Genetic%20Evaluation%20of%20Recruitment%20Success%20of%20Deployed%20Domesticated%20Crassostrea%20virginica%20Oysters%20on%20a%20Man-made%20Reef%20in%20the%20Great%20Wicomico%20River,%20Virginia.

1
Genetic Evaluation of Recruitment Success of
Deployed Domesticated Crassostrea virginica
Oysters on a Man-made Reef in the Great Wicomico
River, Virginia.

• J. Cordes, J. Carlsson, M. Luckenbach, S.
  Furiness, and K. Reece.
• Virginia Institute of Marine Science

2
Background

• Decline of the Eastern oyster in the Mid-Atlantic
  Bight
• Severe over-fishing
• Habitat destruction
• Pollution
• Disease impacts

Dermo found in Bay
MSX found in Bay

• Restoration strategies
• in Virginia
• Harvest limits
• Reef restoration
• Oyster translocation
• Supplementation

Dermo intensifies
3
Background

• Restoration through oyster augmentation in
  Virginia
• Both dredged adult oysters (translocation) and
  hatchery-propagated local oysters
  (supplementation) have been transplanted to
  various parts of the Chesapeake Bay.
• Early restoration efforts using these wild
  transplants were initially successful but later
  hampered by mortality presumably caused by Dermo
  and MSX (Southworth Mann, J. Shellfish. Res.
  1998).
• This prompted an unconventional approach to
  restoration that included the use of domesticated
  oyster strains for seeding reconstructed reefs.

4
Background

• Restoration through oyster augmentation in
  Virginia
• In 2002 the Army Corps of Engineers (ACOE) the
  Chesapeake Bay Foundation (CBF) adopted the use
  of domesticated, disease-selected aquaculture
  oysters (the Andrews DEBYTM strain) for
  deployments on natural and man-made reefs
  throughout the Virginia portion of the Chesapeake
  Bay.
• DEBY line established and bred for disease
• resistance and rapid growth (Ragone Calvo
• et al. 2003) by Dr. Gene Burreson and Lisa Calvo
• and currently maintained by Dr. Stan Allen at the
• VIMS Aquaculture Genetics and Breeding Center
• (ABC).
• It was hoped that DEBYs would survive disease
• challenges, reproduce, and pass on disease
• resistance to wild populations through
• introgression.

VIMS Gloucester Pt. Hatchery
5
Background

• Possible drawbacks to restoration using hatchery
  stocks
• Little data existed on long-term performance of
  DEBYs in the wild.
• Little data existed on the reproductive success
  of DEBYs in the wild.
• DEBYs were known to have reduced genetic
  variability (Carlsson et al. 2006), raising
  concerns regarding negative genetic impacts on
  wild oysters.

6
The mtDNA Experiment
2002 Experimental seeding of reefs in
Virginias Great Wicomico River using DEBY
oysters was initiated to study how this
disease-selected aquaculture line would perform
in a restoration setting.

• Considered a trap estuary w/ high larval
  retention (Southworth Mann 1998).
• No significant oyster populations
• thought to exist in the system.

Potomac R.
Rappahannock R.
York R.
James R.
Great Wicomico R.
7
Methods

• ACOE CBF deployed 15.5 million DEBYs between
  2002-2006. (gt 90 on Shell Bar Reef, adjacent to
  a historical oyster bed).
• Annual recruitment monitored
• at Shell Bar Reef using spat
• collectors from early spring to
• late fall 2002-2007.

Year DEBY Deployments Spat Sampled
2002 79,5700 1281
2003 292,060 286
2004 1,410,000 109
2005 6,071,648 889
2006 6,928,352 2721
2007 --------------- 2197
Combined 15497760 5286
Great Wicomico River
8
Methods

• To determine the self-recruitment success and
  genetic impact of deployed DEBYs on wild Eastern
  oysters at Shell Bar Reef
• Sampled newly recruited spat from the spat
  collectors deployed at Shell Bar Reef .
• Used mtDNA and nuclear markers to distinguish
  among wild, DEBY, and wild/DEBY hybrids.
• Determined the percentage of the annual spat
  fall attributable to hatchery-reared oysters
  transplanted into the system.

9
Methods

• Distinguishing among wild, DEBY, and wild/DEBY
  hybrids
• Amplified two mitochondrial genes (COI and
  COIII) using PCR.
• Used DNA Fingerprinting (RFLP analysis) to
  determine haplotypes of each spat

Individual COI COIII
Spat 1 A A
Spat 2 A B
Spat 3 B A
Spat 4 B B
Spat 5 A C
Most common haplotype in wild and DEBYS
Rare in wild, up to 45 in DEBYS
Rare in wild and DEBYS
10
Results
Genetic impact of deployed DEBYs- mtDNA data
GWR Wild Baseline GWR Wild Baseline GWR Wild Baseline GWR Wild Baseline GWR Wild Baseline GWR Wild Baseline
Collection Date AA RARE BB Total Scored BB
2002 38 0 1 39 2.48
2004 35 0 0 35 0
Totals 73 0 1 74 1.33
Spat Collections 2005-2007 Spat Collections 2005-2007 Spat Collections 2005-2007 Spat Collections 2005-2007 Spat Collections 2005-2007 Spat Collections 2005-2007
Collection Date AA RARE BB Total Scored BB
2002 1259 13 9 1281 0.70
2003 282 3 1 286 0.35
2004 109 0 0 109 0
 2005 857 13 19 889 2.14
2006 2633 19 52 2704 1.92
2007 2140 25 32 2197 1.45
Totals 5630 63 97 5790 1.68
11
Results
Genetic impact of deployed DEBYs- mtDNA data
2002 2003 2004 2005 2006 2007
Spat w/ BB haplotype presumed DEBY and/or hybrid 0.70 0.35 0 2.14 1.92 1.45
Subtract wild baseline 1.33 1.33 1.33 1.33 1.33 1.33
Spat w/BB from DEBYs ----- ----- ----- 0.81 0.59 0.12
Adjust for percentage DEBYs w/ BB (35) ----- ----- ----- 2.31 1.69 0.34
Double to account for male contribution ----- ----- ----- 4.62 3.38 0.68
12
Conclusions

• Based on the mtDNA data
• No noticeable increase in the BB haplotype was
  observed between 2002-2004 some DEBY spawning
  seems to have occurred in 2005-2006 but there is
  no increasing trend (yet?).
• High spat falls in the GWR in 2005-2007 were
  largely a result of reproduction in wild
  populations.
• The standing stock of wild oysters in the GWR had
  probably been underestimated more recent
  estimates suggest around 10-15 million (R. Mann,
  VIMS, pers. comm.) prior to 2002 onset of DEBY
  supplementation.

13
Caveats

• The PCR/RFLP mtDNA analysis has been criticized
  because
• The BB haplotype isnt found in all DEBYs (not
  diagnostic)- we have to adjust numbers based on
  relative frequencies in DEBY and wild
  populations.
• The BB haplotype is maternally inherited- we
  dont detect the male contribution to hybrid
  spat, so we have to assume 11 sex ratios and
  random mating to adjust.
• The BB haplotype may be selected against-
  therefore pure DEBY and hybrid spat carrying it
  would not survive and we would not detect them in
  our sampling.

14
The Microsatellite Experiment

• To address these issues we reexamined the data
  using eight newly developed nuclear
  microsatellite loci
• Bi-parentally inherited, so male and female DEBY
  contribution can be directly assessed.
• Presumably neutral markers not subjected to
  selection.
• Allows for the use of powerful discrimination
  analyses based on Bayesian assignment tests to
  distinguish both pure DEBY and hybrid offspring.

15
Methods

• Randomly selected 100 spat from each of the 2006
  and 2007 collections for testing. Also tested all
  individuals from 2006 and 2007 with BB mtDNA
  haplotype.
• Genotyped these oysters as well as samples of
  wild and hatchery oysters using eight nuclear
  microsatellite loci .
• Assigned individuals as wild, hatchery, or
  hybrid using the program STRUCTURE.

16
Results

• 2006 no spat w/o BB were DEBY, 2 were hybrid

• 2006 20 of spat with BB haplotype were
  DEBY or hybrid

• 2007 no spat w/o BB were DEBY or hybrid

• 2007 no spat with BB haplotype were DEBY, 5
  were hybrid

17
Results
mtDNA Data
Microsatellite Data
2006 2007
Spat w/ BB haplotype presumed DEBY and/or hybrid 1.92 1.45
Wild Baseline - 1.33 - 1.33
Spat w/BB from DEBYs 0.59 0.12
Multiply by percentage DEBYs w/ BB (35) 1.69 0.34
Double to account for Male contribution 3.38 0.68
2006 2007
spat w/ BB haplotype assigned as DEBY and/or hybrid 5.00 1
spat w/o BB haplotype pure DEBY and/or hybrid 1.00 0
DEBY contribution 6.00 1.00
18
Conclusions

• Results of the microsatellite experiment
  consistent with mtDNA data and work by others
  (Hare et al. 2006).
• No apparent selection against the BB mtDNA
  haplotype.
• No apparent effect of differential reproductive
  success between males and females.

19
Dispersal Experiment

• Genetic impact of deployed DEBYs on neighboring
  reefs
• In 2007 sub-market (25-75 mm) and
• market (76-110 mm) sized oysters
• were sampled from late Spring to
• early Fall from five sites in the GWR
• during disease monitoring studies
• conducted by Ryan Carnegie.
• Oysters from each site, as well as
• samples of wild and hatchery
• oysters , were genotyped using
• four microsatellite loci.
• Oysters were assigned as wild,
• hatchery, or hybrid using the
• program STRUCTURE.

20
Results
Genetic impact of deployed DEBYs on neighboring
reefs

• No hybrids found on any of the other sites

21
General Conclusions

• Both mtDNA and nuclear microsatellite data
  suggest some small amount of DEBY spawning and
  self-recruitment to Shell Bar Reef occurred in
  2005-2007, but there was no obvious increasing
  trend (yet?).
• High spat falls in the GWR in 2005-2007 were
  largely a result of reproduction in wild
  populations standing stock of wild oysters in
  the GWR were probably underestimated.
• Deployed DEBY oysters are surviving to market
  size in the system.
• Preliminary data show no evidence of DEBY
  recruitment to other sites in the system.
• The lack of DEBY impact in the GWR- too early to
  tell? Looking in the wrong place?

22
Acknowledgements
VIMS Elizabeth Francis Georgeta Constantin-
Reece Lab P.G. Ross- Eastern Shore Lab Mellissa
Southworth- Mann Lab Dr. Ryan Carnegie- Burreson
Lab Dr. Stan Allen- ABC CBF Tommy Leggett