We are currently seeking a post-doctoral scientist to join our research group where they will help lead efforts focusing on the impact of environmental change on DNA methylation in marine invertebrates. One of the primary projects is an effort examining how ocean acidification impacts genetic and epigenetic variation in oysters. This person should have experience working with marine invertebrates and high-throughput sequencing analysis. Prior experience with RAD-seq and bisulfite sequencing would be preferred. The candidate should also be enthusiastic about open science. For more information on ongoing research efforts and products please visit robertslab.info. The term of this appointment will be for one year with the possibility of extension to two years. Interested applicants should submit a curriculum vitae, a succinct description of their doctoral research, and list of three references to Steven Roberts (email@example.com).
Below is a post from Yaamini's Notebook
Today we went out to NOAA's Manchester Facility to sample our oysters before placing them in our ocean acidification system! We collected length and weight information for twenty oysters, and extracted adductor, ctenidia (gill), mantle and gonad tissues. Gonad tissue was put in histology cassettes and then placed in fixatives.
Here are some photos from our time at Manchester!
Getting excited to shuck Pacific oysters as large as my face!
We layed out each oyster to weigh, measure and extract tissue.
Some of the oysters were so large we had to hammer them open.
Once open, we extracted four tissue types. Then we removed the body and reweighed the empty shell.
These are the histology cassettes we used to hold gonad samples. Pictured here are the samples for the Olympia oysters. The Pacific oyster samples were tissue pieces, as opposed to the whole gonad.
Megan, Laura and Olivia working on Olympia oysters!
Dead oysters are gross.
Meanwhile, Steven worked on setting up some plumbing.
You can find the rest of the photos I took on my Flickr.
Overall it was an extremely productive day! We'll be back out on Feb. 8 to set up the ocean acidification system.
We recently finished and UW Royalty Research Fund Project on Maturation processes in the marine mollusc, Panopea generosa. Here is a summary of internal report.
The overall goal of this project is to develop a fundamental understanding of processes controlling marine mollusc reproductive maturation. In order accomplish this goal the specific research objectives of this proposal were to 1) characterize tissue specific transcriptomic resources for the geoduck and 2) identify proteins that play a role in geoduck reproductive maturation.
The first step in this project was collecting clams at different reproductive stages as determined through histological analysis. Gonadal tissue from 70 geoducks was sampled in batches of about eight per week over the span of two months from November 2014 to early January 2015. Hundreds of images were analyzed and reproductive status was determined for each individual.
Based on histological determination of reproductive maturational stage, seven female and six male paraffin-embedded gonad samples were selected for construction of RNA-seq libraries. A total of 443,468,476 reads were obtained and the de novo assembly resulted in a total of 153,982 transcript contigs with a mean contig length of 660 bp and an N50 value of 1015 bp. In comparing our contigs with oyster sequences whose expression changed during gonad development in 161 matched including geoduck sequences corresponding to genes expressed in gonads in early gonad developmental stages (7), with increasing expression during spermatogenesis (44), with increasing expression during oogenesis (31) and genes with varying expression level during gonadogenesis in both sexes (79).
Proteomic profiles were determined for the primary reproductive maturation stages in both male and female clams using data dependent acquisition (DDA) of gonad proteins. This approach yielded 3,627 detected proteins across both sexes and three maturation stages. This is a significant escalation in the understanding of proteomic responses in maturation stages of marine mollusks. Based on the DDA data, 27 proteins from early- and late-stage male and female clams were chosen for selected reaction monitoring (SRM). The SRM assay yielded a suite of indicator peptides that can be used as an efficient assay to non-lethally determine geoduck gonad maturation status.
Non-metric multidimensional scaling plot (NMDS) of geoduck gonad whole proteomic profiles generated by data dependent acquisition. Gonad proteomes differ among clams by both sex (male = orange, female = blue) and stage (early-stage = circles, mid-stage = squares, late-stage = triangles; p<0.05). data-preserve-html-node="true"
Impact of Award
Beyond contributing to the fundamental knowledge of marine mollusk reproduction, this award produced numerous publications and provided basis for further funding and proposal submissions. In addition the transcriptomic data was the basis for the course: Bioinformatics for Transcriptomic and Epigenomic Analyses - Centro de Investigación Científica y de Educación Superior de Ensenada, B.C. (CICESE) 19-24 October 2015
Currently two projects have been funded that were based on this project and others have been submitted. Funded projects include: Proteomic response of shellfish to environmental stress; Department of Natural Resources $107,805 and Elucidating the physiological and epigenetic response of tetraploid and triploid Pacific Oysters to environmental stressors; NOAA $178,898. Submitted proposals include one to NOAA on the development of new clam species for aquaculture.
Crandall, Grace; Roberts, Steven (2016): Reproductive Maturation in Geoduck clams (Panopea generosa). figshare. https://dx.doi.org/10.6084/m9.figshare.3205975.v1 Retrieved: 14 41, Dec 23, 2016 (GMT) This fileset includes a research paper describing reproductive maturation in geoduck clams with 200 images of gonadal histological sections and associated datasheets. Downloads = 1761.
Emma B. Timmins-Schiffman, Grace A. Crandall, Brent Vadopalas, Michael E. Riffle, Brook L. Nunn, Steven B. Roberts (2016) Integrating proteomics and selected reaction monitoring to develop a non-invasive assay for geoduck reproductive maturation bioRxiv 094615; doi: https://doi.org/10.1101/094615
[Data] Transcriptomic profiles of adult female & male gonads in Panopea generosa (Pacific geoduck).
Panopea gonad transcriptome
Open Science Framework Project
[Data] Geoduck (Panopea generosa) gonad DDA LC-MS/MS https://www.ebi.ac.uk/pride/archive/projects/PXD003127
[Code] Source Code for GO Analysis in Geoduck Gonad Background https://github.com/yeastrc/compgo-geoduck-public
[Data] Geoduck (Panopea generosa) gonad DIA LC-MS/MS https://www.ebi.ac.uk/pride/archive/projects/PXD004921
[Data] Selected reaction monitoring of geoduck gonad peptides to develop biomarkers of reproductive maturation status http://www.peptideatlas.org/PASS/PASS00943
[Data] Selected reaction monitoring of geoduck hemolymph peptides to develop biomarkers of reproductive maturation status http://www.peptideatlas.org/PASS/PASS00942
Below is a copy of the final report submitted to SeaGrant
Alleviating Regulatory Impediments To Native Shellfish Aquaculture
A significant impediment to sustainable aquaculture is the lack of proper information to predict the impacts of culturing native shellfish species for restoration and commercial production. As a result, expansion and growth of domestic aquaculture is constrained and may be halted by management directives that restrict distribution of hatchery derived native shellfish until the potential interactions are better understood. This project set out to provide essential information on local population structure of Olympia oysters such that restoration efforts can be carried out in an efficient and environmentally sustainable manner. The overarching experiment associated with this project included a reciprocal transplant of oysters among three sites across Puget Sound, along with the three populations also being grown out at a fourth site. In addition to the array of data we produced about differences in populations of oysters in Puget Sound, we also developed procedures and resources that will contribute to aquaculture practices and fundamental biological investigations. Specifically, as part of the research activities we were able to
- Develop an anesthesia procedure for non-lethal sampling
- Define improved breeding procedures for conservation aquaculture
- Develop significant genomic resources for the Olympia oyster
As for the characterization of local populations of Olympia oyster in Puget Sound, we identified numerous differences that are critical for consideration of conservation efforts. For this final report we will outline the primary findings from a number of studies carried out during the tenure of this project including
- Field performance of oysters in the transplant experiment
- Differential response to stress of oyster populations as measured by gene expression
- Growth and survival of F2 oysters under hatchery conditions
- Epigenetic and genetic degree of local population differentiation
In the remainder of this report we will first describe the findings of these four core studies followed by how these combined data will be used be resources managers (Section A). Then we will describe the procedures and resources developed as part of work that have immediate impacts beyond our study area (Section B).
A.1. Field performance of oysters in transplant experiment
For this study three geographically separated, discrete groups of O. lurida within Puget Sound were selected. These animals were brought to a hatchery, spawned, and the offspring from each population outplanted back to the bays selected. This approach enables observations about how differing natural environments with resident oyster populations may affect both local and non local populations over time.
Three bays (ie. Fidalgo Bay, Dabob Bay, and Oyster Bay) within Puget Sound were selected for this experiment based on presence of resident O. lurida populations, distance from other bays, and latitudinal position. Fidalgo Bay is the most northern site and experiences cooler year round conditions. This bay is also more directly influenced by the Strait of Juan de Fuca, allowing colder sea water directly from the Pacific to mix with bay waters. Dabob Bay is located within Hood Canal, an area of Puget Sound distinctly separated from the rest of the sound. Oyster Bay is the southern most site and known for its historically large numbers of O. lurida.
In summary, oysters from Dabob Bay had higher survival at all sites but lower reproductive activity and growth (Figure 1 and 2). Oysters from
Oyster Bay demonstrated greater reproductive activity at all sites with moderate growth and survival (Figure 1). Together these data suggest the existence of oyster population structure within Puget Sound and provide information on how broodstock should be selected for restoration purposes.
Figure 1. Proportion survival for three oyster populations at four locations; Southern site (A), Central site (B), Hood Canal site (C), and Northern site (D). Lowercase letters (a, b, c) are significant differences.
Figure 2. Percent Brooding Females from Each Population at Each Sample Date at Oyster Bay (top), Fidalgo (middle), and Manchester (bottom).
Percent determined by number of brooding females (Br) divided by number of open oysters (T) or %=(Br/T)*100.
Differences in life history traits among oyster populations grown in different locations within Puget Sound, WA suggest adaptations possibly linked with environmental cues. High survival, low growth, and low reproductive activity of the Dabob Bay population is likely due to extreme environmental variation at their home site leading to improved stress resilience. The greater proportion of brooding females in the Oyster Bay population and reduced environmental energy needed to induce peak spawning may be related to positive selection pressure for early spawners due to warmer temperature trends at their home site. Findings from this study indicate possible local adaptation in two of the three populations observed but there may be other factors dictating observed phenotypes, including the fact that we do not know broodstock used here were from local parents.
This complete work is published: Heare JE, Blake B, Davis JP, Vadopalas B, Roberts SB. (2015) Evidence of Ostrea lurida (Carpenter 1864) population structure in Puget Sound, WA. PeerJ PrePrints 3:e1986 https://doi.org/10.7287/peerj.preprints.704v4
A.2. Differential response to stress of oyster populations as measured by gene expression
Based on the results of the transplant experiment and apparent differences in stress responses, we examined expression of genes associated with growth, immune function, and gene regulatory activity in oysters from the three populations following temperature and mechanical stress. We found that heat stress and mechanical stress significantly changed expression in molecular regulatory activity and immune response, respectively. We also found that oysters from Oyster Bay had the most dramatic response to stress at the gene expression level.
More specifically oysters from each population were brought to the lab and subjected to acute temperature stress (38°C water for 1 hour; n=14 per population), mechanical stress (1000 rpm x 5 min; n=14) or served as controls (maintained at 8°C; n=14). Oysters were placed back in 8°C seawater and sampled at 1 hour post stress (n=8). Six oysters were also monitored daily for 14 days to assess survival. Ctenidia and mantle tissue was dissected and stored in RNAzol RT (Molecular Research Center, Inc.) at -80°C for later analysis. Quantitative PCR was carried out on the nine genes listed in table 1.
Table 1. Table of qPCR Primers for genes of interest
Contrary to our hypothesis, oysters from Oyster Bay demonstrated the greatest difference in response to stress compared to the other populations. Specifically oyster originally from Oyster Bay, had an increase in H2AV expression during heat stress as compared to control, a decrease in BMP2 and GRB2 upon mechanical stress, and differences in HSP70 expression between the two treatments. While only speculation at this point, the gene expression pattern differences observed here with oysters from Oyster Bay coupled with corresponding field-based observation that this population has the greatest reproductive activity, could indicate this population has a greater ability to effectively respond to energy demands. This trait could certainly be perceived as advantageous for restoration purposes. On the other hand, the results of the gene expression analysis does suggest population differences. Caution should be used in using non-local stocks when structure exists, as it is possible to have supplemented oysters outcompete the native population or to create hybrids that are ultimately less fit than the native counter parts.
This complete study is published: Heare JE, White SJ, Roberts SB. (2015) Differential response to stress in Ostrea lurida (Carpenter 1864) as measured by gene expression. PeerJ PrePrints 3:e1994 https://doi.org/10.7287/peerj.preprints.1595v1
A.3. Growth and survival of F2 oysters under hatchery conditions
To test if differences in fitness observed in the reciprocal transplant experiment were consistent in F2s raised in controlled, common conditions, we conducted a common garden study in Summer 2015 with ~100 F1 oysters from each population at the K. K. Chew Center for Shellfish Restoration and Research in Manchester, WA. Concerns over transgenerational effects would be mitigated if F2 animals reared in the same environment showed similar interpopulation differences as the F1s. Oysters from each population were spawned in 5 groups of 20 oysters to promote genetic diversity. Newly released larvae were combined based on population and reared in 100 L tanks. After reaching >160 μm in size (as determined by filtering over mesh screens), larvae were moved into a second tank and raised until close to metamorphosis (> 224 μm). Juveniles reaching metamorphosis at the same time were established on 10 cm x 10 cm PVC tiles before being placed in nearby Clam Bay to grow for 10 weeks.
Reproductive output and growth rate were used as fitness proxies. Reproductive output was estimated by counting the number of spawned larvae from each group (Figure 3). Similar to the reciprocal transplant experiment, oysters from Oyster Bay showed the earliest and greatest reproductive output. To measure larval growth rate, ~700 one-day old larvae were raised in 1 L beakers for 2 weeks with daily water changes, with 6 replicates per population. Samples of 100 larvae were taken 3 times throughout this period and measured using a microscope and ImageJ. In analysis of protoshell length in 20 seven-day-old larvae from each population, size varied significantly among populations and the larvae from Oyster Bay were significantly larger than those from Oyster Bay (Figure 4). After establishing on the PVC tiles, juveniles were measured at three time points over 10 weeks, from August 2015 to November 2015. A measurement of approximately 30 juvenile oysters from each population in October was consistent with the larvae measurements and reciprocal transplant results in that oysters from South Sound were significantly larger than those from Hood Canal (Figure 5). These results are concordant with the reciprocal transplant indicating that phenotypic differences in size may indeed be due to genetic factors and not environmental effects.
Figure 3. Reproductive output as measured by number of larvae released.
Figure 4. Protoshell length in 27 day old larvae.
Figure 5. Size measurements of oysters on tiles.
A.4. Epigenetic and genetic degree of local population differentiation
In conjunction with examining growth and survival of F2s in order to determine if population difference were independent of environmental factors, we also characterized genetic and epigenetic differences in our target populations. To examine genetic differentiation of the three populations we characterized single nucleotide polymorphisms (SNPs). Using a 2bRAD reduced-representation sequencing approach, we sequenced 279 individuals with roughly equal representation of all three populations. To date we have identified 3,510 SNPs confidently genotyped in at least 50% of samples examined.
To get an estimate of population differentiation among our three populations, we thinned our dataset and selected 882 SNPs genotyped in at least 70% of 59 individuals. Using Weir and Cockerham's 1984 FST estimator for each SNP, we found a mean FST of 0.0528 for all 59 individuals as three subpopulations. Eight SNPs had elevated values of FST over 0.4. Average pairwise FST was highest between the Oyster Bay and Hood Canal populations (0.0668), with 24 SNPs having an FST over 0.4. Pairwise FSTs in comparing Fidalgo to Hood Canal and Oyster Bay to Fidalgo were 0.0433 and 0.0485, respectively.
Together these results indicate there is population differences, though some gene flow could exist. There are also specific loci that are segregating more among populations and could be associated with population specific traits.
For the first time in this species, we also characterized the potential epigenetic component of population differences. To confirm that the epigenetic signature of DNA methylation persists independent of environment, we characterized whole genome DNA methylation via bisulfite sequencing of Oyster Bay and Hood Canal oysters both grown out in Clam Bay. Nine individuals were sequenced and there is a clear population specific signature that likely contributes to phenotypic differences in populations (Figure 6 & 7).
Figure 6. CpG methylation clustering cladogram of oysters from Oyster Bay (blue) and Hood Canal (red) grown in Clam Bay.
Figure 7. CpG methylation PCA analysis of oysters from Oyster Bay (blue) and Hood Canal (red) grown in Clam Bay.
A.5 Implications for resource managers
Our results certainly have important implications for restoration of Olympia oysters within Puget Sound, WA. There are a number of ways that these findings could be used in generating restoration strategies specific to Puget Sound and in the face of climate change. Based on the fact that Dabob Bay oysters had the lowest mortality, use of the most robust population for broodstock may increase chances for outplant survival. Generally, this approach would dictate organisms should be used from home environments that experience persistent stressful conditions. An alternative approach managers might take given the current findings is to take the population with the greatest reproductive output (Oyster Bay) and use it as a source of broodstock. This would increase the likelihood of juvenile recruitment and ultimate restoration of the species, while also producing more offspring for outplant. Having a strong understanding of population related phenotypes creates another option for restoration efforts. An assisted gene flow strategy that incorporates the outplanting of populations known to contain phenotypes fit for the new environmental parameter and have them interbreed with resident populations. It is highly debated whether such a strategy would have benefits that outweigh the drawbacks, such as possible outbreeding depression, but should be considered for restoration efforts facing a variety of climate change scenarios.
Ultimately, what these studies demonstrate is that population structure can and does exist on a relatively small geographic scale and thus moving oyster populations to locations where remnant stocks exist could be disadvantageous. When population structure exists, there should be caution with respect to moving populations as: 1) transplanted populations could overwhelm locally adapted remnant resident populations, and possibly not persist themselves, 2) transplanted populations might not survive in new location and thus wasting resources required for restoration, and 3) transplanted populations could interbreed with remnant population and thus result in overall reduced fitness through outbreeding depression.
B. Additional Products
In addition to the array of data we produced concerning differences in populations of oysters in Puget Sound, we also developed procedures and resources that will contribute to aquaculture practices and fundamental biological investigations.
B.1. Develop an anesthesia procedure for non-lethal sampling
One key metric to understand adaptive divergence in Olympia oysters is reproduction, a fitness component. Reproductive timing and effort differences among populations reared in a common environment can indicate local adaptation. Targeting these metrics heretofore required sacrificing many Olympia oysters to find a subset of active brooders. We developed a simple, nonlethal field procedure to anesthetize Olympia oysters, causing them to gape sufficiently to ascertain brooding status, enable collection of broods, and/or facilitate tissue sampling. Briefly, the method involves 45 min exposure to air, followed by immersion in 83 mM MgSO4 for 45 min. After testing the method in the lab, the method was used in our field study on a weekly basis to obtain reproductive timing and population proportion data. Repeated exposure of Olympia oysters to the anesthetic resulted in no detectable mortality or adverse effects. The anesthesia method has been used in at least 3 other Olympia oyster research projects (B. Becker, UW Tacoma, personal communication; J. Barber, Swinomish Tribe, personal communication; D. Zacherl, CSU Fullerton, personal communication). The method and field application are currently being prepared for submission for publication.
Figure 8. Photograph of oysters subjected to anesthesia procedure prior to sampling.
B.2. Define improved breeding procedures for conservation aquaculture
Olympia oyster restoration in Puget Sound can in some cases be best realized using hatchery produced seed. It is important, however, to minimize genetic differences between hatchery seed and the wild population targeted for supplementation. A breeding plan was set up to try to ensure low differentiation between seed oysters derived from wild South Puget Sound broodstock, and a random sample of the population in South Puget Sound. The breeding plan involved creating a series of small breeding groups of wild adults, thus ensuring genetic diversity in the population of larvae released over a period of about 2 months. To further optimize genetic diversity, the larvae/juveniles from the breeding groups were reared separately until outplanting to enable even contribution from each breeding group.
We used 7 microsatellite loci to compare diversity metrics between a random wild sample and the seed destined for restoration. We also compared commercial hatchery production (Taylor Shellfish) to the wild sample, expecting to see lower diversity. We detected no significant differentiation between the south sound wild Olympia oysters and the Taylor hatchery produced seed. In contrast, we detected low level differences between the restoration seed and the wild adults. The same pattern was evident for the restoration seed produced in the following year. Based on these results, the currently adopted method for restoration seed production mimics the commercial production method used by Taylor Shellfish. Approximately 1200 broodstock are held in a common tank and all released larvae are collected. The primary difference between the commercial and restoration method is the period of collection: for the former, collection is terminated once sufficient larvae are released for production goals, while for the latter, the restoration hatchery collects released larvae over an extended period (ca 2 mo) to capture and retain genetic variation in spawn timing which is likely of adaptive significance.
B.3. Develop significant genomic resources for the Olympia oyster
As part of this project and complementary efforts we have been able to generate a wealth of genomic data including RNA-Seq, RAD-Seq, Bisulfite Sequencing, and Whole Genome Shotgun data. One transcriptome has been published (Timmins-Schiffman EB, Friedman CS, Metzger DC, White SJ and Roberts SB. (2013) Genomic resource development for shellfish of conservation concern Molecular Ecology Resources. doi:10.1111/1755-0998.12052) with all data publicly available at http://owl.fish.washington.edu/nightingales/O_lurida/. We have also developed a webpage to make these data more easily accessible (http://oystergen.es/olympia/). In addition we have created a genome browser where anyone can visualize these data or combine with other data (Figure 9). While much of these data provided core information for us to be able to characterize local population of Olympia oysters, we suspect these data will provide important resources for others involved in aquaculture, biological, and environmental studies.
Figure 9. Screenshot of Olympia oyster genome browser available at https://genomevolution.org/coge.
Over the next few weeks we expect a flood of Ostrea lurida DNA sequence data.
Some of the first to be available is raw data produced in order to generate a draft genome.
Files can be downloaded here via links below. Over the next month we will elaborate on the effort but wanted to make this available ASAP.
Please feel free to ask any questions in the comments.