Aquaculture Innovation 1.2
Experimental production of tetraploid oysters for use as broodstock for commercial hatchery production of triploids
| ID Number | Project Number | Manager | Start Date | End Date | Total Project Funds ($) |
| 1994/081 | Li, Xiaoux | * | * | * |
*Some IP issues remain. Project has commercially in confidence information.
Outcomes Achieved to Date
Tetraploid oyster embryos were induced by most methods used in this study; including hydraulic pressure shock, thermal shock, chemical treatment and combined chemical treatment. Tetraploid larvae and spat were produced by the combined chemical method developed in this study.
Non-Technical Summary
Triploids (3n) are organisms with three sets of chromosomes, while the normal diploid (2n) organisms have only two sets. Over the past two decades, triploids have been studied in more than 20 species of molluscs. The main interests of those studies have primarily focused on their sterility and improved growth rates. These are mainly due to that in the normal diploid commercial stocks the animals expend considerable energy on gametogenesis and become watery in the summer months making them less desirable in the market. In Pacific oysters, for example, they release approximately 50% of their body mass during spawning, affecting their meat quality and acceptance by consumers for an extended period of time. In a sterile stock, on the other hand, they could partially redirect this energy to growth and maintain their meat quality during these periods, allowing them to be marketed year round. Triploids, in most species studied so far, experience significantly higher growth rates than their diploid siblings. In bivalve species, triploids have showed 10 to 80% faster growth than their diploid siblings. However, the expression of the triploid advantage in growth can be influenced by genetic and environmental factors. In addition, production of triploids from normal diploid stock directly is technically difficult in most bivalve species; often resulting in inconsistent percentage of triploid and high larval mortality in the first few days. The commercial use of triploids may, therefore, ultimately depend on the development of tetraploids, which can produce 100 % pure triploids simply by mating with normal diploids.
The establishment of tetraploid breeding stock however, is still a major challenge in most molluscan species. Prior to 1997, tetraploid bivalve spats have only been produced in three species: mussels (17.2% in one month old); Manila clam (3 spat detected); and Pacific oysters (67% in juveniles). The tetraploid mussels and clams were induced using gametes from diploid males and females and the tetraploid Pacific oysters were produced using eggs from triploid females and sperm from diploid males.
The main objectives of this project were to evaluate and develop techniques for the production of tetraploid broodstock in Pacific oysters and to investigate the potential to produce triploids by crossing tetraploids with diploids. Throughout this project (March 1995 to February 1998), most techniques developed to induce tetraploids from diploid stock in fish and shellfish, have been attempted; new techniques were investigated; and tetraploid spats were produced. The techniques published by Guo and Allen (1994) were not tried because no mature triploid broodstock were available in South Australia.
The first objective “the experimental production of tetraploid (4n) oyster embryos, larvae and spat” was achieved. Tetraploid embryos were induced by most methods used in this study; including, 1) electrofusion of cells in two cell stage embryos; 2) thermal, and heat + caffeine treatments, to inhibit first mitotic division; 3) hydraulic pressure treatments to prevent first mitotic division; 4) Cytochalasin B (CB) inhibition of first polar body formation or both polar body formations; 5) CB and 6-dimethylaminopurine (6-DMAP) inhibition of first mitotic division; and 6) combined chemicals treatment. The majority of tetraploid embryos produced in this study (29 % or more) were produced by the hydraulic pressure treatment and the combined chemical methods. Flow cytometric analysis was used to identify tetraploid spat. Analysis from experiments in the second (23 February 1996) and third (25 February 1997) year of the project identified 1 tetraploid in 28 and 79 spat tested respectively. In general, tetraploid levels in embryos were initially high, however, these were not stable; as embryos developed, the ploidy levels decreased.
The second objective “on-growing of tetraploid oyster spats to adulthood and reproductive capability” was partially achieved. In the third year of the project, March 1997, approximately 2 million, eight-day-old larvae (8 % tetraploids) and 150 twelve month old spats (4% tetraploids, as analysed by flow cytometry; n = 28), were sent to the South Australian Oyster Hatchery for grow-out. A sub-sample from the larval batch was also reared at Flinders University. The spat (12 months old) from this batch were sampled towards the end of the third year of the project (January 1998) using flow cytometric analysis. Non-destructive ploidy assessment of spat is not possible and therefore precise estimates of the proportion of tetraploids in the spat being grown out on oyster farms are not available. The percentage of tetraploids growing out on farms can only be estimated based on the samples analysed at Flinders University. At the time of writing the initial report draft, August 1999, the reproductive capability of oysters grown out at the South Australian Oyster Hatchery had not been obtained, however they are being maintained and checked periodically for indications of sexual maturity.
During the second year of the project (July 1997), the industry was consulted about the direction of oyster research at the South Australian Oyster Growers Association (SAOGA) Field Day, Smoky Bay. At this time the industry indicated that grower demand for triploid oysters had fallen considerably in the light of poor growth rates and meat quality of chemically produced triploid oysters on commercial leases. As a result, plans for continued research into production of tetraploid broodstock were abandoned.
The last objective “hybridization of diploid gametes (from tetraploid broodstock) with 1n gametes (from “normal” diploid broodstock) to produce triploid embryos, larvae and spats,” was not achieved due to non-completion of the second objective.
Unfortunately, the ploidy levels and the performance of the stocks sent to the South Australian Oyster Hatchery for grow-out were not collected. If enough tetraploids were produced and both male and female existed in the tetraploid stock, the breeding tetraploid line could be established by mating between them. In United States the second generation Pacific oyster tetraploids have already been established by mating the tetraploids produced using the eggs from triploid females.
Although very low percentages of tetraploid Pacific oyster spat were induced in some experiments, the success of this study also indicates that the development of zygotes, produced by fertilising eggs from diploid females with sperm from diploid males, could tolerate tetraploid genome in their cells.