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

Virginia Oyster Landings 1880 - 2005

What is the best approach to restoration, protection and preservation of the oyster resource?

What is the goal of oyster restoration? 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 To rebuild a sustainableharvest fishery Develop a new oyster industry-aquaculture Native oyster Non-native oyster Not necessary exclusive approaches, but emphasis and measures of success may differ

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

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?

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?

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? wild selected VIMS I like a pale ale- 10 ppt Make mine a stout-30 ppt

Genetic variation Natural spatfall- natural populations High 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: 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).

New stress/challenge: 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? Environmental change New stress/challenge: 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

Genetic diversity (higher effective population size) can be important for survival of a species Environmental change = new stress/challenge and can results in elimination of some genetic types : Others may survive: Some “natural” populations are demonstrating disease tolerance. Maybe these are a better source for supportive breeding broodstock

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

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

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 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 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?

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 Microsatellite- simple sequence repeats often varying lengths among copies (alleles) ATCTATATATATATATATATATATCGTGG TCGATATATATATATATATATATAGCACC Chromosome (allele) from ♀ (TA)10 ATCTATATATATATATATATCGTGG TCGATATATATATATATATAGCACC Chromosome (allele) from ♂ (TA)8

Populations may be genetically different. 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.

Pairwise Comparisons of 10 Chesapeake Bay Populations Is structure relevant? Are populations locally adapted? 4 microsatellite markers Carlsson et al.

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.

Planted hatchery stocks Wild stocks Spat population: Progeny from wild, hatchery or hybrids? Are they more or less fit than wild? 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.

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). 7 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.

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.

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 Hinf I digest of mt coIII in DEBY strain A B Hinf I Digest of mt coIII in a Natural Population 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.

Example Rappahannock River, Drumming Ground Microsatellite markers allow clear discrimination between hatchery lines and natural populations Deployed spat-on-shell - blue Rappahannock wild – yellow Example Rappahannock River, Drumming Ground

Have the deployed DEBYs contributed significantly to spat production in GWR? PRIOR TO DEPLOYMENT DEPLOYED DEBY PRODUCED SPAT   Rare BB AA Mt DNA Analyses Carlsson et al. Great Wicomico 2002-2006 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 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?

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)

There is evidence of reduced genetic variation in hatchery lines of C There is evidence of reduced genetic variation in hatchery lines of C. virginica Allelic diversity of microsatellites reduced in DEBYS compared to natural populations Natural population DEBY strain

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?

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

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 Guo’s group-Rutgers

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 YR 3 of 6 DR  IR KR YR DR - 0.022 0.014 0.026 <0.001 0.01* 0.007* 0.025  Population pairwise Fst (above) and P-values (below). * indicates Non-significant values.

Genetic Variation among Wild Populations Factorial Correspondence Analysis (FCA) by Individuals KR YR DR IR

US Hatchery Stocks F1 “Oregon Strain” F1 WCA WC P1 Pacific Northwest, USA “Oregon Strain” Japan TUI SCA99 SCA00 NCA + F1 Yellow River Beihai China

Genetic Variation in Hatchery Stocks vs. Wild Populations KR Factorial Correspondence Analysis (FCA) by Population YR IR DR TUI WCA SCA NCA

Oregon strain is relatively highly inbred 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 YR 3 of 6 DR Allelic richness for four hatchery strains and four wild populations of C. ariakensis. 5 10 15 20 25 TUI WCA NCA SCA IR KR YR DR CarG110 CarG4-60 Car119-6a Car11-70 Hatchery Strains Wild Stocks 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

Acknowledgements US National Sea Grant-ODRP Elizabeth Francis Georgeta Constantin Jie Xiao Qian Zhang Gail Scott Cheryl Morrison Pat Gaffney Sharon Furriness Stan Allen Roger Mann Missy Southworth Juli Harding Aimim Wang Dr. Wu Dr. Ahn Junya Higano Francis O’Beirn 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

Research Assistant Scientists JAC ARSs Jan F.A. Cordes Jens A. Carlsson Research Assistant Scientists