Generating Triploid Green Sea Urchins for Aquaculture in Near Shore Lease-Sites in the Northeastern United States
We supported three students who have confirmed that our laboratory procedures can be replicated by those initially unfamiliar with the procedures.
We also confirmed that sufficient numbers of triploid embryos can result from a scaling up of these laboratory procedures to commercial levels. Additionally, we have cultured embryos to prism pluteus stage in the hatchery.
Brief Description of Work to be Performed
Edible sea urchin fisheries are high value commercial enterprises on all coasts of the United States and their processed gonads (called uni) or whole urchins are sold in Japanese, American and other world markets (e.g., Italian and Belgian). Alternatives to direct harvest of wild sea urchins are vital to sustain this fishery that provides an exportable product for American fishermen and helps the US trade deficit. In this grant proposal for Sea Grant Development Funds, we describe an alternative technology to natural harvest and land-based urchin aquaculture. The aquaculture of sterile triploid sea urchins in near shore lease-sites will utilize urchins with gonads that contain only nutrient storage cells called nutritive phagocytes (NPs). Use of this aquaculture methodology would be of significant interest to commercial aquaculture for the following reasons:
1) Naturally harvested urchin gonads of both sexes that contain predominantly NPs, are preferred by consumers and bring higher dollar value for the commercial product (Walker and Lesser, 1998; Walker et al., 2005, 2006; Unuma and Walker, 2009; 2010). In the green sea urchin, shorter day-lengths characteristic of fall are correlated with the transfer of nutrients from NPs to gametes during gametogenesis and the commercial quality of green sea urchin gonads progressively decreases as gametes of both sexes are produced. Triploid sea urchins will be sterile and will not develop gametes. As a result, the uni that they contain will yield the highest value in the commercial market because of the exclusive presence of NPs in the germinal epithelium in both sexes. These uni will have the additional benefit of extended shelf-like since they will not deteriorate in quality as gametogenesis proceeds like uni from naturally harvested sea urchins.
2) High commercial quality sea urchin gonads are characterized by large size and by the quality of a number of sensory parameters (taste, color, texture and firmness) expected by consumers.Existing formulated sea urchin feeds that are used for land-based urchin aquaculture emphasize maximizing size of the commercial product. However, indiscriminately increasing protein levels can yield bitter tasting gonads (Hirano et al., 1978; de Jong-Westerman et al., 1995; Hoshikawa et al., 1998; Pearce et al., 2002, Böttger et al., 2006). Obviously, large gonads that do not meet the exacting standards of Japanese and other consumers are of little immediate value and could even be counterproductive to the long-term commercial development of a viable sea urchin aquaculture industry.Triploid green sea urchins maintained in near-shore lease-site aquaculture will consume food abundantly available in their natural environment and as a result, their gonads should develop taste and other sensory parameters that are expected by the consumer.
3) Several near shore lease-sites are available in New Hampshire and Maine and are owned by the following stakeholders that have substantial interest in the results of our study: 1) Jay Gingrich in Portsmouth Harbor, New Hampshire, 1) Chris Hill in Kittery, Maine, 3) Jim Wadsworth two in Penobscot Bay, Maine and 4) Peacock Cannery in Cobscook Bay, Maine. Aquaculture of adult triploid sea urchins in near shore lease-sites using naturally available food sources would not rely on the purchase of expensive formulated feeds nor on complex and expensive mechanisms that yield altered photoperiod.
Triploid molluscs, fish and shrimp can be generated by blocking second meiotic division to yield triploid embryos that develop into sterile triploid adults. Since methods that block release of the second polar body are not possible for sea urchins because both meiotic divisions occur within the ovary and prior to ovulation and fertilization, triploids have never been produced before for any species of sea urchin! We have developed an alternative methodology for producing triploid green sea urchin embryos and have maintained resulting triploid embryos to the prism larval stage (n = 63; Eno et al., 2010; Figure 1A-D). In paper from the Walker laboratory under revision at Aquaculture (Böttger et al., in revision, Aquaculture) we describe methodology for producing triploid green sea urchin embryos (n = 63) and maintaining them to the prism pluteus stage with spicules. Our laboratory methods have yielded the only viable triploid embryos ever generated from any species of sea urchin and are a successful first step in an effort to generate adult triploid green sea urchins. In this proposed study for UNH Sea Grant, we will scale up our laboratory methods for generating triploid green sea urchin embryos for transfer to the hatchery environment where they should undergo metamorphosis and yield juvenile urchins. We will also distribute juveniles to two available near shore lease-sites and will assess the triploid green sea urchin gonads for their sensory parameters important to the commercial market. The assessment will be carried out by the largest commercial buyer of urchins in Maine, Mr. Atchan Tamaki of ISF Trading, Portland and by the Food Science Group of the University of Maine under the direction of Dr. Camire. Each step in this scaling up process is discussed below.
Objective I – To Scale up our successful laboratory methods for generating triploid green sea urchin embryos
a) Spawning, Gamete Collection and Removal of the Vitelline Membrane.
This suite of procedures prepares 1n ova (that exist before ovulation and after both meiotic divisions) for fusion and subsequent fertilization. We will use 0.5M KCl to spawn sea urchins as outlined in Foltz et al (2004). In nature, sea urchin ova are fertilized ecto-somatically in the water surrounding feeding adults. As a result, both sexes produce prodigious numbers of gametes (millions) that are more than sufficient to yield hundreds of thousands of embryos using our methods. Removal of the jelly coat and the vitelline membrane are necessary before fusion of ova can take place. We will remove the jelly coat mechanically during filtration. We will also use the Cleland reagent to remove the vitelline membrane.
b) Fusion, Fertilization and Embryo Culture.
Results from the procedures above should yield approximately 1000 ova/tube. To scale up our laboratory procedure, 500 tubes will yield 250,000 triploid embryos. Based on our previous experience, embryos should reach the gastrula stage after 3 days in laboratory culture (8°C) and early prism pluteus after 6 days in the hatchery post fertilization. Green sea urchin embryos begin feeding in the pluteus after the mouth has formed; and appropriate phytoplankton (Isochrysis galbana, Dunaliella tertiolecta and Rhodomonas lens) will be cultured in adequate amounts to supply feeding embryos as soon as the mouth is formed. It is important to point out that the triploids we have produced successfully gastrulate and continue development. In sea urchins, most new expression of zygotic genes begins at gastrulation. Development to the gastrula stage depends upon the expression of genes or use of proteins placed in the ova during oogenesis. Expression of novel, lethal combinations of genes united during fertilization are potentially fatal at gastrulation.
c) Chromosome Counts.
This process will easily differentiate triploids from embryos with an alternative ploidy and provides an easy method for monitoring the success of the fusion process prior to transfer of embryos to a hatchery environment. The protocol is detailed in Eno et al (2010) and employs a simple compound microscope at 60-100 X magnification.
Objective II – To Culture triploid green sea urchin embryos to metamorphosis in a Hatchery Environment
d) Transfer to hatchery, feeding embryos and maintenance to metamorphosis.
This portion of the scaling up procedure will depend on a hatchery environment available at coastal locations. In our casewe will use the Harris and Gingrich hatchery located in the basement of Portsmouth Scuba in Portsmouth, NH. In this facility, 300-liter vats will be employed using filtered and UV-sterilized seawater and maintained at 10oC with aeration provided by an air-stone to create current within the vat. Stocking density will be 4 embryos/ml of medium with a final density expected to be 300,000 to 600,000 settled juveniles per vat. Larvae will be fed a combination of Isochrysis galbana, Dunaliella tertiolecta and Rhodomonas lens daily and overfeeding will be avoided to yield minimal mortality. In this hatchery, to metamorphosis and settlement for diploid green sea urchins is typically 21 days and juveniles are maintained for 5 to 7 days before extraction from the vats and transfer to additional juvenile culture systems at the hatchery. We anticipate similar numbers for our triploid embryos. Urchins produced from a trial will be maintained in a dedicated trough system with flow-through seawater to measure growth and survival before out-planting the following winter.
Objective III – To Transfer resulting triploid sea urchin juveniles to near shore lease sites in New Hampshire and Maine where the value of their gonads as commercial products will be assessed
e) Out-planting and maintenance of juveniles in open-ocean lease sites.
Diploid juvenile green sea urchins are typically maintained in recirculating or flow-through culture systems at land-based facilities until they are approximately 10–20mm in diameter (unpublished data from Dr. Larry Harris, UNH). These juvenile urchins are then transferred to near-shore lease sites until they reach harvest size at 52 mm diameter. Previous and on-going studies show that transfer to lease sites during winter months when predators are dormant results in higher survival and under appropriate culture conditions, juveniles reach transfer size within 6 months. Sea urchins remain within lease sites if adequate food is available (Dumont et al., 2004; Lauzon-Guay et al., 2006). Another approach currently being tested at the lease site of Dr. Harris is to utilize cage systems on the lease site through the juvenile stage to minimize overhead costs required for land-based cultivation of juveniles. We will attempt similar procedures with our triploid juveniles.
f) Histological and sensory evaluation of the commercial quality of triploid green sea urchin gonads.
We will collect triploid green sea urchins by SCUBA from each lease site employed in this study and will sacrifice ten individuals initially and at the completion of the experiment to determine their gonad indices, sex and for histological analysis. Individuals will also be supplied each time for sensory analysis to Mr. Atchan Tamaki at ISF Trading and the Food Science Group of the University of Maine. Mr. Tamaki will rank gonads from 10 individuals on a scale of 1-5 (5 being the highest score) for color, texture, taste and marketability using his knowledge of the Japanese and American sushi markets. The Food Science Group of the University of Maine will perform an extensive sensory analysis on 50 sea urchins using 50 consumers and a nine-point hedonic scale developed by Peryam and Pilgrim (1957) to rate color, appearance, aroma, firmness, flavor and acceptability. The tissue in 50 sea urchins each of which has 5 large gonads is more than sufficient to provide tasting samples for this number of consumers.
Rationale for Seeking N.H. Sea Grant Development Funds
This winter, using Sea Grant Development Funds, we will attempt to scale up our successful small-scale laboratory studies to a commercial scale encompassing steps a-d. This effort will be a proof of concept study that will attempt to demonstrate that this kind of work can be accomplished at a commercial scale. With success of this study, we would then apply for full Sea Grant and/or USDA grants to further support this work and carry out all steps outlined above from a-f.
Böttger, S.A., Eno, C.C., Walker, C.W., (in revision at Aquaculture). Methods for generating triploid green sea urchin embryos: An initial step in producing triploid adults for near-shore aquaculture.
Böttger, S.A., Devin, M.G., Walker, C.W., 2006. Suspension of annual gametogenesis in North American green sea urchins (Strongylocentrotus droebachiensis) experiencing invariant photoperiod – Applications for land-based aquaculture. Aqua. 261: 1422-1431.
de Jong-Westman, M., March, B.E., Carefoot, T.H., 1995. The effect of different nutrient formulations in artificial diets on gonad growth in the sea urchin Strongylocentrotus droebachiensis. Can. J. Zool. 73, 1495–1502.
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Unuma, T., Walker, C.W., 2010. The role of the major yolk protein in sea urchin reproduction and its relevance to aquaculture. In: Echinoderms: Durham, Harris, L.G., Böttger, S.A., Walker, C.W., Lesser, M.P. (Eds), Balkema, the Netherlands, pp. 437-444.