Final report for FW15-035
Farming of the Pacific Oyster (Crassostrea gigas) is an important part of the economies and cultures of the West Coast states. Oyster culture supports thousands of jobs in rural areas and provides locally produced seafood at a time when the U.S. imports approximately 90% of its seafood. Oysters are also critical in maintaining water quality and provide other important ecological services in coastal areas. The West Coast oyster industry faces numerous challenges such as ocean acidification impacts, rising labor costs, and permitting difficulties, but now faces a challenge that could almost immediately undermine the entire industry as a key patent expires. The patent (#US5824841 A) on the use of tetraploid oysters expired in January 2015, an event which might otherwise go unnoticed, were it not for the fact that the West Coast industry is heavily dependent on the use of tetraploid oysters to produce triploid oysters, the mainstay of the industry. Triploid oysters (which contain three sets of chromosomes) are essentially sterile, a characteristic that makes them harvestable year-round. These generally sterile oysters do not become “spawny” (e.g. full of eggs or sperm) or flaccid and thin during the warmer months, as do fertile diploid oysters. Without triploid oysters, farmers will not be able to harvest during much of the year, thus threatening the economic viability of the farms, hatcheries, and processors. While the importance of the future availability of triploid oysters cannot be underestimated, neither can the current difficulty and cost of obtaining triploid oyster seed. While triploid oysters can in theory be produced using a combination of chemicals, pressure, heat, and other inducers at a precise moment in oyster embryo development, these methods are not efficacious enough to obtain the benchmark of at least 90% triploid progeny to assure summer harvests. The only reliable method of producing greater than 90% triploid oyster progeny is to utilize tetraploid oyster sperm to fertilize diploid eggs. In order for oyster hatcheries to obtain tetraploid oysters for this purpose, a license must be obtained from the 4C’s company. This company was created by Rutgers University, the assignee of the patent for tetraploid oysters, to manage this valuable intellectual property.
To fully understand the difficulty in which the West Coast oyster industry now finds itself, a bit of history must be recounted. In 1993, Rutgers University was awarded a patent for the tetraploid oysters with Drs. Ximing Guo and Standish Allan as co-inventors. At this point, the evolution of the use of tetraploid oysters on the East Coast and the West Coast diverged. The two inventors went on to develop thriving research programs at Rutgers University and the Virginia Institute of Marine Science (VIMS), respectively. One of the outcomes of their continued research on the genetics of the Eastern Oyster (Crassostrea virginica), East Coast farmers, and hatcheries now has access to improved lines of diploid and triploid oysters that demonstrated high performance and disease resistance in both high and low salinity environments. VIMS regularly produces and distributes genetically selected tetraploid and triploid oysters for use in hatcheries and on farms. In contrast, the intellectual property represented by the patented tetraploid oyster languished on the West Coast. The reasons for this are subject to conjecture, but the outcome is that one partner hatchery has been used by 4C’s to produce and distribute tetraploid oysters to the rest of the industry. The results have been less than ideal. For example, even once a license for tetraploid use has been granted to a West Coast hatchery, obtaining verified tetraploid broodstock from the distributor is difficult. Often none is available. The tetraploids also produce low-viability sperm and the resulting triploid progeny is weak and difficult to raise to maturity in the hatchery. The triploid progeny also exhibit early mortality, often dying en masse by the end of the second summer of growth. The cause of this mortality is unknown, but is suspected to be related to the probable inbreeding of the tetraploid stock. No information is released on the genetics of the broodstock. Moreover, licensees are required to propagate their own tetraploid broodstock after receiving the first animals, which is difficult due to their fragility.
With the expiration of the patent, a team of university and private sector partners came together to conduct research and development efforts to advance the development and use of tetraploid Pacific Oysters, thus making production of triploid oysters more efficient, reliable, and available. The goal was to develop reliable methods of tetraploid production to ensure a readily available supply of higher quality broodstock and thus be able to produce a continuous supply of triploid oysters. This clearly benefits farms that would otherwise have trouble obtaining triploid seed, and moreover, provides some degree of control over the quality.
An additional benefit to developing the capability to produce tetraploids de novo rather than continuing to inbreed existing stocks is that researchers and hatchery managers can now improve tetraploid lines and integrate tetraploids into breeding programs. This will benefit all stakeholders in the West Coast Shellfish industry which depend on the availability of triploid seed, but it also improves the resilience of the industry since breeding can help adapt oysters to climate change impacts and disease and optimize performance traits. The final result of the research and development efforts supported by WSARE is a pool of oysters, of which about 30% will be tetraploid adults that can be used to produce triploids and tetraploids.
Hawaiian Shellfish LLC and Paepae o He`eia are partnering with the Pacific Aquaculture and Coastal Resources Center (PACRC) at the University of Hawaii Hilo (UHH) to execute this work. Hawaiian Shellfish also supplies over 20 West Coast and Hawaii farms with seed, thus multiplying the benefits of the work. The team is fortunate in obtaining the technical assistance of Dr. Ximing Guo, co-inventor of the tetraploid production methods, and Dr. Anu Frank-Lawale, who most recently served as the lead Breeding Manager for the oyster improvement program at VIMS.
The overall goal was to conduct research to develop more reliable methods and refine existing methods for de novo tetraploid oyster production suited for commercial and research hatcheries. This will ensure that tetraploid and triploid oyster supply will continue and increase access for farmers, hatchery managers, and researchers. It will also allow tetraploids to be integrated into West Coast breeding programs which currently focus on diploids. Hatchery managers, researchers, and technicians associated with the Hawaiian Shellfish and the PACRC hatcheries were trained in methods to produce tetraploids by the co-inventor of the original methods. A manual will be produced to explicitly detail these methods and how to adapt to variable conditions between hatcheries. These actions constitute an important component of a broader effort between Hawaiian Shellfish LLC, PACRC, and the Molluscan Broodstock Program (MBP) at Oregon State University to develop an oyster industry breeding plan for the benefit of the Western Region (including Hawaii), as well as to incorporate tetraploids into the MBP’s existing genetic selection program.
Objective 1: Conduct research to develop reliable and effective methods for tetraploid oyster production. This research will be conducted with the assistance of Dr. Ximing Guo (Rutgers University), one of the co-inventors of the original patent for the tetraploid oysters. He will be assisted by Dr. Maria Haws (PACRC) who has also conducted trials on de novo tetraploid production and methods for production of triploids. Dr. Frank-Lawale will advise on issues related to breeding.
Objective 2: Grow out and monitor performance of tetraploid oysters with associated producers. Given that genetic selection for oysters is complicated by the magnitude of genetic X environmental interaction found in this species, i.e. the same strain of oyster may perform very differently in different environments, it is important that the tetraploid specimens be monitored under the conditions in which their progeny will develop. We will grow out and monitor tetraploids in four locations: Goosepoint Farm in Washington, He`eia Hawaiian Fishpond in Oahu, Keawanui Fishpond in Molokai, and at the PACRC. Partners will be trained in monitoring and data collection methods.
Objective 3: Produce a manual and video clips that describe tetraploid production methods in sufficient detail that other hatchery operators or researchers can replicate them. Although an extensive body of scientific literature exists for tetraploid and triploid production methods, extensive trials at the PACRC hatchery demonstrates that the methods contained within are not detailed or complete enough to allow for replication. We will produce a manual that contains complete and detailed methods to allow other hatchery operators to more easily replicate these critically needed methods.
- - Technical Advisor (Educator and Researcher)
- (Educator and Researcher)
Tetraploids are created using eggs from ripe triploids and sperm from diploids to produce larvae that have a percentage of tetraploids in the population. Ripened triploids are first shucked, sexed under microscope, and then flowed with a Ploidy Analyzer, only keeping oysters confirmed to be triploid with viable eggs.
- 5 liter jugs
- 20 and 75 micron screens
- 200L of 25˚ C filtered salt water
- Graduated cylinders
- Cafeteria trays
- Exam gloves
- Micro capillary tubes or tooth picks
- Counting slides
- Disposable pipettes
- Marker pens
- Oyster knives
- Spray bottles
- Paper towels
- 6-DMAP stock solution
- Make stock solution concentration: 2mg/mL. 6DMAP (sigma D2629 – 16)
- Add 1 g of room temperature 6DMAP to 50 ml of DI water along with 2ml of DMSO and mix well. Add additional 450ml of DI water. Mix well until completely dissolved.
- Treat eggs at 50-70mg / liter (25-35ml of stock solution per liter)
(Store at 4˚C , last about 1 year) In freezer if it re-crystalizes, make sure it re-dissolves before use.
Tetraploid Induction Process
The methods developed for tetraploid induction do not deviate significantly from the methods described previously by scientists. It is critical however, that users be very familiar and practiced with standard oyster hatchery methods, particularly for spawning and early larval rearing. Great care must be taken to perform each step carefully to obtain optimal results. The usual “quick and dirty” hatchery spawning methods will not suffice in this case. We do not provide detail on the flow cytometery methods since each user will have access to different instrumentation. It is important however, that the flow cytometer (FC) be equipped to detects polyploidy. Partec makes a model that is specifically equipped for detection of polyploidy. Barring access to this, other FC’s that are designed for measuring polyploidy will work, but the user should become conversant with their use prior to attempting induction. Failure to understand and be able to practice standard FC methods will lead to confusion and wasted time if novices make their first attempts in conjunction with the induction trials.
The user must obtain a source of large triploid oyster specimens. Expect to spend most of one day conducting the induction trials. Some diploids may be mixed with the triploids, hence, each putative triploid female must have a tissue sample removed and the ploidy level validated using FC. Any diploid oysters are discarded. Any males or hermaphrodites detected in the pool of triploids are also discarded. Care must be taken to avoid cross-contamination between oysters when making the microscopic assessment of sex.
Triploid females are strip spawned individually. Strip spawning is conducted using a clean scalpel blade. The eggs are delicately scraped from the gonad of the oyster taking care not to puncture the gut (the enzymes in the gut can decrease fertilization rates of the eggs and damage sperm). The eggs are normally in little pockets that have a “popcorn” appearance throughout the region normally containing gametes in diploid oyster. A clean spray bottle filled with 25˚ C salt water can be used to irrigate the scraped eggs from the oyster. The eggs are passed through a 75-um screen to remove large pieces of tissue and the eggs are then caught on a 20um screen. The eggs are then placed in a container and filled with 25˚ C salt water to a density no greater than 2million eggs/Liter and allowed to hydrate for 40-60 minutes.
Fertilize 3N eggs with 2N sperm at a density of at least 10-15 sperm around each egg. The timer is started immediately upon initial fertilization. At 1-5 minutes post fertilization, treat the fertilized eggs with 50-70 mg/liter previously prepped stock 6-DMAP solution. Mix egg, sperm, and 6-DMAP solution extremely well and let sit with gentile additional mixing every 2 minutes. Treat eggs for 15 to 20 minutes and stop the induction processes by rinsing the eggs gently on a 20-um screen. Immediately after rinsing onto the sieve, stock the eggs into an incubation tank at density no greater than 50 eggs/ml. Reserve an aliquot of the fertilized eggs to observe development. Do not add algae to the tank as the trochophores and young larvae will not feed.
When the larvae reach the D-stage, which will be on the day after the induction trials are conducted, a sample is removed and will be tested using FC to determine the percentage of tetraploids. Be sure to use the appropriate standard (i.e. larvae standard for larvae tests). Cohorts which do not exhibit at least 50% tetraploidy should be discarded to avoid wasting time in raising larvae which are destined to have increasingly lower levels of tetraploidy over the development period. Larvae should be tested at the D-stage and just prior to metamorphosis. Spat should be tested once they reach 1 mm and about every other month during the growout period. Again, cohorts with low levels of tetraploidy should be discarded. Any cohort with 70% tetraploidy should be kept. Putative adult tetraploids should be tested prior to using as broodstock.
Dr. Ximing Guo, one of the co-inventors of the original methods, visited the PACRC on February 27th through March 5th 2017. He trained PACRC and Hawaiian Shellfish staff and students on the de novo technique for tetraploid and triploid oyster production. Approximately 12 inductions were done during his visit, with mixed success rates as there is a high number of variables in each induction.
Results are measured by the percentage of tetraploids present in the population, which is determined by a flow cytometer machine. Dr. Guo recommended that a population with less than 50-70% tetraploids should be discarded.
Over the next 7 months, staff at Hawaiian Shellfish performed 150 de novo inductions, and 20 of them were successful and contained enough tetraploids in the population to be grown out for production in the following years. This is a 13.3% success rate, which is considered a success for this type of induction.
Objective 1: Conduct research to develop reliable and effective methods for tetraploid oyster production.
Over the course of the project, 150 induction trials were conducted in the attempt to induce polyploidy in oyster larvae. Although there is an extensive body of literature describing many methods of producing tetraploids, it rapidly become clear that a high level of effort would be needed to validate nearly every step of the induction procedure. In part this is due to the high degree of natural biological variability between oysters, which includes seasonal effects. Methods for each step of the procedure (e.g. conditioning, selection of females, chemical dosages, timing of chemical administration) were systematically tested until optimal results were achieved. Additionally, flow cytometery is used to monitor the results of the induction with samples being taken two days after induction, a few days prior to metamorphosis and several times in the early spat stage. Flow cytometry methods must be developed for each individual flow cytometer used, which will vary according to the instrumentation available to hatchery operators. Multiple testing events are required since there is a tendency for tetraploid individuals to revert to lower states of polyploidy or become mosaics. This means that some batches may need to be discarded and that even successful batches are labor intensive to rear and test. By early 2017, methods were sufficiently well-developed so that relatively high levels of tetraploidy were induced on a regular basis.
Objective 2: Grow out and monitor performance of tetraploid oysters with associated producers.
The animals are being reared and monitored at the PACRC (Hawaii) and the Goosepoint farm (Willapa Bay, Washington). The Hawaiian Shellfish team designed and built a Floating Upweller System (FLUPSY) which is being used to safely contain the oysters under high water flow conditions to promote good growth. The FLUPSY is the result of an earlier WSARE grant made to the PACRC five years ago and represents one of the long-term benefits of the WSARE support to the nascent oyster farming industry in Hawaii.
Oysters have also been transferred to the He`eia fish pond on Oahu, in part for training purposes. Oysters with a higher level of tetraploidy will be transferred to He`eia in early 2018 as training will be completed and there will be a higher level of assurance that the precious tetraploid broodstock will be adequately tended. We also exercised a certain level of caution in transferring the more valuable stock to He`eia (with mutual agreement) since the landward portion of the pond’s rock wall is under repair. Oahu commonly experiences torrential rains during the winter season which floods the pond with freshwater which could kill the oysters if this were prolonged. Thus far the oysters at the various sites have had good growth and survival rates.
Objective 3: Produce a manual and video clips that describe tetraploid production methods in sufficient detail that other hatchery operators or researchers can replicate them.
The team has drafted the training materials and has video clips which are being edited. We expect the materials to be ready within 2 weeks of submitting the final report. The materials and supplies list, and the description of the induction procedure (below) are the key pieces of information needed for successful replication of this work.
Educational & Outreach Activities
Dr. Ximing Guo visited Hawaii twice during the project, the first being in August 2015 where he assisted in training and planning sessions with 6 PACRC and Hawaiian Shellfish staff.
Dr. Anu Frank-Lawale, who most recently served as the lead Breeding Manager for the oyster improvement program at VIMS also visited Hawaii to assist with breeding planning and also gave lectures at Hawaiian Shellfish and PACRC attended by approximately 15 students and staff.
The results of this project are very encouraging for the development of triploid and tetraploid oysters, which are becoming more vital for both Hawaii and West Coast oyster farms. With the development of tetraploid broodstock, Hawaiian Shellfish and PACRC will be able to provide much needed triploid larvae and seed to Hawaii and West Coast farms.
Currently there is a shortage in availability of triploid seed which puts farmers in a poor situation where they cannot plant oysters that they can later harvest in summer months. During the summer, diploid oysters become laden with gametes which is not a desirable condition for most oyster enthusiasts. After they spawn they lose much of their mass and become flaccid and translucent, which is also an undesirable condition for oyster consumers. Triploid oysters maintain their meat quality over these months, allowing producers to harvest during this period.
This work also helps make Hawaii’s aquaculture industry more self-sufficient and sustainable.