Aquacultural Engineering 5 i 1986) 347-355

Biotechnology in Marine Aquaculture* Daniel E. Morse

Department of BioloNcal Sciences and the Marine Science Institute. University of California. Santa Barbara, California 93106, USA

ABSTRACT bztensification of production, modernization of technique, and improvement of cuhivars are needed to increase the economic eJficiency of marine aquacultztre industries. To help achieve such improvements, modern methods of biochemical and genetic engineering can be adapted to control biological processes which intrinsically limit the efficiency attd yield of production. Such applications now have been developed/'or improved production of molhzscs. Critical life-cycle stages of several species of abalones and other commercially valuable molhzscs which thus far have proved amenable to improved control by these means inchzde reproduction, larval settlement, metamorphosis, and acceleration of early growth. Analysis of the physiological and molecular mechanisms ~4zich control reproduction in rnolhtscs reveals" a prostaglandin-dependent regulation of spawning in abalones and certain other species. Spawning of gravid adults can be induced by the addition of prostaglandins to the surrounding seawater, or, more reliably and inexpensively, by activation of the endogenotzs enz)wzatic synthesis of prostaglandin-related spawning triggers" in response to added hydrogen peroxide. Peroxide activation of the prostaglandin-depettdent spawning reaction has been found widely usefid for obtaining synchronous and copious release of fidly competent gametes in a large number of species of abalones', oysters, scallops, mussels, clams, and other vahtable molluscs; a number of these species had not been successfidly spawned by other methods. Settlement and metamorphosis of many molluscan species can be brought utzder similar biochemical control Most ej]~ciently and
* Portions of this report have been published previously (Morse, 1984a, d). 347 Aqzmctdtural Engineering 0 1 4 4 - 8 6 0 9 / 8 6 / S 0 3 . 5 0 - Elsevier Applied Science Publishers Ltd, England, 1986. Printed in Great Britain

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D. E. Morse inexpensively, the natural bioctzerrzical requirement for induction of these critical developmental processes can be met by providing competent larv'ae with unique amino acid constituents associated with the naturally required inducing molecules. Thus, y-aminobu~'ric acid (GABA) can be used sirnpl~, safely and inexpensively to induce cornplete and rapid larval settlement and metamorphosis -- with minimal mortali O' - - i n many commercially important abalone species. This and similar net~rotransmitter-related, amino-acid-derived compounds are proving cornparably effective for the reliable induction of settlement and metamorphosis in a number of'other vahtable rnolhlscatz species. Recent analyses of the requirements Jar rapid early/zlvenile growth in abalones reveals a mechattisnz of growth-control which is Sllsceptible to exogenous hormonal acceleration. Genetic cloning and amplification oJ"recombinam-DNA templates for the efficient production of essential growth-regulating hormones, and substances required for enhanced nutritional efficienQ, and resistance of cultivars, are now in progress. Genetic engineering for t/ze direct improvement of cultivated species soon will be within reach.

INTRODUCTION

Many commercial and developing aquaculture industries face serious economic inefficiencies and losses resulting from insufficient control of reproduction, development, growth, and survival of cultivars; reliance on inefficient, capital-intensive, energy-intensive and/or labor-intensive technologies: and reliance on increasingly unavailable large bodies of unpolluted water. To improve the economic efficiency and yield of marine aquaculture industries, and to adapt these industries to present economic and environmental constraints, significant intensification of production, modernization of technology, and improvement of cultivars are needed. Recently developed methods of biochemical and genetic engineering can be adapted for the improved control of those biological processes which intrinsically limit the efficiency and yield of marine aquaculture production, to help achieve these required changes. As illustrated in recent research and development with abalones and a number of other commercially valuable marine molluscs, such critical processes including reproduction, development, larval settlement, metamorphosis, and the acceleration of growth have proved amenable to improved control by these new methods (Morse, 1984a, b; Hooker and Morse, 1985).

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Identification of the underlying molecular mechanisms controlling reproduction, development, metamorphosis and growth in abalones and other species has revealed several essential processes which can be controlled easily and efficiently by provision of the required biochemical regulatory substances or their inexpensive natural analogs (Morse et al., 1977a; 1979a). These biochemical methods are reliable, safe and convenient, making possible the low-cost improvement of massproduction and efficiency (Morse et al., 1977b, 1978. 1979b; Morse, 1984a). Such methods have proved widely applicable to the improved production of a large number of species of abalones, oysters, scallops, mussels, clams and other valuable molluscs under cultivation for food and other products worldwide (Morse, 1984a). Other methods for the acceleration of growth, improvement of food-conversion efficiency, and the enhancement of the resistance of cultivars which are presently under development involve the use of genetic engineering for the amplified production of hormones and related growth-regulating molecules, production of vaccines, manipulation of genetic content, and clonal production. These and similar methods, including techniques for controlled gene amplification and transfer, are under investigation for the enhancement of production of a number of important marine plant and animal species.

CONTROL OF REPRODUCTION Absolute control of reproduction is one of the most central and basic processes required for efficient aquaculture. Analyses conducted in our laboratory revealed that reproduction in abalones and a large number of other commercially valuable molluscs is controlled by hormones known as prostaglandins (Morse et al., 1977a, b; 1978; Morse, 1984a). In a number of these species, prostaglandins released in the spawn of one animal will trigger the spawning of other gravid animals nearby. Taking advantage of this observation, we found that we could induce spawning in gravid male or female abalones simply by adding very low concentrations of prostaglandins to the surrounding seawater (Morse et al., 1977a). However, this procedure was dependent upon the use of rather expensive hormones, and proved less than perfectly reliable. In searching for a more reliable and less expensive method that could be used for the control of reproduction in a large number of

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species, we found that the enzymatic synthesis of prostaglandins in the reproductive tissues of molluscs (and certain other species) was naturally dependent upon -- and limited by -- the rate of synthesis of minute amounts of hydrogen pero~de (Morse et al., 1977a, b). This hydrogen peroxide is made naturally, in the reproductive tissues, by the biosynthetic enzyme which catalyzes the rate-limiting reaction in the biosynthesis of the prostaglandins; the peroxide then is used by this enzyme as an obligatory or required co-substrate in the prostaglandin biosynthetic reaction. Based upon this finding, we developed a convenient and inexpensive method for reliably inducing spawning, simply by adding a small amount of hydrogen peroxide to the seawater surrounding the animals to be reproduced (Morse et al., 1977a, b; 1978). This method conveniently and reliably activates the synthesis of the endogenous prostagtandin spawning trigger, and thence induces the animals to spawn. Copious spawning of both males and females is induced synchronously. The eggs and sperm obtained by this method prove fully competent for normal fertilization and development (Morse et al., 1977b; 1978). This method for the direct molecular control over the production of the natural spawning trigger has proved to be rapid, reliable, inexpensive and widely applicable to many species (including a growing number which could not reliably be spawned by other methods). As a result, this method has been widely adopted in research and industrial applications, for improved control of reproduction of a large number of species of abalones, oysters, scallops, mussels, clams, and other commercially valuable molluscs under cultivation in the US and abroad (Morse, 1984a). CONTROL OF LARVAL SETTLEMENT, METAMORPHOSIS AND EARLY POSTLARVAL SURVIVAL Serious economic and operational inefficiencies and high losses typically are found in conventional aquaculture industries resulting from poor control of processes dependent upon larval metamorphosis (Morse, 1984a, b; Hooker and Morse, 1985). By analyzing the natural requirements for the induction of larval settlement and metamorphosis in a number of abalone species, we found that these processes are strictly dependent upon specific biochemical substances which the

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larvae normally contact only at the surfaces of certain algae in their natural environment (Morse et al., 1979a; Morse and Morse, 1984). This process ensures the substrate-specific recruitment of abalone larvae to suitable habitats in coastal waters. In cultivation, however, if the naturally required inducing molecules are not adequately provided, the larvae show very. poor success at metamorphosis, with subsequently resulting very low survival and poor growth as juveniles typically observed in traditional low-technology aquaculture operations (Morse et al., 1979a, b; Morse, 1984a, b; Hooker and Morse,

1985).
Although the naturally required algae proved to be unsuitable for direct use in aquaculture operations (specimens collected from the field frequently carry many small predators, and the algae grow too slowly for large-scale growth of predator-free surfaces), characterization of these algae has made direct biochemical control feasible (Morse et al., 1979b). From analyses of the naturally required inducing molecules available at the algal surface, a number of simple and inexpensive biochemical inducers capable of satisfying the larval requirement was found. Most efficiently and inexpensively, the natural biochemical requirement for induction of settlement and metamorphosis in abalones can be met by providing the larvae in culture with 7-aminobutyric acid (GABA), or similar readily available amino acid constituents associated with the naturally required inducing molecules (Morse et al., 1979a, b). Thus, GABA can be used simply, safely, and inexpensively to induce complete and rapid larval settlement and metamorphosis- with minimal m o r t a l i t y - in many commercially important abalone species (Morse, 1984a). The contrast in results obtained by the use of GABA and by cultivation in the absence of this naturally required inducer is dramatic. When GABA is added in low concentration to competent abalone larvae, virtually all of the larvae settle, attach to natural or artificial substrates provided, and rapidly commence their genetically programmed metamorphosis to the juvenile form. Following this synchronous induction of metamorphosis, the resulting healthy juveniles grow relatively rapidly with very high survival. In contrast, larvae cultivated in the absence of GABA or other added inducer show a very low percentage of settlement, retardation or complete failure of metamorphosis, and slow and asynchronous juvenile development and growth, with very high mortality (Morse, 1984a, b; Hooker and

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Morse, 1985). Typically, survival of juveniles one month after metamorphosis induced with GABA is observed to be in the range of 90-100%, whereas survival of siblings cultivated in parallel without GABA is typically ~<1% (Morse, 1984a). It should be noted here that provision of GABA or similar neurotransmitter-related, amino-acid-derived compounds (such as DOPA) also is proving useful for improving the efficiency of induction of settlement and metamorphosis of a number of other valuable molluscan species (including various oysters and clams)(Morse et al., 1979a; Morse, 1984a).

ACCELERATION OF GROWTH, IMPROVEMENT OF NUTRITION, AND RESISTANCE T H R O U G H GENETIC ENGINEERING A major remaining limitation to the efficiency of aquaculture for many animal species is a complex of problems related to their relatively low food-conversion efficiency, with consequent high costs of feeding, relatively slow growth and sensitivity to trauma, stress and disease (Morse, 1984a, b; Hooker and Morse, 1985). Through the ability to induce the synchronous start of juvenile growth under defined conditions (as described above), it has become possible to dissect this problem by analysis of the hormonal requirements for optimal growth, nutritional efficiency, and resistance of abalones and other molluscs in cultivation. Preliminary studies reveal that growth can be significantly accelerated by provision of exogenous insulin and related peptide growth hormones (Morse, 1984a, b; Hooker and Morse, 1985). These studies, and those of our colleagues in Europe, have established that abalones and other molluscs produce their own insulin and growth hormones. Such hormones prove to be essential to the molluscs for: (a) the efficient uptake and utilization of nutrients from food, (b) the rapid conversion of these nutrients into essential enzymes, other proteins, and glycogen (meat constituents of principal nutritive, flavor, and commercial value); (c) rapid growth; and (d) resistance to trauma, disease, and stress under cultivation conditions. It should be possible, then, to use these hormones to increase the rate of growth, the economic efficiency of feeding and the nutritional and commercial value of the meat of shellfish under cultivation. It is

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important to note that because these hormones are peptides and proteins, they are naturally and rapidly degraded by enzymes, and not stored in animal tissues as steroids are. Thus, in contrast to the steroids, peptide hormones can be used safely to accelerate growth. However, industrial applications of these findings will require inexpensive supplies of these hormones from homologous tissues (i.e. from the molluscs themselves). Because molluscs make only minute quantities of these hormones, the necessa W supplies can be produced most efficiently and inexpensively by applications of recently developed genetic engineering technology, using the cloned molluscan hormone genes as templates. For this purpose, our laboratory' has begun experimental work on the cloning and amplification of the molluscan (abalone) genes coding for growth-accelerating hormones (Morse, 1984a). Similar hormones now are being produced from the cloned genes of humans and terrestrial animals, for use in medicine. pharmaceutical production, and agriculture. There is every reason to expect that such techniques should prove equally useful for improvements in aquaculture as well. FUTURE PROSPECTS From the cloned gene-banks now under construction from marine food resource species, still other useful genes and gene products can be conveniently selected for the analysis and improved control of nutrition, growth, resistance, reproduction, and development. More advanced applications of genetic engineering, including the selective modification, amplification, and reintroduction into the genome of specific genes for the direct improvement of cultivated species of marine plants and animals, and the production of vaccines, soon will be within reach. In addition, various strategies for the clonal propagation of marine plants and animals presently' are under investigation. The newly developed biochemical and genetic engineering technologies described here for improved food production also are being applied to the analysis and control of marine fouling (Morse, 1984< 1986); to the enhanced production of medically and other commercially valuable compounds from marine organisms: and to the improved processing of metals, hydrocarbons, and polymers by marine microorganisms (Morse, 1986).

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ACKNOWLEDGEMENTS T h e research reported here was s u p p o r t e d by the US D e p a r t m e n t of C o m m e r c e ( N O A A ) - University of California Sea Grant College Program (Grants R/A-43 and R/A-51), the US Navy, Office of Naval Research (Contract No. N 0 0 0 1 4 - 8 0 - C - 0 3 1 0 ) , and contributions from C h e v r o n USA, Inc., and the Atlantic Richfield Foundation.

REFERENCES Hooker, N. & Morse, D. E. (1985). Abalone: the emerging development of commercial cultivation in the United States. In: Crustacean and Mollusc Aquaculture in the United States, eds J. V. H u n e r & E. E. Brown, AVI Publishers, Westport, 365-413. Morse, A. N. C. & Morse, D. E. (1984). Recruitment and metamorphosis of Haliotis larvae induced by molecules uniquely available at the surfaces of crustose red algae. J. Exp. Mar. Biol. Ecol., 75, 191-215. Morse, D. E. (1984a). Biochemical and genetic en~neering for improved production of abalones and other valuable molluscs. Aquaculture, 39, 263-82. Morse, D. E. (1984b). Prospects for the California abalone resource: recent developments of new technologies for aquaculture and cost-effective seeding for restoration and enhancement of commercial and recreational fisheries. In: Ocean Studies, ed. S. Hansch, California Coastal Commission, San Francisco, 165-89. Morse, D. E. (1984c). Biochemical control of marine fouling. In: Marine Biodeterioration, eds R. Tipper & J. Costlow, Naval Institute Press, Annapolis, 134-40. Morse, D. E. (1984d). Biochemical and genetic engineering in marine aquaculture: the role of modern biotechnology in the production of food from the oceans. Oceans '83, Mar. Tech. Soc., San Francisco, Vol. 2, pp. 1009-11. Morse, D. E. (1986). External molecular signals controlling reproduction, settlement, and metamorphosis of benthic marine invertebrates. In: Biology of Benthic Marine Organisrns, eds M.-F. Thompson, R. Sarojini & R. Nagabhushanam, Oxford Press, New Delhi, 379-86. Morse, D. E.. Duncan, H., Hooker, N. & Morse, A. (1977a). Hydrogen peroxide induces spawning in molluscs, with activation of prostaglandinendoperoxide synthetase. Science, 196, 298-300. Morse, D. E., Duncan, H., Hooker, N. & Morse, A. (1977b). An inexpensive chemical method for the control and synchronous induction of spawning and reproduction in molluscan species important as protein-rich food

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resources. United Nations Food and Agricultural Organization Fisheries Bulletin, 200, 291-300. Morse, D. E., Hooker, N. & Morse, A. (1978). Chemical control of reproduction in bivalve and gastropod molluscs, Iii: An inexpensive technique for mariculture of many species. Proc. World Maricult. Soc., 9,543-7. Morse, D. E., Hooker, N., Duncan, H. & Jensen, L., 1979ai. y-Aminobutyric acid, a neurotransmitter, induces planktonic abalone larvae to settle and begin metamorphosis. Science, 2 0 4 , 4 0 7 - ! 0. Morse, D. E., Hooker, N., Jensen, L. & Duncan, H. (1979b). Induction of larval abalone settling and metamorphosis by ;/-aminobutyric acid and its congeners from crustose red algae, II: applications to cultivation, seedproduction and bioassays; principal causes of mortality and interference. Proc. World Maricult. Soc., 1O, 81-91.