Progress 07/01/20 to 02/28/21
Outputs
Target Audience:The immediate target audience for this project are Manila clam farmers in Washington State. As introduced in our proposal, our customer archetype is Bill Dewey, a spokesperson for the shellfish industry and the owner and operator of Chuckanut Shellfish, who provided a letter of support. Bill primarily produces Manila clams under 8 miles of clam netting in Samish Bay, WA, and pioneered mechanized clam farming methods. Taylor Shellfish Farms has 80 miles of clam netting in Samish Bay; over 100 M Manila clams are cultivated just in this one area of WA. Currently, the Pacific Coast Shellfish Growers Association lists 35 Manila clam producers, who are potential customers. We will license triploid, female-biased seed clams for a share of the increased revenues resulting from higher yields and elimination of post-harvest losses. Secondary customers are the major shellfish hatcheries, to whom we will eventual license use of tetraploid male and diploid female broodstock for production of female-biased triploid Manila clam seed. Changes/Problems:The COVID-19 pandemic of 2020 presented enormous obstacles for this project. Pacific Hybreed's facility in Manchester, WA, is located at the NOAA Manchester Research Station, under the terms of a Cooperative Research and Development Agreement (CRADA). During the pandemic, access to this station by researchers and vendors was highly restricted by NOAA. Only the technician on the project and our hatchery manager had permission to access the facility; co-PDs Hedgecock and Davis could not access the facility nor were they able to provide in-person supervision or contributions. PDs and staff had contact weekly by virtual meetings, but virtual meetings are a poor substitute for in-person observation and training. Pandemic restrictions were a major factor preventing the completion of all proposed technical objectives. In view of this severe and unforeseen impediment, we are pleased to be on the cusp of producing commercial quantities of triploid Manila clam seed, a product that is not presently available to West Coast growers and one that is quite likely to solve a major problem, post-harvest spawning. What opportunities for training and professional development has the project provided?The technician working on this project, who has a MS in Biology, has been trained in all aspects of shellfish biology and culture. In addition, the PD gave a biweekly series of virtual lectures, with assigned readings, on shellfish genetics and breeding. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The main purpose of this project was to eliminate the loss of farmed Manila clams that results when already-harvested clams spawn. Spawned out clams cannot be replanted and, worse, if this occurs after sale to a customer, the fetid, decomposing spawn can greatly harm growers' reputations. Secondarily, we sought to increase yield by producing faster-growing, all-female seed, which if realized by the entire industry would be worth about $5 M annually. Clam farmers would be most immediately helped by these advances, but in the longer term commercial hatcheries which produce clam seed would benefit, as well. These goals were to be achieved by bundling three technical advances: (1) implementation of strip-spawning methods to facilitate pair crosses of select males and females; (2) induction of triploidy (three sets of chromosomes, 3N), which renders clams effectively sterile, and of tetraploidy (four sets of chromosomes, 4N), which enables production of triploid clams by a simple cross of gametes from tetraploid and diploid parent stocks; and (3) identification of primary sex-ratios in full-sib clam families, at the 2- or 4-cell embryo stage, and rearing of selected families with contrasting, female biased or male-biased sex-ratios. We were only partially successful with objective 1. After multiple trials, using a matrix of serotonin and potassium chloride concentrations, we found that strip-spawning is simply not a viable way to obtain fertilizable eggs. The highest return to the so-called D-hinge larval stage (reached at 24-48 h after fertilization), using stripped eggs, was <1% (i.e. fewer than one in one hundred fertilized eggs reached the D-hinge stage). On the other hand, we developed methods for strip-spawning males and found stripped sperm to be effective in fertilizing naturally spawned eggs. In an experiment performed in late February (well outside the Manila clam's natural reproductive season), stripped sperm resulted in D-hinge returns comparable to those achieved with naturally spawned sperm (14% and 13% respectively). Significant time and energy burdens will be lifted from commercial propagation, using stripped sperm. In addition, we gained experience with more traditional methods of isolating spawning clams of each sex to achieve pairwise crosses, including methods for biopsy of gonadal tissue to confirm sex, so that clams can be isolated by sex prior to induction of spawning, reducing the likelihood of uncontrolled fertilizations. We have also increased the reliability and frequency of successful thermal spawns via tightly controlled conditioning in the hatchery. With these new protocols in place, controlled pair crosses are possible, and accidental mass spawns are eliminated. To achieve objective 2, we performed triploid induction experiments as often as gamete conditioning allowed, in order to optimize triploid percentage and D-hinge returns. The only treatments that resulted in >4% D-hinge return and >85% triploidy (3N) were exposure of fertilized eggs to (1) a 10oC cold shock combined with 100uM 6DMAP at 22oC, at 10 minutes post-fertilization (4.3% D-hinge return, 100% 3N) or 12 minutes post-fertilization (5.2% D-hinge return, 95% 3N) post-fertilization, for 10 minutes, and (2) a 10oC shock plus 100uM 6DMAP at 18oC and dosed at 15 minutes post-fertilization, for 15 minutes (5.0% Dhinge, 100% 3N). Continuing experiments will aim to replicate another 100% triploid run with higher D-hinge returns, by treating the developing embryos for a shorter amount of time at higher incubation temperatures. Applying what was learned from experimental trials, we attempted to produce triploid Manila clams at a commercial scale. In the first attempt, we successfully induced 100% triploidy in Manila clams, using a combination of 100uM 6-DMAP and a 10oC cold shock applied at 15 minutes post-fertilization. D-hinge returns at 48 hrs were 5%, compared to 13% in the control diploid batch. After 22 days, we set about 40,0000 3N clams in downwelling screens. These are presently being reared for proof-of-concept field trials and to confirm their lack of reproductive development in summer of 2021, compared to diploid controls. In the second attempt, we used the same procedure but only managed to produce 50% triploids. It is likely that egg quality influenced the timing of embryonic development, resulting in asynchrony between polar body extrusion and our calculated dosage timing. D-hinge returns at 48 hours, on the other hand, were higher at 17%, which resulted in ~200,000 setters at day 16. These clams have been planted in Thorndyke Bay, WA, for evaluation of reproductive status during summer 2021; in this regard, the roughly 50:50 split of diploids and triploids in this group provides a superb internal control. With all effort directed at developing a protocol for commercial production of triploid clams, we did not have the time or resources to investigate methods for the induction of tetraploidy. For objective 3, identifying primary sex-ratios in 2- and 4-cell stage embryos by visualizing the fate of mitochondrial DNA, we obtained and made operational a fluorescent microscope (Nikon 80i) outfitted with a camera for capturing low-light images. We have successfully stained Manila clam sperm with MitoTracker dye, utilizing the protocols of Milani et al. 2012. We have so far been unable to identify and image the sperm mitochondria in embryos, but these efforts continue. In the meantime, we did create six full-sib families and rear them through the larval stages to settlement; these families are also being reared in Thorndyke Bay, WA, for assessment of sex ratios during summer 2021.
Publications
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