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