Local Adaptation in Olympia Oysters: HSP70 mRNA Expression Response to

Mild Heat Stress

Olympia oysters in the Puget Sound• 100 years of Anthropogenic

Influence• Declines in most native

species• Olympia oyster

• Historically Important• Culturally Important• Commercially Harvest• Population Decline

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Life history of Olympia Oysters• Mexico to Alaska• Cold Water Adapted• Small Size• Salinity requirements• Threatened By

• Pollution• Fresh Water Inundation• Overharvest• Ocean Acidification• Climate Change

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Rehabilitation Efforts• Extensive Rehabilitation Efforts

• Puget Sound Restoration Fund• NOAA/NMFS• Private Producers

• Limited Success• Poor Location Selection• Poor outplanting timing• High Predation• Other?

Photo: Puget Sound Restoration Fund

Local AdapationLocal adaptation is a phenomena in which geographically separated populations of a native species are better adapted to the habitat parameters of their specific location.(Kawecki and Ebert, 2004).

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Photo: Alaskan Cruises

Local Adaptation Cont.• Differing Environment Conditions

and Selection• Temperature• Salinity• DO• Etc.

• Genetic and Phenotypic Differences

• Size, Shape, Color, Etc• Growth/Development• Response Mechanisms Photo:

retrieverman.net

Response Mechanisms• Many pathways effected

• Energy Metabolism• Reproduction• Homeostatic Regulatory Enzymes

• Affected Regulatory Enzymes• Copper Toxicity Response (Ritter et al., 2010)• Phosphoglucose isomerase (Dahlhoff and Rank, 2000)• HSP70 (Feder and Hofmann, 1999)

• Effects• Downregulation• Isoform Concentrations• Priming

HSP70• Maintain Protein Function• Biomarker for Stress• Affected by Local Adaptation• Well Studied

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HypothesisPopulations from warmer southern regions will be better adapted to handle heat stress than those from colder northern regions.

1. Does HSP70 response vary between populations during mild heat stress events?

2. Can we measure this response through mRNA concentrations for HSP70 transcripts?

Experiment• Oysters

• Offspring from 3 distinct populations• Collected in Port Gamble, WA

• Set up• 1 tank• Cold Room at 12 C• Acclimated 1 week• Fed once every 24 hours

• Experiment• Time 0 (12 C)• Time 24 (24 C)• Fed once

• Sampling• Measured Length• Dissected out 50-250 ug whole body tissue• Flash Frozen• Stored in TriReagent Photo:

Christine Savolainen

Processing/Testing• Phenol/Chloroform RNA

isolation• cDNA created • Primers Designed

• HSP70 (C. gigas)• Actin (O. lurida,

Normalizing)

• PCR• qPCR

Photo: Christine Savolainen

Results—PCR• Products in

Agarose Gels• Strong Banding• Low Base pair

sizes• ~100 bp Actin• ~200 HSP70• 113 bp expect for

Actin• 208 BP expected

for HSP70

Results—qPCR Amplification CurveHSP70

Actin

Results—qPCR Melt CurveHSP70

Actin

Results—qPCR Data TableHSP70 C(t) Crude Analysis Normalized Info Normalized Difference Fold DifferenceNorth 0 30.05 241.5127149 2.000783052 4.115736639North 24 27.19 1755.383452 0.574169343 -1.426613709 1.181102468South 0 27.23 1707.355665 0.486129526 0.999999026South 24 25.39 6116.908723 0.76301311 0.276883583 1.56956598Hood 0 28.46 727.5251313 0.562346505 1.156782146Hood 24 27.56 1358.088276 0.61968645 0.057339945 1.274734022

Actin C(t) Crude AnalysisNorth 0 31.05 120.7090967North 24 26.39 3057.257366South 0 26.19 3512.141461South 24 25 8016.780634Hood 0 27.63 1293.731046Hood 24 26.87 2191.573296

Results—qPCR C(t) Normalized Values• Fluorescence Measured In

Samples• No Fluorescence from

Control• C(t) between 26-30• Later than expected• C(t) converted and

normalized• =10^(-(0.3012*Ct)+11.434)

• Normalized =V(h)/V(a) North 0 North 24 South 0 South 24 Hood 0 Hood 240

0.5

1

1.5

2

2.5

2.00078305215239

0.5741693427228280.486129526474855

0.763013109973937

0.5623465045692090.619686449978381

Normalized HSP70 Values

Parent Pop/Time Point

Ratio

of H

SP70

to A

ctin

Tran

scrip

ts

Discussion• PCR results good

• Bands approximately the same size as expected

• No indiscriminate binding

• qPCR results mirror PCR• Amplification later than expected• Normalized values show equality

between Actin and HSP70 concentrations

• Slight differences between individuals not statistically significant

• Needs more replicates

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Conclusion• More replicates• More Time Points• Varied primers

Photo: Christine Savolainen

Thanks• Steven Roberts• Claire Olson • Mackenzie Gavery• Sam White• Ryan Crim PSRF• Christine Savolainen• And Viewers Like You!

Photo: Christine Savolainen