Genetic Considerations in Broodstock Selection for Oyster Restoration, Aquaculture Development, and Non-native Species Introductions

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Genetic Considerations in Broodstock Selection
for Oyster Restoration, Aquaculture Development,
and Non-native Species Introductions
Kimberly S. Reece
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Virginia Oyster Landings 1880 - 2005
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What is the best approach to restoration,
protection and preservation of the oyster
resource?
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Preferred Approach May Depend on Motivations and
Perspectives
What is the goal of oyster restoration?
Industry Restoration-objective to become
profitable and self-sustaining
Ecological Restoration To restore habitat
and populations of native oysters
Develop a new oyster industry-aquaculture
To rebuild a sustainableharvest fishery
Non-native oyster
Native oyster
Not necessary exclusive approaches, but emphasis
and measures of success may differ
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Possible Solutions

1. Oyster reef restoration- build/restore habitat
   (reefs) and establish sanctuaries.
2. Reefs provide substrate for natural spatfall,
   sanctuaries protect from fishing pressure.
3. Stock reefs with oysters from hatcheries-goal
   self-sustaining
4. wild broodstock
5. selected / domesticated strains?
6. Aquaculture development through improved
   selective breeding practices
7. Enhanced disease tolerance
8. Enhanced growth rate
9. Consideration of alternative Crassostrea species
   for Chesapeake Bay aquaculture and maybe
   restoration of the fishery (or ecological
   restoration).
10. Asian oysters are significantly more resistant
    (tolerant) to MSX and Dermo.
11. Crassostrea ariakensis tested in Chesapeake Bay
    has shown
12. rapid growth
13. taste that is acceptable to market
14. disease tolerance in field trials

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Genetic Considerations
Stocking reefs with hatchery oysters Does 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 Development Which 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?
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Genetic Considerations (Restoration)
Stocking reefs with hatchery oysters Does 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?
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Should Reefs be Stocked?

• 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?

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Genetic variation

• The answer to the questions of whether to stock
  and with what, depends on
• The genetic structure of the historical and the
  extant populations.
• The phenotypic relevance of any detected genetic
  variation. Is there local adaptation?
• The genetic impact of hatchery (planted) oysters
  on the wild populations and overall genetic
  variance (Ne).

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Do the disease-tolerant oysters, selected lines
have a better chance of survival in the face of
disease challenges?
Maybe yes, in the short term, but what about
longer term? Risks of inbreeding?
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
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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
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Shell Bar Reef, Great Wicomico River

• June-September 2006 biweekly analysis of P.
  marinus in samples (each n 25) of deployed
  DEBYs and naturally recruited C. virginica

P. marinus Weighted Prevalence
Carnegie and Burreson
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York River-Disease Data

• 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
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• 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.

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Need Basic Genetic Data Chesapeake Bay What 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 Hares presentation
on Thursdayhigh 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
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What is the genetic structure of the extant
native oyster populations?
What historically was the genetic structure of
the native oyster populations?
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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?
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Microsatellites High power of discrimination
for populations genetics and restoration
monitoring Highly variable High
throughput Nuclear marker-biparentally
inherited
Microsatellite- simple sequence repeats often
varying lengths among copies (alleles)
ATCTATATATATATATATATATATCGTGG TCGATATATATATATATATA
TATAGCACC
Chromosome (allele) from ? (TA)10
ATCTATATATATATATATATCGTGG TCGATATATATATATATATAGCAC
C
Chromosome (allele) from ? (TA)8
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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. 97158-170
Populations may be genetically different. There
is evidence that more distant populations are
more distinct.
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Pairwise Comparisons of 10 Chesapeake Bay
Populations
Is structure relevant? Are populations locally
adapted?
4 microsatellite markers
Carlsson et al.
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• 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.

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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.
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Genetic analyses tracking the success of reef
stocking
Objective Monitoring the breeding success, and
longer-term relative survivability, of oysters
planted on reefs
Experiment designed for the Great Wicomico River
system using the genetically unique, disease
tolerant aquaculture strains (DEBYs).
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6
5
4
3
2
1
Spat collected at sample sites every 2 weeks from
June -October for genetic typing in the years
2002-2006.
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GWR has been seeded multiple times over the years
with several different stocks
-06
Since 2002 primarily DEBY deployments as part of
the experimental design to track success of
planted oysters.
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Why did we choose DEBYs for the GWR
experiment? DEBYs are genetically unique.
Maternal signal-mtDNA.
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 lt2. Frequency of the B
alleles is much higher in the DEBY stock,
generally ranging from 25-50 depending on the
spawn.
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Microsatellite markers allow clear discrimination
between hatchery lines and natural populations
Deployed spat-on-shell - blue
Rappahannock wild yellow
Example Rappahannock River, Drumming Ground
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Have the deployed DEBYs contributed significantly
to spat production in GWR?
Carlsson et al. Great Wicomico 2002-2006
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.
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Genetic Considerations (Aquaculture)
Aquaculture Development Which 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 But Is this a concern for
aquaculture development in oysters?
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Little evidence of genetic impact to date
Analysis of oysters collected near two farms
growing DEBYs
Site 1
4 microsatellites 2 mtDNA genes Over 85
significantly not assigned to DEBY 1 individual
assigned to DEBY
Site 2
4 microsatellites 2 mtDNA genes Over 90
significantly not assigned to DEBY No individuals
assigned to DEBY
1 DEBY (natural collection)
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There is evidence of reduced genetic variation in
hatchery lines of C. virginica
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Genetic Considerations (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?
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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.
ICES Protocols
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.
EIS Currently Being Drafted
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Genetic Analyses of Crassostrea ariakensis

• 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

Jan Cordes and Jie Xiao Ximing Guos group-Rutgers
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There is Genetic Variation among Wild C.
ariakensis Populations
linkage disequilibrium, and significant
deviations from Hardy-Weinberg Equilibrium (HWE)
Sample LD HW E
IR 0 of 6 none
KR 1 of 6 none
YR 3 of 6 none
DR 0 of 6 none

IR KR YR DR
IR - 0.022 0.014 0.026
KR lt0.001 - 0.01 0.014
YR lt0.001 0.007 - 0.025
DR lt0.001 lt0.001 lt0.001 -

Population pairwise Fst (above) and P-values
(below). indicates Non-significant values.
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Genetic Variation among Wild Populations
Factorial Correspondence Analysis (FCA) by
Individuals
KR
YR
DR
IR
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US Hatchery Stocks
Japan
China
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Genetic Variation in Hatchery Stocks vs. Wild
Populations
KR
Factorial Correspondence Analysis (FCA) by
Population
YR
IR
DR
TUI
WCA
SCA
NCA
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• Hatchery Stocks show reduction in genetic
  diversity compared to wild populations
• Oregon strain is relatively highly inbred

Wild 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
Allelic richness for four hatchery strains and
four wild populations of C. ariakensis.

Hatchery Stocks
Sample LD HW E
TUI 1 of 6 4
WCA 3 of 6 2
NCA 5 of 6 1
SCA 0 of 6 1
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Acknowledgements
Stan Allen Roger Mann Missy Southworth Juli
Harding Aimim Wang Dr. Wu Dr. Ahn Junya Higano
Elizabeth Francis Georgeta Constantin Jie
Xiao Qian Zhang Gail Scott Cheryl Morrison Pat
Gaffney Sharon Furriness
Francis OBeirn Tommy Leggett Ryan Carnegie Mark
Luckenbach Ken Paynter Matt Hare Don
Merritt Wendi Ribeiro
US National Sea Grant-ODRP NOAA/NMFS Chesapeake
Bay Program Office Virginia Sea Grant College
Program Chesapeake Bay Foundation US Army Corps
of Engineers
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JAC ARSs

• Jan F.A. Cordes Jens A. Carlsson
• Research Assistant Scientists