Progress 09/01/23 to 08/31/24

Outputs
Target Audience: During this reporting period the target audience has been commericial aquaculture operations and the scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training has primarily involved two undergraduate students and that of a post-doctoral research associate. How have the results been disseminated to communities of interest? Primarily through online notebooks as well as direct conversations with commercial hatchery operators. Results will be presented at a regional aquaculture meeting next month. What do you plan to do during the next reporting period to accomplish the goals?Present results, continue to analyze data from controlled experiments, monitor commerical grow-out trials, and outplant more oysters for assessment.

Impacts
What was accomplished under these goals? During this reporting period a series of hatchery-based environmental conditioning activities (referred to as hardening) were completed with oysters out-planted onto three farms to evaluate performance. Additionally, climate resiliency was assessed in hardened oysters under controlled conditions. Early in the season, a cohort of oysters were subject to (a) daily thermal hardening for approximately two weeks prior to out planting in hanging bags on a farm in Sequim Bay, alongside a control group. These oyster will be assessed in early Autumn 2024. Later in the season oysters were subjected to a more diverse series of hardening conditions with oyster split between two commercial farms, one located in Willapa Bay and a second in the San Juan Islands. The combination of age classes and hardening conditions were as follows: b) small seed / daily thermal hardening, c) adult / daily thermal hardening, d) small seed / weekly thermal hardening, e1) large seed / weekly thermal hardening, e2) large seed / weekly zero salinity hardening. These hardening activities spanned from several weeks to months. For all hardening efforts controls were also outplanted. Oysters out planted in Willapa Bay will be assessed for survival and growth in early September 2024. Oysters out planted in the San Juan Islands have be subject to more routine monitoring. At this site we are currently observing a trend where oysters subjected to the daily hardening regime are surviving better than controls. To assess the effectiveness of hardening within a hatchery setting, four ages classes of oysters were examined: adults (40-100mm), juveniles (20-55mm), seed (6-15mm), and spat ()1-6mm). In order to assess hardening potential and environmental memory, oysters were subject to an initial chronic temperature stress, then subjected to a secondary stress to see if the response to secondary stress was impacted by initial exposure. During the chronic temperature stress period, oysters from each age class were exposed to a daily 25ºC temperature spike. This repeated for 14 days starting on October 1. Control oysters from each cohort were maintained at an ambient temperature of approximately 17ºC during the same time period. Adult oysters in stress conditions were also subject to mechanical stress at day 1, 7, and 48, which was induced by 15 minutes. At day 147 (7 weeks after initial chronic stress), both stressed and non-stressed adult, juvenile, spat, and seed were subjected to a secondary stress of 30 minutes in 32ºC. Gill tissue was sampled immediately after and placed into RNAlater. Spat and seed that were too small to dissect gill tissue were sampled whole with all tissue used in gene expression assays. To date we have analyzed gene expression data from seed and spat. There was no mortality during this trial. Hardening increased growth (compared to controls) in spat. Further there was a reduced transcriptional response in spat. Thesedata suggest temperature hardening of spat could result in a resiliency to later temperature stress whereby energy resources could favor growth. In a second experimental regime, juvenile oysters were subjected to four hardening conditions 1) high temperature (35°C), 2) high temperature and freshwater, 3) freshwater, and 4) poly I:C immersion. These were exposures that occurred every other day for about 3 weeks. Approximately 1 month after hardening, oysters were brought to the lab and subjected to metabolic assessment using resazurin at 18°C and 42°C. Resazurin is a dye that is commonly used in cell viability, and it works by changing color as an organism consumes oxygen. Survival was assessed during the resazurin assay and 24 hours later. To date, our analysis suggests metabolic activity of the 42°C oysters was higher in the control oysters than previously hardened oysters. In the near future we will assess the relationship of metabolic activity and survival however it appears higher metabolic rate directly correlates with mortality. Taken together these sets of controlled experiments suggest hardening does have an impact on resiliency, though dependent on age, hardening duration, and subsequent environmental conditions.

Publications

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:During this reporting period the target audience has been commericial aquaculture operations and the scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training has primarily involved that of a post-doctoral research associate. How have the results been disseminated to communities of interest?Primarily through online notebooks as well as direct conversations with commercial hatchery operators. What do you plan to do during the next reporting period to accomplish the goals?We plan to provide seed that have experienced different environmental conditions, and/or are offspring ofbroodstock have experienced differential conditions to commercial operations for assessement.

Impacts
What was accomplished under these goals? Primary activity associated with project goals included construction of rearing facilities, brood stock and larval exposure trials, and a lab-based immune response experiment. In January of 2023, our team visited the USDA's Agricultural Research Service (ARS) Pacific oyster genome selection (POGS) program, located at the Hatfield Marine Station, Newport, OR. While there, we received a demonstration of the newly developed Hatfield Ultra-Density Larval Systems (HUDLS) that the Pacific Shellfish Breeding Center is using to rear oyster larvae. During the months of February and March of 2023, we constructed our own HUDLS system at the Jamestown Point Whitney Shellfish Hatchery, located in Quilcene, WA, in coordination with our industry partners. Our system consists of 24 replicate tanks that are capable of rearing oyster larvae through their pelagic developmental stages. The temperature and seawater carbonate chemistry of each replicate can be independently controlled and each unit is capable of rearing approximately 250,000 spat. Using the hatchery system we initially conducted a bloodstock exposure trial. Broodstock were haphazardly split into one of four treatment groups: (1) heat shock, (2) mechanical stress, (3) immune priming, or a (4) control. Treatments were applied to broodstock before conditioning and spawning to assess their impact on reproductive success and larval performance. Eggs from 4-6 females were collected from each treatment group and mixed with the combined sperm of 4 males from the same treatment at a ratio of one egg to 3-5 sperm. Samples of pooled sperm from each treatment were flash frozen for later analyses. Ultimately larval output was minimal, however we developed several new methods for broodstock manipulation. Subsequently we focused on larval rearing using and have had several successful runs that were comparable to our commercial partner production percentages. In the last month we have made a few improvements on the production system allowing us to run units under different feeding regimes. To investigate whether the immune priming solution used in hatchery experiments elicits a molecular response in oysters from the Pacific Northwest, a laboratory experiment was undertaken wherein diploid and triploid juvenile oysters from a single family were either injected with or submersed in a bath containing a known amount of poly(I:C). For injections, diploid and triploid oysters were anesthetized through immersion in seawater with 50 g/L MgCl, adjusted to a salinity to 30 ppt; 80% of diploids and 73% of triploids had opened their shells after 18 hours. An immune priming solution was prepared by adding 4 ml of sterile, 0.2 um filtered seawater to a glass vial with 10 mg of lyophilized poly(I:C) powder and incubating at 65C for 10 minutes. Oysters were injected with 100 ul of the prepared poly(I:C) solution in the adductor muscle (0.25 mg per animal), allowed to sit open for 10 minutes, and then were returned to seawater without MgCl for recovery. A subset of oysters within each ploidy that were anesthetized with MgCl but not injected were returned to seawater to serve as a control. For the submersed immune treatment, naive oysters were placed in 500 ml of seawater containing 10 ug/ml of poly(I:C) with a bubbler for 24 hours. One day after oysters recovered from injection and submersion immune priming treatments, gill tissue samples from a subset of oysters in each treatment and the control were collected, flash frozen in liquid nitrogen, and stored at -80?. The remaining oysters in each treatment were then maintained within static laboratory conditions at 12? for 30 days before another sampling gill tissue again; these samples were collected to investigate whether any observed molecular responses persisted after treatment. To date a portion of the samples have been characterized using quantitative PCR, indicating that at least within diploids, there is a immune response signature.

Publications