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
NOAA Chesapeake Bay Office
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 Virginia’s 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.
Tracking study using dyes by Mann et al. (year?)
Rappahannock R.
York R.
James R.
Great Wicomico R.

7. Methods
ACOE & CBF deployed ~15.5 million DEBYs between (> 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
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
5286
Great Wicomico River
Shell Bar Reef

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
Spat 2 B
Spat 3
Spat 4
Spat 5 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
Collection Date AA
RARE BB
Total Scored
% BB
2002 38 1 39
2.48
2004 35
Totals 73 74
1.33
Spat Collections
Collection Date AA
RARE BB
Total Scored
% BB
2002
1259 13 9
1281
0.70
2003
282 3 1
286
0.35
2004
109
 2005
857 19
889
2.14
2006
2633 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%
2.14%
1.92%
1.45%
Subtract wild baseline
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 ; some DEBY spawning seems to have occurred in but there is no increasing trend (yet?).
High spat falls in the GWR in 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 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 isn’t 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 don’t detect the male contribution to hybrid spat, so we have to assume 1:1 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%
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%

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 ( 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.
Shell Bar Reef
Cranes Creek
Hilly Wash
Rogue Point
Sandy Point

20. Results Genetic impact of deployed DEBYs on neighboring reefs
At least 40% of adult oysters on Shell Bar Reef are deployed DEBYs
Found market-size remnants of DEBY deployments at Rogue Pt. (2004) & Crane Pt. (1998)
Two sub-market size (0.8%) oysters assigned as hybrids on Shell Bar Reef
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 , but there was no obvious increasing trend (yet?).
High spat falls in the GWR in 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