WERA-099 Meeting Notes –2006 (Monterey, CA) March 25-26, 2006

An introduction to the two day meeting was given by the chair, Ford Evans. There was a discussion of the agenda and fees ($30.00) to support this program. A list was circulated to get participants’ names and email addresses. We then began with individual presentations:

Ford Evans (OregonStateUniversity) summarized the work to date with the Molluscan Broodstock Program (MBP). Nineteen cohorts were produced with sixteen harvested to date (60 families per cohort) from seven test sites in four states. The average yield was 40% higher than the unselected wild oysters after two generations of selection (three generations of breeding). They are planning to utilize Japanese oysters (previously obtained by Joth Davis and Chris Langdon in 2004) in future breeding. With the fourth generation looming and inbreeding becoming a concern, plans are to replace the line breeding scheme with rotational breeding with 6 cohorts available. Concerns within the program include the need to reduce the cost of field trials and more investigations into genotype by environment interactions. Also, beginning with Cohort 18 the historical practice of mating one male with four females to constitute a line will be replaced by single pair matings to produce full sib families. This change in approach will likely decrease within-family variation in increase intra-class correlations in future analyses.

Mark Camara (USDA ARS) described current projects in his laboratory with Pacific oysters with a focus on assessing the genetic basis of cadmium accumulation (along with other metals), and QTL mapping in full sib families for survival at various sites (DabobBay, and upper and lower YaquinaRiver). This is a course mapping approach using bulk segregation analyses. In addition, he has other studies focusing on Kumamoto oyster broodstock assessment utilizing microsatellites (10 of 40 from C. gigas working) to address the question whether current stocks used in the industry are genetically similar. Finally, he described a population genetics project assessing the genetic variability of Ostreola conchaphila from various locations in eastern Pacific region.

Dennis Hedgecock (USC) briefly described the results from the recently completed WRAC project focused on determining the genetic basis for heterosis and crossbreeding in Pacific oysters. Key findings included the observation that superior hybrid performance relative to inbred performance relies on a combination of additive and dominance effects. Life after WRAC funding for the project now involves working with Taylor Resources to include changing the genotyping platform to the ABI 377 and focusing more on the development of microsatellite markers for Manila clam pedigree analyses. Other ongoing projects at USC include QTL mapping utilizing the Taylor Resources 2x10 F2 family, modeling larval physiology and genetics (QTL mapping and expression analyses) with Eric Powell and Donal Manahan. Future projects for the coming year include ODRP research looking at QTL mapping in F2 hybrid families of Pacific oysters subjected to mortality pressure in southern Puget Sound bays.

Kristie Straus discussed her work with Pinto abalone population genetics, mentioning that the work quantifying QPCR with direct counts of abalone larvae was recently published. Discussion of current status of work describing phylogenetic relationships between pinto and other northeast Pacific abalone followed.

Joth Davis (Taylor Shellfish) gave a brief update on breeding work there, focusing on a new program in manila clam breeding. The ongoing project has a critical need for genetic markers to conduct pedigree analyses. Results to date indicate lots of genetic variability in size and shell pattern available for selection. The project is ongoing at Taylor Shellfish with new cohorts planned for production in 2006.

Ximing Guo discussed ongoing programs at RutgersUniversity, mainly taking place at the Haskins Laboratory. Projects include the following: (1) Oyster breeding for both disease resistance (Dermo, MSX) and fast growth (added to selection criteria this past year), (2) evaluation of inter-strain triploids eastern oysters grown out in different sites to address question of whether we can combine two families each with disease resistance to one disease, to produce triploids with resistance to both. Results indicate:

  • Triploidy has been successful, but growth rates are very different between sites. At two sites, triploids grew only 15% faster than diploids. At one site (KatamaBay), triploids grew 50% faster than diploids in height, length and width. Whole weight triploids are 175% bigger than diploids at 4 months.
  • Juvenile Oyster Disease (JOD) mortality is much lower in triploids.

The explanation of superior growth in triploid oysters was discussed based on variety of hypotheses.

  • There may be energy relocation in triploids: Energy relocation may result in sterility, less energy to gonad development, more to growth. This hypothesis may be a misconception because the researchers already saw growth differences at 4 months between diploids and triploids, prior to gametogenesis.
  • Heterozygosity: The researchers saw a positive correlation with metabolic efficiency and growth as a contributing factor, but not fundamental to superior growth in the triploids.
  • Larger cells in triploids due to large nuclei as compared to cell size in diploids best explains the differences in growth rate and constitutes the most fundamental and plausible hypotheses.

Molecular tools being used in the Haskins lab were discussed and include:

  • Fluorescence in situ hybridization
  • Physical mapping—cytogenetic map for C. virginica
  • Linkage mapping—AFLPS

Progress to date mapping disease resistance genes in C. virginica includes:

  • 108 putative host defense genes identified: 9 mapped.
  • 9101 ESTs were downloaded for MS and SNP discovery.
  • 200 MS containing ESTs identified: 53 MS were developed.
  • 120+ SNP containing ESTs identified: 32 were evaluated: 12 mapped
  • 60+ MS were added to the AFLP maps

Kim Reece (Virginia Institute of Marine Science) discussed work in her laboratory, which is mainly focused on phylogenetics and the genetics of disease resistance in eastern oysters.

  • Marker development is ongoing but they are seeing many null alleles. They have observed:
  • EST-linked microsatellites (10 loci)
  • Anonymous microsatatellites from microsatellite-enriched libraries. They are looking to minimize null allele problems.
  • They are examining the breeding success of hatchery-reared, planted oysters with the following findings to date:
  • They planted DEBY oysters in NY (Delaware Bay, disease resistant cultured oyster line).
  • COI and coi MtDNA / microsatellites
  • There are no significant contribution of DEBY’s found in native oysters to date; thus dilution, predation, sex ratio, maladapted phenotypes are the questions being posed for reasons that introgression has not yet observed.
  • Population genetic structure studies of C. virginica are ongoing in Chesapeake Bay (two cohorts per location, 50 individuals per cohort).
  • They have found microsatellites (4 loci).
  • Significant genetic population structure is indicated from preliminary Fst analyses of Wild vs. DEBY and among wild populations.

Phylogenetics and species identification of Crassostrea oysters constitute other ongoing projects in Reece laboratory.

  • They are establishing the phylogenetic relationships among Pacific Crassostrea spp. oysters based on ITS-1 and COI gene regions and are developing PCR/RFLP identification keys for Indo-Pacific Crassostrea species. Recent oberservations and conclusions are that species found southwest of China that have been sent to VIMS as Crassostreaariakensis are many other things….but not ariakensis!!Recent applications of techniques in the Reece laboratory show that oysters suspected to be diploid C. ariakensis were in fact C. virginica. A Japanese import of C. ariakensis for disease study was overset with C. gigas and C. sikamea.Oysters suspected to be diploid C. ariakensis collected in WillapaBay were C. gigas.
  • The laboratory is pushing limits of the identification keys. Oysters collected from Tunisia thought to be C. gigas probably represented a native species not found in the key. Oysters sent from Korea labeled as C. ariakensis were almost entirely at least two unknown species, probably of the genus Saccostrea. Live oysters found in VA were not C. virginica or C. ariakensis, bit were identified as Ostrea edulis.

Hard Shell Clams, Quahogs, Mercenaria mercenaria, were next discussed with the following details:

  • Wild harvest of clams has declined, but culture activity has grown very rapidly.
  • There is a QPX (quahog parasite unknown) problem for clams in the Northeast.
  • The key to sustainability of wild and aquacultured clam industries is preservation of genetic diversity and preventing negative interactions between wild and cultured clams.
  • Molecular markers (microsatellites, SNPs)
  • A total of 31 primer sets were initially designed but major problems ensued.
  • Four-five microsatellite markers are working fairly well to date.
  • There are extremely low allele numbers, only 2-4 per locus per population.

Chris Davis (Pemaquid Oyster Company and University of Maine) described an ongoing collaboration between Paul Rawson (UMO) and a ten farm hatchery liaison, with the farms contributing $500-$1500 each to help support the eastern oyster breeding program.

Current Projects include the following collaborations with industry:

  • There is a stock evaluation trial for oyster lines currently produced based on the Flowers line.
  • The laboratory is continuing with a selective breeding program for higher yield in Maine produced oysters.

Their current concerns are:

  • A low parentage through several generations with Flowers line has been a problem.
  • UMFS developed and tested primarily with the DamariscottaRiver.

Stock evaluation trial—results to date include the following:

  • Most of the variation in growth and yield associated with SITE (sites with temps above 20 degrees perform the best).
  • The Flowers Pure line performed about 20% better than the UM-Flowers line. The Rutgers line underperformed, and the hybrids generally performed better but weren’t consistent.
  • The UM-Flowers line had the lowest mortality—so in respect to yield, UM-Flowers performed the highest (3 generations in Maine).
  • The second cohort gave similar results to first cohort.
  • There is some advantage to interline crossing, but this is not consistent among sites.
  • There were significant stock-by-site interactions in the trial.
  • There also appear to be substantial stock-by-year interactions.

Stan Allen [VIMS and ABC (Aquaculture Genetics and BreedingTechnologyCenter)] followed with an update on activities in his laboratory.

  • They are trying to foster selective breeding for shellfish, promote the industry, and develop new feeds for restoration purposes.
  • Their new facility will provide quarantine facilities for sensitive strains or non-natives. It is good for an on-land broodstock conditioning facility.
  • Two approaches have been used in selection for disease resistance in C. virginica. In both cases, they want to start with dermo resistance and overlay MSX resistance on top.
  • They are proceeding with Line (variety) development.
  • Nine lines, 7 replicates, 4 sites vary in salinity and disease.
  • Selection is different at each site.
  • Family selection is taking place.
  • Louisiana stocks (no resistance to MSX, some resistance to dermo) do worse than other stocks.
  • Family selection
  • They work with 50 families every year, and 100 out in the field right now.
  • There is nice variation between families, and the mean survival is similar to the lines.
  • As a service, they make the broodstock that they’ve improved available to the industry as they need them.

There is remote setting on cultch for public-private oyster restoration. The goal is to get the industry involved with restoration.

The objectives in the Allen Laboratory in 2005-2006 are:

  • Remote setting at two industry sites and VIMS.
  • Plant the shell and determine the survival rate of the spat.
  • Establish 4 more settling stations.
  • Season synopsis—In all they have produced:
  • 39 million eyed larvae
  • 2 setting stations and VIMS
  • 21% setting rate—6.1M spat have been deployed.
  • There is a 40% survival rate in the field (14 days), approximately 2.4million 5mm spat
  • 2006-07 goals are:
  • 270M eyed larvae with increased hatchery capacity
  • 6 setting stations plus VIMS
  • With similar results, 21million 5mm spat should survive.

Economics

  • NOAA pays hatcheries “incentives” to get them involved with this program.
  • The projects keep people employedin the aquaculture industry by providing seed for restoration.
  • Hatcheries have to sign up for a 2 year stint in which they receive subsidies. VIMS will work with individual hatcheries to assist in eyed larvae production
  • After two years, market rates are charged.
  • For whole program (including monitoring and oversight), each 5mm spat that is set costs 1.2 cents.

Kim Reece (VIMS) initiated a general discussion on Genetic Considerations in Shellfish Restoration – a very timely topic with activities of various types ongoing around the country. She initiated the discussion with a prepared presentation outlining the general concerns with a series of questions and areas for research.

Shellfish vs. other taxa

Are the restoration genetic issues for shellfish different than they are for restoration of other organisms including finfish?

  • The answer appears to be “Yes.” Shellfish fecundity can be orders of magnitude greater than that in finfish and the mutational load is much higher.
  • It is harder to flood a river with the offspring of a single salmon.
  • Shellfish have less local adaptation than finfish. There is nothing close to the salmonids, but not panmictic in Chesapeake Bay.

We have no idea what our effective population size is. It could be as low as 40 or as high as thousands or hundreds of thousands.We need to look at selection, hatchery bottlenecks, and hatchery effective population size.

  • Founder effect and drift transiently generate correlations among alleles at unlined genes (gametic phase disequilibrium) exists.
  • Magnitude of correlations depends on Ne (Waples 1991 Method, 8 microsat loci binned into biallelic data).
  • Even in the primary lines in DEBY, we see very small effective sizes (4-6)!
  • 2 million seed oysters.
  • The contribution is proportional to the numbers of seed planted, but we’re not planting by scattering them, but clustering them to increase fertilization efficiency to get a better outcome for the money and time spent.
  • We’ll get more oysters out there but we’ll also have more than a 1-2% contribution within the river, per generation…more like a 10% contribution.
  • If the effective population size in the river was 1500, it drops down to 500 after only one generation.
  • This may look bad, but its local and its temporary.
  • If we think of a river as an experiment with an oyster an that we can track, it doesn’t look so bad…If it had a negative effect, it is local and it will eventually recover. Could this be better than using ariakensis…..?
  • Are there differences in life histories and reproductive biology that affect how we view the genetic impacts?
  • What can we learn from previous restoration efforts with other species in the marine environment?

How are the genetic issues in aquaculture or restoration similar? Are they different?How much genetic variation is appropriate for each application?

Olympia Oyster (Ostrea conchaphila)

  • Our native oyster on the west coast
  • We don’t know the population structure. The more we look, the more remnant populations we find.
  • We have a colorful history of translocating entire populations from one bay to another bay.
  • Restoration is going on right now.
  • Our current focus isto put out habitat for natural spat to settle.
  • Other focus is supplementation with relatively local broodstock.
  • Question: Have our bays have changed so much and does it matter if our spat come from ancestral populations?

DEBY oysters

  • are a disease resistant cultured line of eastern oysters from Delaware Bay.
  • They were used in a small initial restoration project—not because they are the super restoration oyster, but because they have a genetic signature and could be tracked.
  • Should they be deployed in all these reefs in all these other bays?
  • Do well in aquaculture—would they for restoration? They are great for aquaculture, that is what they were developed for.
  • The growers didn’t do a good job of telling the general public what these disease resistant strains were for….the general public made a jump….if they have better oysters for aquaculture, why don’t we use these for restoration?
  • We need to make sure people understand these issues.

Any time we perform restoration, we need to ask whether we are :

  • Swamping the natural population,
  • Supplementing the natural population, or
  • Losing the supplemented animals as background noise to the natural population?

Meeting Notes for Sunday, March 26th recorded by Kristie Strauss (University of Washington)

Sunday March 26th was the second day of the meeting. Kim Reece was named as Chair for next year, with Kristie Strauss named as Secretary. We continued with additional reports by the laboratories.

Marta Gomez Chiarri (University of Rhode Island) began the session with her topic, Host-Pathogen infection in Oysters. She spoke about:

  • Mechanisms of disease resistance
  • Evolution and resistance in oyster strains (natural and experimental infections)
  • Identification molecules differentially expressed in infected/ non infected; resistant/ susceptible lines (genomics, proteomics, candidate approach)
  • Molecules in immune response (inhibition, over-expression)
  • Mechanisms of pathogenesis (histology, immunological assays)
  • Her goal is to integrate all of the above with mapping to identify markers for selection and to develop of new, disease resistant oyster strains.

She has carried out testing for disease resistance in oysters in RI.

  • 3 strains have been tested at 3 farms plus in the laboratory.
  • Experimental infections have been tested at 3 different sizes (larvae, 5-9mm, 20-27mm).
  • 3 bacterial pathogens were used for challenge: (Vibrio, (JOD),
  • Not all oysters created equal---There is a huge difference in time to mortality depending on size.
  • It took 24 hours for larval oysters to die, and 13 weeks for 22-27mm size.
  • Lines were evaluated for time to 80% mortality. Differences between the lines—NEH larvae did better than the local strain (>48h vs 24h). NEH also did better (to 43 days versus 8 days) than 5-9mm animals for 22mm animals, NEH and flowers were the same (>13 weeks) but were better than the local strain.
  • Survival was determined by
  • Temperature
  • Pathogen
  • size/stage of oyster
  • line of the oyster

Oysters have definite responses to infection.