Post on 24-Feb-2016
description
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
Photo: thurstontalk.com
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
Photo: John Rowley/ Flickr
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).
Photo: Baja Travel Tours
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
Photo: Matt DiLeo/thescientistgardner blog
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
Photo: Charlie Day/Pacific Rim/ Legendary Pictures
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