Draft Summary of project needs to include:
Overview:
Intellectual Merit
Broader Impacts
Draft summary & outline submitted for class, 01-16-2017
Overview: This project seeks to elucidate Ostrea lurida's potential for phenotypic plasticity by examining transgenerational inheritance in response to dual climate stressors. By examining how genetic and epigenetic modifications over three generations correspond to climate resilience, we will reveal whether O. lurida populations may be able to persist in our rapidly changing world. We will condition O. lurida broodstock of known lineages in elevated temperature and dissolved CO2, breed them, rear the progeny in a common garden, then repeat the previous steps with the progeny, and continue through a third generation. We will compare overall larval quality, production and development, and will measure differential gene expression (transcriptome) and epigenetic markers (methylation, histone modification, transposable element expression) in broodstock gonads and larvae for each generation. We anticipate that O. lurida broodstock conditioned in elevated pCO2 and temperature will: differentially express genes compared to oysters reared in ambient conditions; present different rates and loci of histone modification, DNA methylation, or transposable element expression; produce a greater percentage of progeny that are more resilient to the dual climate stressors, as determined by growth rate and survival; and transfer heritable, climate stress related epigenetic markers to successive generation.
 
Intellectual Merit: A broadening body of work indicates that low pH and high temperature negatively affect fertilization and early life stages of many marine invertebrates. Oyster may, however, contain a unique capacity to keep pace with rapidly shifting climate stressors. The oyster genome is highly polymorphic, communities may differ significantly between generations via the Sweepstakes Reproductive Success hypothesis, and the newly emerging field of epigenetics suggests that adult responses to environment conditions can be passed on to offspring. Indeed, offspring of Sydney rock oysters (Saccostrea glomerata) exposed to elevated pCO2 during reproductive conditioning performed better in OA conditions compared to larvae of broodstock conditioned in ambient pCO2 levels. Still unknown, however, are the persistence of these adaptations through the generations, and the underlying mechanisms by which inheritance occurs. This project seeks to answer these questions, and will be the first to explore mechanisms underlying potential differential gene expression and transgenerational inheritance in oysters reared in dual climate stressors. Results will have implications on future community-level resilience in O. lurida, and other invertebrates with similar genetic polymorphism and epigenetic mechanisms. 
 
Broader Impact: The ecologic and economic void left by the near collapse of Ostrea lurida, the Northeast Pacific Ocean’s only native oyster, has spurred major efforts to restore populations along its historic distribution from Alaska to Baja California. While shoreline enhancement and seeding projects are making headway, there is growing concern that changing ocean conditions further threaten existing populations and may stymie restoration investments. This research will explore new territory of O. lurida epigenetic and transgenerational inheritance of climate stressor resiliency, with the primary goal to inform commercial and restoration hatchery breeders to select for OA- and high temperature-resilient oysters, or to induce an multi-generational “immune-like” response by exposing broodstock to future climate conditions. Secondly, results will inform further climate-related reproduction research in other species of wild and farmed bivalves such as Crassostrea virginica and Crassostrea gigas. Thirdly, a deeper understanding of CO2 and temperature related drivers in O. lurida reproduction will help to expose its vulnerability or adaptability to the climate model predictions of steadily decreasing ocean pH and increasing temperatures, and could have profound implications for wild community-level population dynamics. Finally, results from this project would need to be considered in ecological models, and restoration groups could refine selection processes for shoreline enhancement and seeding processes, or amplify efforts in locations with variable pH and temperature swings to allow for maximum adaptability.
 
Draft Proposal Outline
 
Cover Page:  Working title: Transgenerational epigenetic inheritance of climate adaptability in Ostrea lurida
Summary (1 page)
Overview: This project seeks to elucidate Ostrea lurida's potential for phenotypic plasticity by examining transgenerational inheritance in response to dual climate stressors.
Intellectual merit: A broadening body of work indicates that low pH and high temperature negatively affect fertilization and early life stages of many marine invertebrates. Oyster may, however, contain a unique capacity to keep pace with rapidly shifting climate stressors. Still unknown, however, are the persistence of these adaptations through the generations, and the underlying mechanisms by which inheritance occurs. This project seeks to answer these questions. 
Broader Impacts:This research will explore new territory of O. lurida epigenetic and transgenerational inheritance of climate stressor resiliency, with the primary goal to inform commercial and restoration hatchery breeders to select for OA- and high temperature-resilient oysters, or to induce an multi-generational “immune-like” response by exposing broodstock to future climate conditions.
Project Description (4 pages)
Background
- Oysters are ecologically vital to intertidal communities; oysters and ecosystem engineers
- Oysters are economically vital to many coastal communities in the United States, France, Australia, etc.
- Oysters aquaculture is extremely sustainable, and the industry has been identified as a NOAA priority, necessary to provide sufficient, efficient, and healthy protein sources in the future. See NOAA Marine Aquaculture Strategic Plan 2016-202
- Ocean acidification and rising ocean temperatures threaten many marine organisms. Calcifying invertebrates are at risk, and there has been a surge of research seeking to understand how populations will change in response to climate projections. Most research identifies fertilization and larval stages as the “bottleneck.” Oysters are intertidal animals, experiencing extreme environmental changes. As such, researchers are exploring how oyster genetic polymorphism, generational shifts due to Sweepstakes Reproductive Hypothesis, and epigenetic transfer of environmental memory may allow for rapid adaptation and resiliency to climate stressors.
Rationale: Researchers hypothesize that epigenetic mechanisms (methylation, histone modification, non-coding RNA, transposable elements) may increase the plasticity of oysters. Unknown is a direct linking of differential epigenomes, induced by climate stressor treatments, and rates of survival, reproductive success, and subsequent generational transfer of these successful epigenetic markers.
Goals/Questions 
- Do brooding oysters respond to climate stressors via epigenetic mechanism? If so, which ones? DNA Methylation, Histone Modification, lncRNA, transposable element expression, or other?
- Do different epigenomes (in response to OA and high T) correspond to fecundity?
- Are epigenetic traits passed on to their progeny?
- Can memory of environmental stress persist through generations? If so, what is the mechanism? Do they increase or decrease fecundity and resiliency in first or second, and/or third generations?
Approach
- In early winter 600 O. lurida hatchery-born (F1) individuals from known lineages will divided into 2 separate flow-through seawater culture tanks, and conditioned for ~6 weeks in two temperatures: ambient (~8degC), and high (~12degC).
- Directly subsequent to the temperature treatments, individuals will be moved into two pCO2 treatments and conditioned for ~6 weeks @ ambient temperature (~8degC): ambient (~400ppm), and high (~1000ppm). In both treatments water temperature will be gradually increased to ~10degC.
- Directly subsequent pCO2 treatments, oysters will be conditioned in “separate but equal” culture tanks, fed well, and temperature gradually increased to ~14degC, and monitored for spawning activity. Larvae from each treatment group will be collected and reared. Post settlement, oysters will be grown off-dock in ambient conditions, until the following season when the experiment will be repeated with these experimentally produced F2 individuals.
- Broodstock gonads & whole-body larvae will be sampled for:
* DNA
* Genetic variation
* Protein?
* RNA
Transcriptome
Bisulfite treated for methylation
Histone Modification
Transposable element location and expression
Outputs
- SAFS Thesis
- Publication
- Guideline memo to restoration and commercial hatcheries on potential method of selecting for climate-resilient family lines.
Budget: TBD
Budget Justification (1 page): TBD
References: TBD