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Genetic Considerations in Broodstock Selection for
Oyster Restoration, Aquaculture Development, and
Non-native Species Introductions
Genetic Considerations in Broodstock Selection for
Oyster Restoration, Aquaculture Development, and
Non-native Species IntroductionsKimberly S. Reece
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Virginia Oyster Landings 1880 - 2005
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Chesapeake Bay Market Oyster Landings 1931-2005
Mortality from H. nelsoni (MSX)
begins
P. marinus (Dermo)
intensifies
What is the best approach to restoration, protection and preservation of the oyster resource?
Develop a new oyster industry-
aquaculture
Preferred Approach May Depend on Motivations and Preferred Approach May Depend on Motivations and PerspectivesPerspectives
Preferred Approach May Depend on Motivations and Preferred Approach May Depend on Motivations and PerspectivesPerspectives
Ecological Restoration
To restore habitat and
populations of native oysters
To rebuild a sustainable
harvest fishery
Industry Restoration-objective to
become profitable and self-sustaining
What is the goal of oyster restoration?What is the goal of oyster restoration?
Native oyster Non-native oyster
Not necessary exclusive approaches, but emphasis and measures of success may differ
1. Oyster reef restoration- build/restore habitat (reefs) and establish sanctuaries.
1. Reefs provide substrate for natural spatfall, sanctuaries protect from fishing pressure.
2. Stock reefs with oysters from hatcheries-goal self-sustaining
1. wild broodstock
2. selected / domesticated strains?
2. Aquaculture development through improved selective breeding practices:
1. Enhanced disease tolerance
2. Enhanced growth rate
3. Consideration of alternative Crassostrea species for Chesapeake Bay aquaculture and maybe restoration of the fishery (or ecological restoration).
1. Asian oysters are significantly more resistant (tolerant) to MSX and Dermo.
2. Crassostrea ariakensis tested in Chesapeake Bay has shown:
1. rapid growth
2. taste that is acceptable to market
3. disease tolerance in field trials
Possible SolutionsPossible SolutionsPossible SolutionsPossible Solutions
Genetic ConsiderationsGenetic ConsiderationsGenetic ConsiderationsGenetic Considerations
Stocking reefs with hatchery oystersDoes it work?
Is it a good idea from the genetics point of view?Which oysters to use? Wild or Selected?
What is the genetic impact on extant natural populations?
Ultimate goal = self-sustaining populations, but of what genetic make-up.
Aquaculture DevelopmentWhich oyster stocks to use? Diploids or triploids?
Special genetic lines might be selected for particular traits of interest.
Maintain genetically healthy lines.Is there any genetic impact on extant natural
populations?Introduction of a Non-native Oyster
Aquaculture or on bottom fishery?Which species? Genetic identification needed.
Which stock? Broodstock source?Oregon strain too genetically bottlenecked?
Genetic ConsiderationsGenetic Considerations(Restoration)(Restoration)
Genetic ConsiderationsGenetic Considerations(Restoration)(Restoration)
Stocking reefs with hatchery oystersDoes it work?
Is it a good idea from the genetics point of view?Which oysters to use? Wild or Selected?
What is the genetic impact on extant natural populations?
Ultimate goal = self-sustaining populations, but of what genetic make-up?
•Supportive breeding - adding hatchery broodstock to reefs to
supplement natural populations.
•If we do stock, what is the best broodstock?
• Hatchery oysters from wild broodstock too wimpy? ie. Subject to
high disease mortality?
•Any selected line?
• Different lines (or wild broodstock) be used for different
systems/environments?
I like a pale ale- 10 ppt
Make mine a stout-30 pptwild selected
Should Reefs be Stocked?
VIMS
Genetic variation
Natural spatfall- natural populations
Hatchery oysters from wild broodstock
Selected lines
Highly inbred lines
High
Low
The answer to the questions of whether to stock and with what, depends on:
1. The genetic structure of the historical and the extant populations.
2. The phenotypic relevance of any detected genetic variation. Is there local adaptation?
3. The genetic impact of hatchery (planted) oysters on the wild populations and overall genetic variance (Ne).
Maybe yes, in the short term, but what about longer term?Risks of inbreeding?
Do the disease-tolerant oysters, selected lines have a better chance of survival in the face of disease challenges?
Selected stock may not be able to survive different challenges-may really be “wimpy” under a different set of conditions.Inbreeding may lead to increasing deleterious allele frequencies = line crash
Environmental changeNew stress/challenge:
Genetic diversity (higher effective population size) can be important for survival of a species
Some “natural” populations are demonstrating disease tolerance.Maybe these are a better source for supportive breeding broodstock
Environmental change = new stress/challenge and can results in elimination of some genetic types :
Others may survive:
Shell Bar Reef, Great Wicomico RiverJune-September 2006: biweekly analysis of P. marinus in samples (each n = 25)
of deployed DEBYs and naturally recruited C. virginica
P. marinus Weighted Prevalence
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6/8/06 6/22/06 7/6/06 7/20/06 8/3/06 8/17/06 8/31/06 9/14/06
NaturalDEBY
Carnegie and Burreson
York River-Disease Data
Mortality, York River, 2006
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DEBYs, Resistant
Ross Rock, Susceptible
Aberdeen Natives
Wreck Natives
OctSeptAugJulyJune
•Cumulative mortality higher in Ross Rocks -- approaching 100% by September -- than in DEBYs (63% in October)•Cumulative mortality in Aberdeen Rocks (58% by October) similar to DEBYs; Wreck Shoals slightly higher (72%; MSX disease?)
Carnegie and Burreson
Motivations for, and the risks of, supportive breeding-
using selected/hatchery stocks for restoration efforts.
Motivations
•Increase the chances of survival/reproduction in the face of disease.
•Genetic rehabilitation-introgression of “disease resistance” alleles into
natural populations.
•Ability to genetically track the success and dispersal patterns at
restored sites-experiments to help design/improve restoration strategies.
However, (the risks)
•Calculations and analyses indicate population bottlenecking possible by
deploying highly inbred selected lines (Hare and Rose)
•Little evidence to date that the selected lines are doing well and
reproducing. Are we wasting $? (Carlsson et al.--stay tuned)
•Evidence of resistance (tolerance) in natural populations (Carnegie and
Burreson), which are genetically more diverse and therefore risk can be
reduced by using wild broodstock.
Need Basic Genetic Data
Chesapeake BayWhat is the Crassostrea virginica population genetic
structure?Ongoing studies-published and preliminary results
What is the effective population size in CB and how would selectively bred stock impact this?
Matt Hare’s presentation on Thursday:high risk with current selected highly inbred lines with low Ne.
What are the larval dispersal patterns around restored reefs?
Ongoing studies-published and preliminary results
What is the genetic structure of the extant native oyster populations?
What historically was the genetic structure of the native oyster populations?
The BAYLOR SURVEY of OYSTER GROUNDS
1892 survey of most productive oyster grounds in Virginia (8 million bushels/year)
Chesapeake Bay Oysters
One panmictic population
OR
Isolated, genetically distinct populations?
One population, which over time declined to an extent that there are now individual populations that have become genetically isolated?
Retentive/trap-like estuaries with low gene flow among systems?
Microsatellites
+High power of discrimination for populations genetics and restoration monitoring
+Highly variable
+High throughput
+Nuclear marker-biparentally inherited
ATCTATATATATATATATATATATCGTGG
TCGATATATATATATATATATATAGCACC
ATCTATATATATATATATATCGTGG
TCGATATATATATATATATAGCACC
Chromosome (allele)
from ♀ (TA)10
Chromosome (allele)
from ♂ (TA)8
Microsatellite- simple sequence repeats often varying lengths among copies (alleles)
Evidence of Genetic Structure in the Bay using Microsatellite Markers
But Weak Structure
Buroker et al. 1983. Evidence of differentiation using allozyme markers
Rose, Paynter and Hare. 2006. J Hered. 97:158-170
Populations may be genetically different.
There is evidence that more distant populations are more distinct.
FST 1 2 3 4 5 6 7 8 92 0.000233 0.00010 0.000334 0.00102 0.00167 0.001765 0.00129 0.00092 0.00033 0.001216 0.00051 -0.00049 -0.00010 0.00124 0.002237 0.00070 0.00050 0.00025 0.00160 0.00119 -0.000128 0.00101 0.00111 0.00121 0.00062 0.00171 0.00094 -0.000619 0.00018 0.00005 0.00034 0.00097 0.00072 0.00052 0.00065 0.00102
10 -0.00055 -0.00057 0.00027 -0.00039 0.00089 -0.00050 0.00075 0.00121 0.00030
P 1 2 3 4 5 6 7 8 92 0.42683 0.5576 0.39164 0.1162 0.0186 0.05865 0.0762 0.1846 0.4902 0.21586 0.3262 0.8965 0.6260 0.1621 0.03327 0.2168 0.2793 0.4277 0.0713 0.2022 0.66418 0.0488 0.0557 0.1162 0.3574 0.0781 0.1787 0.93269 0.5557 0.6826 0.5352 0.3057 0.4453 0.4854 0.3848 0.2285
10 0.9102 0.8916 0.4482 0.7666 0.2763 0.8271 0.2382 0.1445 0.5479
Pairwise Comparisons of 10 Chesapeake Bay Populations
Carlsson et al.4 microsatellite markers
Is structure relevant? Are populations locally adapted?
What happens to the oysters deployed on reefs?
Molecular markers to track deployed oysters.
•Do they reproduce? •Genotype (genetically fingerprint) the spatfall.
•Are progeny purebred deployed or wild oysters? AND/OR
•Hybrids?
•Do the deployed oysters survive? How long? •Yearly genetic assessments of oysters at experimental deployment sites.
•What impact do they have on surrounding populations?•Screening populations-follow through time.
Molecular markers can help us discriminate among stocks/lines and allow us to learn more about the reef recruitment shadow and the results of the inter-breeding of wild and hatchery stocks.
Planted hatchery Planted hatchery stocksstocks
Wild stocksWild stocks
Spat population:Spat population:Progeny from wild, Progeny from wild, hatchery or hybrids? Are hatchery or hybrids? Are they more or less fit than they more or less fit than wild?wild?
Spat collected at sample sites every 2 weeks from June -October for
genetic typing in the years 2002-2006.
Experiment designed for the Great Wicomico River system using the genetically unique, disease tolerant aquaculture strains (DEBYs).
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Genetic analyses tracking the success of reef stocking
Objective: Monitoring the breeding success, and longer-term Objective: Monitoring the breeding success, and longer-term relative survivability, of oysters planted on reefsrelative survivability, of oysters planted on reefs
Year Number Stock1996 750000 Tangier Sound1997 150000 Tangier Sound1998 150000 Deep Rock1998 2500 Tangier Sound1999 5000 Tangier Sound2000 24750 CROSBreed2000 210000 Deep Creek2001 10000 CROSBreed2001 300000 Deep Creek2001 200000 Lynnhaven/Plantation Creek2002 13500 CROSBreed2002 795700 DEBY2003 292060 DEBY2004 18000 CROSBreed2004 1400000 DEBY2005 15000000 DEBY
Year Number Stock1996 750000 Tangier Sound1997 150000 Tangier Sound1998 150000 Deep Rock1998 2500 Tangier Sound1999 5000 Tangier Sound2000 24750 CROSBreed2000 210000 Deep Creek2001 10000 CROSBreed2001 300000 Deep Creek2001 200000 Lynnhaven/Plantation Creek2002 13500 CROSBreed2002 795700 DEBY2003 292060 DEBY2004 18000 CROSBreed2004 1400000 DEBY2005 15000000 DEBY
GWR has been seeded multiple times over the years with GWR has been seeded multiple times over the years with several different stocksseveral different stocks
Since 2002 primarily DEBY deployments as part of the experimental design to track success of planted oysters.
-06
DEBYs Show High Frequency of Mitochondrial Haplotypes (DNA fingerprint patterns) that are Rare in Natural Chesapeake Bay Populations
Frequency of the B alleles is relatively low in natural populations: <2%.
Frequency of the B alleles is much higher in the DEBY stock, generally ranging from 25-50% depending on the spawn.
Hinf I digest of mt coIII in DEBY strain
A B
Hinf I Digest of mt coIII in a Natural Population
A
Why did we choose DEBYs for the GWR experiment?Why did we choose DEBYs for the GWR experiment?
DEBYs are genetically unique. Maternal signal-mtDNA.DEBYs are genetically unique. Maternal signal-mtDNA.
Rappahannock wild – yellow
Deployed spat-on-shell - blue
Example Rappahannock River, Drumming Ground
Microsatellite markers allow clear discrimination between hatchery lines and natural populations
Have the deployed DEBYs contributed significantly to spat production in GWR?
Carlsson et al. Great Wicomico 2002-2006
PRIOR TO PRIOR TO DEPLOYMENTDEPLOYMENT
DEPLOYED DEPLOYED DEBYDEBY
PRODUCED PRODUCED SPATSPAT
RareRare
BBBB
AAAA
Mt DNA Analyses
Hare et al. 2006- form Great Wicomico River 2002
Mt DNA and microsatellite analyses
•1579 spat collected in the summer of 2002•1 individual confidently assigned to DEBY
•~10% DEBY/WILD hybrids
Overall, data to date suggest that the DEBY contribution has been low: predation, poor survival and reproduction? Recently there have been much larger deployments with efforts and protecting plants and genetic signal needs to be followed over several years.
Genetic ConsiderationsGenetic Considerations(Aquaculture)(Aquaculture)
Genetic ConsiderationsGenetic Considerations(Aquaculture)(Aquaculture)
Aquaculture DevelopmentWhich oyster stocks to use? Diploids or triploids?
Special genetic lines might be selected for particular traits of interest.
Maintain genetically healthy lines.Is there any genetic impact on extant natural
populations?
Genetic impact of aquaculture lines on natural populations is a concern in many aquatic systems. Eg. Salmonids
ButIs this a concern for aquaculture development in oysters?
Little evidence of genetic impact to date
Analysis of oysters collected near two farms growing DEBYs
Site 14 microsatellites4 microsatellites
2 mtDNA genes2 mtDNA genes
Over 85% Over 85% significantly not assignedsignificantly not assigned to DEBY to DEBY
1 individual assigned to DEBY1 individual assigned to DEBY
Site 24 microsatellites4 microsatellites
2 mtDNA genes2 mtDNA genes
Over 90% Over 90% significantly not assignedsignificantly not assigned to DEBY to DEBY
No individuals assigned to DEBYNo individuals assigned to DEBY
1 DEBY (natural collection)
There is evidence of reduced genetic variation in hatchery lines of C. virginica
Allelic diversity of microsatellites reduced in DEBYS Allelic diversity of microsatellites reduced in DEBYS compared to natural populationscompared to natural populations
Natural population DEBY strain
Genetic ConsiderationsGenetic Considerations(Introduction)(Introduction)
Genetic ConsiderationsGenetic Considerations(Introduction)(Introduction)
Introduction of a Non-native Oyster Aquaculture or on bottom fishery?
Which species? Genetic identification needed.Which stock? Broodstock source?
Oregon strain too genetically bottlenecked?
1995 Virginia House of Delegates Resolution no. 450
“Requesting the Virginia Institute of Marine Science to develop a strategic plan for molluscan shellfish research and begin the process of seeking the necessary approvals for in water testing of non-native oyster species.”
The International Council for the Exploration of the Seas (ICES) Code of Practice on Introductions and Transfers of Marine Organisms (ICES, 1994): “…prior to any introduction a detailed analysis should be conducted on the ecological, genetic and disease relationships of the species in its natural range and environment.”
ICES ProtocolsICES Protocols
EIS Currently Being DraftedEIS Currently Being Drafted
Objectives:
• Inventory of germplasm resources in the species, Crassostrea ariakensis- Correct
identification of the species became a large concern.
• To examine genetic variation and differentiation (population structure), among
natural populations of the C. ariakensis from China, Korea and Japan
• To examine genetic variability.
• In US hatchery stocks (Oregon Strain)
• Compared to wild source populations
Genetic Analyses of Crassostrea ariakensis
Jan Cordes and Jie Xiao
Ximing Guo’s group-Rutgers
Population pairwise Fst (above) and P-values (below). * indicates Non-significant values.
IR KR YR DR
IR - 0.022 0.014 0.026
KR <0.001 - 0.01* 0.014
YR <0.001 0.007* - 0.025
DR <0.001 <0.001 <0.001 -
There is Genetic Variation among Wild C. ariakensis Populations
Sample LD HW E
IR 0 of 6 none
KR 1 of 6 none
YR 3 of 6 none
DR 0 of 6 none
linkage disequilibrium, and significant deviations from Hardy-Weinberg
Equilibrium (HWE)
Factorial Correspondence Analysis (FCA) by Individuals
IR
DR
YR
KR
Genetic Variation among Wild Populations
US Hatchery StocksUS Hatchery Stocks
Japan
China
SCA99 SCA00
NCA
+
F1
F1
Yellow
River
Beihai
WCA
F1WC
P1
TUI
Pacific Northwest, USA
“Oregon Strain”
TUI
NCA
WCA
IR
KRYR
DR
SCA
Factorial Correspondence Analysis (FCA) by Population
Genetic Variation in Hatchery Stocks vs. Wild Populations
Allelic richness for four hatchery strains and four wild populations of C. ariakensis.
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TUI WCA NCA SCA IR KR YR DR
CarG110CarG4-60Car119-6aCar11-70
Hatchery Strains Wild Stocks
•Hatchery Stocks show reduction in genetic diversity compared to wild populations•Oregon strain is relatively highly inbred
Wild Populations
Hatchery Stocks
Sample LD HW E
IR 0 of 6 none
KR 1 of 6 none
YR 3 of 6 none
DR 0 of 6 none
Sample LD HW E
TUI 1 of 6 4
WCA 3 of 6 2
NCA 5 of 6 1
SCA 0 of 6 1
AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgementsElizabeth Francis
Georgeta Constantin
Jie Xiao
Qian Zhang
Gail Scott
Cheryl Morrison
Pat Gaffney
Sharon Furriness
Francis O’Beirn
Tommy Leggett
Ryan Carnegie
Mark Luckenbach
Ken Paynter
Matt Hare
Don Merritt
Wendi Ribeiro
US National Sea Grant-ODRPNOAA/NMFS Chesapeake Bay Program OfficeVirginia Sea Grant College ProgramChesapeake Bay FoundationUS Army Corps of Engineers
Stan Allen
Roger Mann
Missy Southworth
Juli Harding
Aimim Wang
Dr. Wu
Dr. Ahn
Junya Higano
JAC ARSs
Jan F.A. Cordes Jens A. Carlsson
Research Assistant Scientists