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

NOAA Chesapeake Bay Office

NOAA Chesapeake Bay Office

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

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

Dermo intensifie

s

Dermo found in

BayMSX

found in Bay

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.

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 Calvoand currently maintained by Dr. Stan Allen at theVIMS Aquaculture Genetics and Breeding Center (ABC).

• It was hoped that DEBYs would survive disease challenges, reproduce, and pass on disease resistance to wild populations throughintrogression. VIMS Gloucester Pt. Hatchery

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.

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 populationsthought to exist in the system.

Potomac R.

James R.

York R.

Rappahannock R.

Great Wicomico R.

• ACOE & CBF deployed ~15.5 million DEBYs between 2002-2006. (> 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

DeploymentsSpat

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

Shell Bar Reef

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.

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

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

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

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%

Genetic impact of deployed DEBYs- mtDNA data

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.

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.

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.

• 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.

0

10

20

30

40

50

60

70

80

90

100

2006 Total 2006 BB 2007 Total 2007 BB

% In

d.

Year

2006-07 Shell Bar Reef Spat Assignments

% Wild

% Deby

% Hybrid

% Unassigned

2006-07 Shell Bar Reef Spat Assignments

0

10

20

30

40

50

60

70

80

90

100

2006 Total 2006 BB 2007 Total 2007 BB

Year

# In

d.

Wild

Deby

Hybrid

Unassigned

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

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

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

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

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%

mtDNA Data

2006 2007

% spat w/ BB haplotype assigned as DEBY

and/or hybrid5.00% 1%

% spat w/o BB haplotype pure DEBY and/or hybrid 1.00% 0%

% DEBY contribution 6.00% 1.00%

Microsatellite Data

• 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.

Shell Bar Reef

Cranes

Creek

Hilly Was

h

Rogue Point

Sandy

Point

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 GWRduring disease monitoring studies conducted by Ryan Carnegie.

• Oysters from each site, as well assamples of wild and hatchery oysters , were genotyped using four microsatellite loci.

• Oysters were assigned as wild, hatchery, or hybrid using the program STRUCTURE.

2007 GWR Percent Assigned Adult Oysters

0

20

40

60

80

100

120

Sandy Point Rogue Point Hilly Wash Shell Bar Reef Crane's Point

Site

% In

divi

dual

s

Wild

DEBYHybrid

Unassigned

2007 GWR Assigned Adult Oysters

0

50

100

150

200

250

300

350

400

450

500

550

600

Sandy Point Rogue Point Hilly Wash Shell Bar Reef Crane's Point

Site

# In

d.

Wild

DEBY

Hybrid

Unassigned

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

• 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?

VIMS

Elizabeth Francis & Georgeta Constantin- Reece LabP.G. Ross- Eastern Shore LabMellissa Southworth- Mann LabDr. Ryan Carnegie- Burreson LabDr. Stan Allen- ABC

CBF

Tommy Leggett