Location: Forage Seed and Cereal Research Unit
2018 Annual Report
Objectives
The overall goal of this project is to improve stocks of Pacific oysters with desirable performance traits for U.S. West Coast hatchery and farm production under increasingly difficult environmental conditions of ocean acidification and the threat of the introduction of new, highly pathogenic variants of the oyster herpes virus (OsHv-1 µVar).
Objective 1: Determine the effects of selective breeding in Pacific oysters for improved larval performance in acidified seawater and resistance to oyster herpes virus (OsHV-1).
Subobjective 1A: Improve performance of oyster larvae in hatcheries exposed to ocean acidification (OA).
Subobjective 1B: Improve resistance of Pacific NW oyster stocks to microvariants of the oyster herpes virus OsHv1.
Approach
Utilize quantitative and molecular genetics techniques to improve the performance of Pacific oysters by 1) including larval traits (growth and setting success) in the selection index of the Molluscan Broodstock Program (MBP), and 2) including individual pedigreed oysters in the breeding program that are selected for high larval growth rates and settlement under potentially adverse commercial hatchery conditions. Collaborate with researchers in France and the Universities of Washington and Maryland to screen selected U.S. West Coast oyster stocks for resistance to new microvariants of the oyster herpes virus (OsHv-1 µVar) that have decimated Pacific oyster farms abroad. Estimate heritabilities of MBP families for resistance to OsHv-1 based on lab exposure experiments and use this information to develop effective breeding strategies to produce OsHv-1 resistant stocks of Pacific oysters for U.S. west coast farms.
Objective 1: Determine the effects of selective breeding in Pacific oysters for improved larval performance in acidified seawater and resistance to oyster herpes virus (OsHV-1).
Subobjective 1A: Improve performance of oyster larvae in hatcheries exposed to ocean acidification (OA). Research Goal: Implement breeding strategies to improve the survival and growth of Pacific oyster larvae in hatcheries that are dependent on sources of acidified seawater. Experimental Design: MBP stocks will be selected for improved larval performance in lab and commercial hatchery conditions. Unselected control populations will be maintained to assess the genetic improvement. Three changes to current MBP practices need to be implemented to meet these objectives: a) include performance traits for larval (hatchery) growth and settlement success in a modified MBP selection index to identify families with high breeding values (BLUPs) for both hatchery and on-farm traits. b) include individual pedigreed oysters as broodstock in the production of MBP’s cohorts that are selected from mixed families for high larval growth and settlement success under commercial hatchery conditions, as well as high growth rates at farm test sites. c) include randomly mated controls in MBP cohorts to determine the response to selection from one generation to the next.
Subobjective 1B: Improve resistance of Pacific Northwest oyster stocks to microvariants of the oyster herpes virus OsHv1. Research Goal: Prepare the U.S. West Coast oyster industry for a possible accidental introduction of a microvariant of OsHV-1 µVar by developing resistant oyster stocks. Experimental Design: a) In laboratory trials, determine the potential for selection for resistance to OsHV-1 µVar infection and compare results with exposure to the Tomales Bay OsHV-1 strain. b) Formulate optimal breeding strategies to develop OsHV-1 and OsHV-1 µVar resistant broodstock.
Progress Report
This report documents progress for project 2072-31000-005-00D, which started in October 2017 and continues research from project 2072-31000-004-00D, “Determine Genetic Diversity and Develop Tools for Genetic Improvement of Oyster Stocks for the Pacific Northwest."
Reduced carbonate saturation states, known as ocean acidification (OA), cause larval oysters in particular to have problems when forming/secreting their shells and when they metamorphose and settle to the substrate. U.S. West Coast commercial shellfish hatcheries are dependent on sources of seawater that are frequently acidified when deep hypoxic seawater is brought to the surface by upwelling or mixing events. Shellfish hatcheries now buffer their seawater by adding carbonate, but larval production is still inconsistent, so other factors associated with the incoming seawater such as microbial flora and other inorganic and organic compounds may also be important. All of these factors contribute to stress during periods of high energetic demand as larvae are building shell or metamorphosing to settle to the substrate and increase mortality at these critical points.
Research for Sub-objective 1A is designed to 1) examine whether family selection can be used to improve general performance under these adverse conditions including acidified water in commercial shellfish hatcheries and 2) incorporate larval hatchery performance traits (growth and settlement success) into the ongoing Molluscan Broodstock Program (MBP) selection program.
The change in genetic composition of larval oyster populations reared in ambient and high partial pressure of carbon dioxide (pCO2) seawater conditions was investigated and comparisons made between selectively bred oysters from the MBP and wild counterparts from Willapa Bay, Washington. A pooled sequencing analyses of genome-wide single nucleotide polymorphism (SNPs) in early veliger larvae (48 hours post fertilization) and settled spat (22 days post fertilization) reared in both conditions suggests that MBP stocks produce more genetically ‘stable’ larvae than wild genotypes overall, with ~20% fewer allele frequency differences in MBP lines relative to wilds across this period. Additionally, wild stocks appeared to be ~two times more ‘genetically sensitive’ to high pCO2 conditions, with ~40% of all SNPs exhibiting a change in allele frequency owing to high pCO2 culture, compared with ~23% of SNPs in MBP groups. These findings are in agreement with the observed phenotypic advantage of MBP stocks in hatchery and laboratory conditions reported earlier and reinforce the value of breeding as a tool to combat the negative effects of OA for commercial shellfish aquaculture.
Additional research to address Sub-objective 1A investigated gene expression associated with shell formation in larval oysters reared in ambient and high pCO2 seawater. The analysis identified 55 genes across two experiments that correlated to shell formation, all of which were under-expressed in high pCO2 treatment groups. These genes were categorized into four major functional groups: 1) Metabolic Functions, 2) Transmembrane Proteins (transporters), 3) Shell Matrix Proteins and 4) Protease Inhibitors. This analysis reveals the highly orchestrated genetic network contributing to this vital developmental process and provides insights into the mechanisms of calcification in bivalves and how these processes are affected by OA conditions.
In order to apply these findings to breeding gains, fertilized eggs from 50 full-sib crosses created in 2018 for MBP’s cohort 27 were proportionally mixed and cultured at ambient (~400 parts per million (ppm)) and high (~1600 ppm) pCO2 levels through settlement. Surviving spat from these cultures were planted alongside the MBP cohort at field sites in Yaquina Bay, Oregon, and Willapa Bay, Washington, in spring 2018. After two years of culture in the field, the largest 200 individuals from these mixed family groups at each site (that are derived from larvae that perform well under OA conditions, as well as post-metamorphic stages that grow well to harvest size) will be genotyped to identify their pedigree and, after inclusion of separate family performance data, will potentially be used as broodstock to create the next MBP generation. This trial will provide additional quantitative genetic information and selected individual broodstock to improve the breeding program for both OA-resistant larvae as well as high-yielding larger oysters.
Outbreaks of Pacific Oyster Mortality Syndrome (POMS) caused by new microvariants of the oyster herpes virus have become a global problem for Pacific oyster farmers, killing large proportions of juvenile and adult oysters in Ireland, New Zealand, Australia and France. Selective breeding efforts in France have been successful in increasing oyster herpes virus (OsHV-1) resistance after four generations of mass selection. Sub-objective 1B seeks to develop similar herpes-resistant Pacific oyster broodstock in the U.S. to mitigate any potential negative economic impacts should an OsHV-1 microvariant be introduced to the U.S. West Coast or the original variant spread beyond California.
To address this issue and take advantage of pre-existing knowledge developed to test oysters in France, where one of these microvariants is present, three-month old oyster spat from 70 bi-parental families raised in the MBP hatchery (Newport, Oregon) were shipped to French Research Institute for Exploitation of the Sea (IFREMER, La Tremblade, France) in May 2018 where plate and bath assays for OsHV-1 mortality were conducted. In the plate assay, spat from each family were placed in plates with either OsHV-1 contaminated seawater or uncontaminated seawater and mortality was measured over seven days. The virus used was the French microvariant of OsHV-1. Genetic variation in mortality among families was found and accounted for 90% of the overall mortality variation. No mortality was observed in control plates not exposed to the virus. In the bath assay, spat from each family were placed in mesh bags and the bags were placed in an aquarium with virus contaminated seawater. The correlation between the bath assay mortality and plate assay mortality was 0.5. This is a very important result because it indicates that some MBP families are more resistant than others and like the previous French tests, it indicates that selection for resistance to OsHV-1 is possible. This represents an important first step towards protecting the U.S. West Coast industry from economic impacts from this potential threat.
Experiments with the French microvariant of OsHV-1 were repeated in the Aquaculture Pathology laboratory at the University of Arizona (Tucson, Arizona) in June 2018. Genetic variation accounted for 40% of the variation seen in overall mortality. There was a weak correlation with the French bath assay mortality and no correlation with the French plate assay mortality. In addition to the bath assay conducted on all 70 families, a plate assay was conducted in Arizona with a subset of the 10 most resistant and the 10 most susceptible families found in the French plate assay. Here, the French microvariant of OsHV-1, the Australian microvariant of OsHV-1, and Tomales Bay, California variant of OsHV-1 were tested. The Tomales Bay strain mortality among families was not significantly different than the control, while the French and Australian strain mortality levels among families were significantly different. The correlation between the French and Australian phenotypes was 0.5, while the correlation between the French OsHV-1 plate assay conducted in France and the French OsHV-1 plate assay conducted in Arizona was 0.4. This suggests that selection for resistance to one microvariant strain will provide resistance to other microvariant strains.
The same 70 MBP oyster families that were tested as spat in the French and Arizona labs have been planted out in Tomales Bay, California, in a field trial to observe mortality of these families under natural conditions. In late July, after a mortality event had been reported in Tomales Bay by Hog Island Oyster Co., mortality data were collected and about 100 tissue samples were taken of live and dying oysters for assessment of OsHV-1 levels. Results from this field trial will determine whether selection for microvariant strain resistance in the lab can be used to select for resistance to the non-microvariant strain endemic to Tomales Bay, under field conditions.
Accomplishments
Review Publications
De Wit, P., Durland, E., Ventura, A., Langdon, C.J. 2018. Gene expression correlated with delay in shell formation in larval Pacific oysters (Crassostrea gigas) exposed to experimental ocean acidification provides insights into shell formation mechanisms. BMC Genomics. 19:160. https://doi.org/10.1186/s12864-018-4519-y.
De Melo, C.M., Morvezen, R., Durland, E., Langdon, C. 2018. Genetic by environment interactions for harvest traits of the Pacific oyster Crassostrea gigas (Thunberg) across different environments on the West Coast, USA. Journal of Shellfish Research. 37(1):49-61. https://doi.org/10.2983/035.037.0104.
