1000 µatm caused decreased growth and calcification in C .gigas larvae through 72 hpf¤  There is evidence of physiological stress ¤  Significant for exposure to other stressors ¤  Significant for continued growth, development, and survival

Thank youEmily Carrington•Matt George•Michelle Herko•LauraNewcomb•Ken Sebens•Richard Strathmann•AdamSummers•Billie Swalla•Brent VadopalasChelsea Farms LLCLittle Skookum Shellfish GrowersRock Point Oyster Co.Seattle ShellfishTaylor ShellfishNOAA AquacultureNSA, Pacific Coast Section

References¤  1Watson et al. 2009. Early larval development of the Sydney rock oyster Saccostrea glomerata under near-future predictions of CO2-driven ocean acification. Journal of Shellfish Research. 28 (3)): 431-437.¤  2 Gaylord et al. 2011 Functional impacts of ocean acidification in an ecologically critical foundation species. J Exp Biol. 214: 2586-2594.¤  3 Parker et al. 2010. Comparing the effect of elevated pCO2 and temperature on the fertilization and early development of 2 species of oyster. Marine Biology. 157(11): 2435-2452.¤  4 Findlay et al. 2009. Post-larval development of 2 intertidal barnacles at elevated CO2 and temperature. Mar Biol. 157: 725-735.¤  5 Todham & Hofmann 2009 Transcriptomic response of sea urchin larvae Strongylocentrotus purpuratus to CO2-driven seawater acidification. J Exp Biol. 212: 2579-2594.¤  6 Stumpp et all. 2011. CO2 induced seawater acidification impacts sea urchin larval development II: Gene expression patterns in pluteus larvae. Comparative Biochemistry and Physiology – Part A. 160(3): 320-330.¤  7 Wong et al. 2011. Response of larval barnacle proteome to CO2-driven seawater acidification. Comparative Biochemistry and Physiology – Part D. 6(3): 310-321.¤  8 Stumpp et al. 2011. CO2 induced seawater acidification impacts sea urchin larval development I: Elevated metabolic rates decrease scope for growth and induce developmental delay. Comparative Biochemistry and Physiology – Part A. 160(3): 331-340.¤  9 Bechmann et al 2011. Effects of ocean aciidification on early life stages of shrimp (Pandalus borealis) and mussel (Mytilus edulis). J Toxicol Environ Health. 74(7-9): 424-438.¤  10 Martin et al. 2011. Early development andmolecular plasticity in the Mediterranean sea urchin Paracentrotus lividus exposed to CO2-driven aciidification. J Exp Biol. 214(8): 1357-1368.¤  11 DuPont et al. 2010. Near future ocean acidification increases growth of lecithotrophic larvae and juveniles of the sea star Crossaster papposus. J Exp Biol Part B. 314B(5): 382-389.¤  12 Kurihara et al. 2007. Effects of increased seawater pCO2 on early development of the oyster C.rassostrea gigas. Aquat Biol. 1:91-98.¤  13 Dupont et al. 2008. Near-future level of CO2-driven ocean acidification radically affects larval survival and development in the brittlestar Ophiothrix fragilis. Mar Ecol Prog Ser. 373: 285-294.¤  14 O’Donnell et al. 2009. Predicted impact of ocean acidification on marine invertebrate larvae: elevated CO2 alters response to thermal stress in sea urchin larvae. 156(3): 439-446.

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