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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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