Contents
-Home
-Welcome Letter
-Purpose of Workshop
-Program
-Presentations

Appendix
-Participants
-Steering Committee
-Program Committee

Workshop Report
-Preface
-Final Report

• We have discovered marine invertebrates that can be sources of new pharmaceuticals, cosmetics, and nutraceuticals—and perhaps can be raised in culture.

• We have experimented with new methods to raise fish, mollusks, crustaceans, and algae in aquaculture.

• We have developed new remote sensing methods to detect marine environmental changes.

But aquaculture is both modern and old.

[SLIDE 2]

This is a photo of a low-tech state fish hatchery facility (the state will remain unidentified), with a gravity feed water supply, indoor troughs, and outdoor pools for the larger fish. There’s no magic here.

Yet this facility successfully releases 800,000 salmonids each year to stock rivers and lakes for recreational fishing. It supplies fish raised in uncontaminated water in a clean facility.

What can biotechnology do for a facility such as this? The answer may be more fish, bigger fish, and, with vaccinations against common diseases, healthier fish.

It has taken us nearly thirty years to travel from the first genetically engineered microorganism to the ability to sequence the full human genome—or any other genome,

Brief history of biotechnology

Let’s begin with a very brief history of biotechnology. One of the most apt definitions is "making money from biology."

[SLIDE 3]

Biotechnology is not new. Fermentation technology—the earliest form of biotechnology—originated with mold-fermented foods in China and beer brewing and bread making, combined, in Egypt.

The history of the field is strewn with milestones. The Biotechnology Industry Association has "A Timeline of Biotechnology" online. This is a selective list of how quickly the field has progressed. The first page begins in 1750 B.C. with beer brewing and ends in 1911 with the discovery of the first cancer-causing virus.

The second page extends through the 1950s. It includes Avery, MacLeod, and McCarty’s discovery of "transforming factor," McClintock’s transposable elements, and Watson and Crick’s discovery of the DNA double helix. 1998, the last year of the timeline, takes up a full page.

During the 48 years since Watson and Crick’s short and succinct paper on what is one of the most historically important scientific discoveries ever, the structure of the DNA molecule, the field of biotechnology has progressed from the world of science fiction to the world of science present.

• In 1973, Cohen and Boyer produced the first recombinant DNA by cloning a gene into a bacterial plasmid. From then on, commercial biotechnology grew in quantum leaps.
• In 1981, the first biotechnology product, a monoclonal antibody-based diagnostic test kit, was approved by the U.S. Food and Drug Administration (FDA).

[SLIDE 4]

• The following year, Genentech’s recombinant human insulin (rh-insulin; Humulin^®) was approved in the U.S. for human use. Rh-insulin is now the primary insulin on the market.

[SLIDE 5]

Ernst and Young reported 305 biotech drugs in Phase II/III or Phase III clinical trials for major diseases in 1999, up from about 150 such trials in 1998. And the number continues to grow.

[SLIDE 6]

Technological advances include the polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis and other means of artificially replicating or analyzing nucleic acids and the ability to determine the genetic makeup of organisms via high-throughput genomic analysis.

The awesome discovery that the human genome is not as large as we had thought is perhaps the most important biological finding of the beginning of this new century. Perhaps more important than the size of the human genome is the new knowledge of protein function: after genes code for proteins in transcription and translation, the proteins are broken into fragments and reaggregated.

Thus, the field of proteomics—the study of the intracellular function of proteins—will be a major research focus in what some are calling the post-genomics era.

The information we glean from our newfound knowledge of the genome and proteins may be made accessible via bioinformatics. This is the maintenance and mining of databases that will, eventually, contain genomic, physiological, and ecological data. These databases, in turn, can be used by researchers for comparative purposes:

• How is this molluscan protein similar to the recorded human protein?

• Can two known proteins interact in a new and unique manner to form a third protein?

• If we simulate a protein, can we determine how it will act in vivo?

These questions were unanswerable as little as a decade ago.

[SLIDE 7]

Our knowledge of evolutionary genetics is growing rapidly. Not only did we first learn of the existence of the Archaea only a bit more than 20 years ago, but we now know from Ed DeLong’s work that they comprise a major portion of the microbial world in the deep ocean and are spread throughout the ocean’s depths (Karner, DeLong, and Karl, Nature 409:507-510, 2001).

These discoveries would have been impossible without the ability to use rRNA probes to identify microorganisms—a genetic technology, born of biotechnology methods. These technologies are adaptable from Archaea to humans. The push to complete the Human Genome Project early led to technologies that are amenable for use with other species. And organizations that were heavily invested in the HGP are looking outside the human genome for other genomes to mine.

Marine Biotechnology

[SLIDE 8]

In 1992, an Organization of Economic Co-operation and Development report on biotechnology defined marine biotechnology as "the application of scientific and engineering principles to the processing of materials by marine biological agents to provide goods and services." (Bull, 1992, as cited in Zilinskas, Colwell, Lipton & Hill, 1995, Status report U.S., Japan, Australia and Norway)

This would include biotechnology based methods for aquaculture, fisheries, and marine natural products.

[SLIDE 9]

Marine organisms are likely to be sources of numerous pharmaceuticals. Although few of them are near commercialization, there is hope that bryozoans, which may be sources of anticancer drugs, will be able to be cultivated via aquaculture, which would greatly relieve environmental harvest pressure on the species.

[Slide 10]

Many of the marine-derived pharmaceuticals are likely to be microbial in origin. Fish and invertebrate toxins are thought, in many cases, to be produced by symbiotic microorganisms. Maintaining these aquatic species in culture may be one way to have a continuing commercial source of toxin, although this will depend upon whether the captive circumstances are conducive to the symbionts’ thriving. Ultimately, culture of the microbial symbiont, with its ability to produce toxin induced, will unlock much of the mystery surrounding marine toxins.

[SLIDE 11]

Marine organisms, many of which can be maintained in culture, are sources of commercial products, among which are food additives such as carrageenan, agar, and chitin; commercial glues; enzymes for detergents and for biological reagents; and other products.

• Nutraceuticals, such as food-based enhancers, and cosmetics also can be produced from marine sources.

• The soft coral, Pseudopterogorgia elisabethae, is the source of pseudopterosin that is used as a cosmetic additive [SLIDE 12]. It has been licensed to Estée Lauder for Resilience^® skin cream.

• At present, Pseudopterogorgia is harvested directly from reefs off the coast of Bermuda. This organism is unlikely to be cultured because the reefs can sustain the harvest, but other marine organisms will be amenable to aquaculture.

Our knowledge of the marine environment is limited. We’ve only recently learned of the extensive distribution of Archaea in the ocean. With use of submersibles, we have discovered fish and invertebrate species we previously had no knowledge existed. But to understand fully the marine environment and the interactions and interrelationships between organisms, we must be able to use tools of genetic mapping and proteomics.

Genomics and aquaculture

[SLIDE 13]

Aquaculture may be defined as the propagation or cultivation of aquatic vertebrate or invertebrate organisms in a controlled or selected environment.

[SLIDE 14]

The goal of aquaculture is a commercial product, although subsidiary goals may be for recreational use such as recreational fishing or restocking. Until recently, almost all fish and other seafood were "wild caught."

• According to the National Marine Fisheries Service, in 1998, the U.S. produced 358,209 metric tons (789.7 million lbs.) of fish and invertebrates from aquaculture, with a total sales value of nearly $1 trillion.

• The UN Food and Agriculture Organization (FAO) estimates worldwide, 30.9 million metric tons of fish and invertebrates were produced by aquaculture.

• Wild caught fisheries supplied 86.3 million metric tons.

Thus, aquaculture is a burgeoning industry, with the U.S. supplying only a small portion of it.

Wild fisheries cannot supply enough seafood products to meet the world’s increasing demand. And as wild fisheries decline or areas are protected, it will become even more essential to expand aquaculture.

[SLIDE 15]

Here are the statistic on the number of each species taken from the George’s Bank fishing area off Cape Cod in 1963 and 1990.

If you saw the movie, The Perfect Storm, you’ll recall that the boat went far beyond George’s Bank because they wouldn’t be able to obtain a sufficient catch there. Note the decrease in valuable fish species and the increase in lower quality fish, such as rays.

[SLIDE 16]

This change in percentages of biomass may be an artifact of the sampling method, but there is no doubt that by the early 90s, fisheries declined in the George’s Bank area. In 1993, the George’s Bank fishing areas were closed, although portions were opened for scallops in 1999 and 2000.

The National Marine Fisheries Service is aware that during the closed period, absolute numbers of fish and otherwise depleted species increased. But should they continue protecting the biodiversity of the area and prohibit many kinds of commercial fishing there?

So although excellent fishing grounds may be closed, the worldwide demand for aquatic organisms—for food, for animal feeds, for nutriceuticals such as eicosapentaenoic acid and shark cartilage, for example—is growing.

Genomics will allow us to identify useful genes in fish, invertebrates, and algae for maintenance in aquaculture. It also will allow us to select with precision genes that we wish to amplify in a particular species in culture. Genome mapping is being carried out in a number of economically important aquaculture species.

[SLIDE 17]

Production of transgenic fish through electroporation and microinjection has been successfully carried out since the mid-1980s. Genomics promises the ability to specifically identify and insert genes that will aid in propagation and in maintaining the animals’ health.

In a number of cases, fish growth hormone genes, along with a reporter gene, which will tell whether the transformation is successful, are inserted into the fish. There are reports—some by Tom Chen, who is here--of good growth in transgenic species such as tilapia, catfish, and rainbow trout, among others.

This is not a panacea. A recent study published in Nature on growth and survival of wild and domesticated transgenic trout, in which a growth hormone gene was inserted, showed that the animals grew more than nontransgenic trout (R. H, Devlin et al., Vol. 409: 781-782).

But, the transgenic fish all died before reaching sexual maturity and showed significant cranial abnormalities. Sufice it to say, we need more research in this area.

Current genomics research on fish and invertebrate species that can be raised in aquaculture was recently summarized in a report on proceedings of an aquaculture genome workshop. It included information on genomics of shrimp, oysters, salmonids, catfish, and tilapia (Alcivar-Warren and Kocher, 1999).

The goals are not simply to map the genomes. They are to identify genetic diversity within stocks and identify genes that can be transferred into fish or invertebrate eggs and result in transgenic animals with improved safety and economic benefit.

We can insert genes that will result in better and faster growth rates, disease resistance, greater toleration for environmental change (e.g., cold water tolerance), increased fecundity, and more edible "meat." We even can insert genes that will lead to manufacturing centers, producing pharmaceuticals for the human or veterinary market!

Manipulation of ploidy in fish and shellfish in culture is essential for guaranteeing that the genetically engineered organisms are sterile and will not be able to reproduce should they escape into the environment. It also allows for reproduction of species during seasons when they would not normally reproduce. As these methods are not always foolproof, research continues on better means of manipulating ploidy.

In these artificial environments, natural reproductive mechanisms often fail. Induction of spawning requires a surge in gonadotrophin, which is absent in some cultured fish. Thus, hormonal manipulation of spawning is an important factor in reproduction of fish in aquaculture.

In the past, hormones had to be administered by injection, a process stressful to the animals. New methods to release gonadotropins or steroidal hormones at appropriate times include slowly released hormonal implants in a polymer system. Low-intensity ultrasound treatment also increases uptake of hormones and other small molecules.

We still need increased knowledge of fish physiology, including information derived from the genome and the proteins it may encode. This would aid us in developing methods to artificially manipulate ovulation and spawning through gene activation and/or suppression.

[SLIDE 18]

Biotechnology methods have resulted in production of new, genetically engineered vaccines for aquaculture, such as the vaccine against infectious hematopoietic necrosis (IHN) virus, which is responsible for the death of trout and other salmonid stock. Of course, the ideal situation would be to genetically manipulate fish so they carry disease-resistance genes and, thus, don’t have to be vaccinated.

[SLIDE 19]

One current need is to identify new sources of food protein for aquaculture. Currently, fish in culture are fed mainly commercial chows composed of fish meal. Were fish to be found to carry infectious prions—similar to those that cause bovine spongiform encephalopathy—fish protein-containing chows could be an expensive liability.

[SLIDE 20]

It may be possible to grow algae in bioreactors to add nutrient value to the algae—making them an appropriate fish food. Microalgae already are grown in bioreactors—in this case, for the purpose of drug discovery. The methodologies are there and the scale can be increased.

[SLIDE 21]

The expanding knowledge of marine organisms allows us to maintain and grow selected commercially valuable species in aquaculture—sometimes in polyculture in which several different species are maintained together.

[SLIDE 22]

Integrated mariculture systems, such as those devised by Muki Shpigel and Amir Neori at Israel’s National Center for Mariculture, allow land-based production of fish, valuable bivalves, shrimp, and abalone in polyculture.

[SLIDE 23]

Here, algae serve to remediate the nitrogenous pollution from the aquaculture waste.

[SLIDE 24]

The algae, in turn, serve as a food source for the bivalves or abalone, which are sold to the tourist trade in Israel.

[SLIDE 25]

Charles Yarish and his colleagues at the University of Connecticut are taking this a step further by genetically engineering the red alga, Porphyra.

[SLIDE 26]

These are used for remediation in aquaculture of salmonids, to better tolerate diverse habitats. The Porphyra can be used to remediate wastes from salmonid culture and, in turn, it produces a valuable product, nori, the seaweed sheets used to wrap the Japanese culinary specialty, sushi.

Despite the increases in the weight and dollar values of aquaculture-based products in the U.S., we still import a total of more than $9 billion worth of seafood—one-third of which is shrimp. But Asian countries—China, India, Japan, Indonesia, Thailand, Bangladesh, and Vietnam—produce much more seafood by aquaculture than we do.

[SLIDE 27]

We need to adapt our technological advances to this field. For example, minor changes in environmental conditions—including the presence of infectious microorganisms—could, in the not-too-distant future, be monitored by array technologies or perhaps nanofluidic sensors, minutely sized biosensors made of silicon.

If we learn what organisms can best maintain closed culture systems—systems where there is no water from outside—we could use biosensors to determine the levels of these organisms in the systems. We could even genetically engineer microorganisms to work best within the systems.

Let me give you an example of direct application of genomic technologies to solve what has the potential to be an aquaculture problem. Pfiesteria is a dinoflagellate that produces a potent toxin during blooms. Aquatic toxins, in general, are concentrated as you move up the food chain. So carnivorous fish along with filter feeders, such as mollusks, tend to demonstrate high concentrations of the toxin during times of the dinoflagellate bloom.

Pfiesteria has been implicated in fish kills and human illness. And the species is close to home: Pfiesteria has been identified in the Chesapeake Bay. Any offshore mariculture facility that is dependent on sea water as a water source in an area where Pfiesteria blooms, is at risk for toxin-related problems and potential economic losses. Researchers have found that in culture without fish present, Pfiesteria loses its toxicity.

What is happening? Is there a change in gene expression in the dinoflagellate or is the toxin produced by a bacterium and is related to the presence of fish?

A team from the University of Maryland along with scientists from North Carolina is planning to use genomics to determine if toxin production is a result of direct production by the dinoflagellate, or is it produced by a microorganism that somehow functions only in the presence of the fish or is contributed by the fish? These types of experiments will help us to control outbreaks of major disease that cause economic losses.

Among the technologies directly applicable to aquaculture are genomics, genetic modification, hormonal control of reproduction, manipulation of ploidy, identification and treatment of disease, and development of vaccines against disease.

Also related to aquaculture are the ability to raise species in polyculture, ability to remediate wastes produced by aquaculture, and development of more sophisticated housing and monitoring methods for fish, invertebrates, and algal culture. The ability to monitor environmental parameters and infections in aquaculture facilities and waste streams from these facilities will require use of sophisticated technologies, such as biosensors and genetic microarrays, meaning chips.

We are ready to take the technological advances that have brought us the successful Human Genome Project and apply them to the study and modification of organisms maintained in aquaculture. There are three especially promising opportunities.

• The first is production of genetically modified fish and invertebrate species for food.

• The second requires research on the genomics, proteomics, and physiology of microorganisms that can be introduced into recirculating systems. We need to identify strains that are effective in circulating systems and determine their parameters of growth and the economics of their use.

[SLIDE 28]

The third is in systems for bioremediation such as those being studied by Yarish and being used now in Israel.

[SLIDE 29]

Biocomplexity

Before we can select species that are amenable to aquaculture, we may first need to study complex environmental-species interactions. We call these interactions between the organism—from the cellular level—and all the environmental factors influencing the organism and the community in which it lives, biocomplexity.

Biocomplexity is a term that describes the dynamic web of interrelationships that arise when living things interact with their environment. at all levels, from the molecular, and intracellular to intraorganismal and environmental.

In the past two years, the National Science Foundation has put a significant amount of funding into research on biocomplexity. In 2000, we granted over $52 million for biocomplexity related studies. Our biocomplexity grants are among our newest multidisciplinary collaborative projects.

The NSF is devoting an increasing amount of our budget to these systemic studies, and we plan to host a workshop on biocomplexity projects this year.

Many of the projects being funded relate directly to our work. Researchers from Cornell University are studying water movement and ecosystems in watersheds connected to Lake Ontario. A consortium led by scientists at University of New Mexico is using mathematical models and empirical measurements to study principles related to life history, abundance, distribution, and species richness of organisms in scale with body size, space, and time.

Two ongoing projects may yield information that could have an effect on aquaculture.

[SLIDE 30]

One is a study by Jesús Pineda of the Woods Hole Oceanographic Institute on effects environmental factors, including currents, predation, food sources, substrate availability, and disturbance on survival of larvae of near shore bottom-associated invertebrates.

[SLIDE 31]

Another, carried out by Peter Verity and colleagues of the Skidaway Institute of Oceanography, is studying organizational and communication processes in solitary cells and colonies of the colonial phytoplankton, Phaeocystis.

[SLIDE 32]

This species is especially important in boreal and arctic waters of both poles. The species can occur in prodigious blooms in temperate regions, such as New England and the North Sea. It is considered to be a nuisance alga—perhaps even a harmful algal bloom (HAB) species in the North Sea, especially on the coast of Norway, where salmon pen farming is widespread.

Biocomplexity studies can be valuable in aquaculture research. They can increase our understanding of interrelationships between aquatic organisms and environmental factors. They would allow study of diseases that can affect cultured aquatic organisms and wild aquatic organisms. Such studies may also aid in identification of new organisms for aquaculture, along with methods for culturing them.

They also may result in identification of new marine biotechnology products or organisms that themselves are sources of products. This may help us learn what keeps marine bacteria alive in the natural environment. This in turn translates finding ways to culture as yet unculturable microbial species, a potential source of new products and processes.

Problems

As in all technologically advanced applications, there are unresolved questions and problems. First, we must deal with the issue of cost versus profitability. Yes, we can successfully treat fish with hormones in a well-controlled manner to regulate reproduction, but the costs must be offset by potential profit or this will remain in the laboratory and not be transferred to industry.

• Can we afford to garner all this technology for aquatic species production?
• Can the market bear the cost?

Next, an educated public is a public that is more likely to support biotechnology initiatives in aquaculture. With any even minor lapses, biotechnology-assisted aquaculture is apt raise the public’s level of concern—and rightly so—about safety, both of the food products and of the environment.

Recent articles in the popular press and other venues warn of "Frankenfish," the fish version of gene-altered plants. Certainly there are reasons for concern.

• Can these fish escape into the environment?

• Although many of these fish are polyploid, and therefore sterile, how do we know they’re really sterile?

• What proof do we have that they cannot mate and pass these genes on?

• What proof do we have that, although unable to produce live offspring, they will not court wild fish, competing with wild animals for mates and potentially affecting the wild population size by this behavior?

• What about parental stocks that are genetically modified but not sterile?

These are questions that are being raised and that research will have to answer. And once we have answers, we must be ready to communicate these answers to the media and the public—whether these answers allay fears or even if they leave more questions.

Communication--with the public, with decision -makers, with industry, with universities and other research institutions—is essential. Not only must we keep the public educated and informed so they support our work, they also need to know that our aquaculture products are available.

For example, a recent "Q&A" to the Science pages of The New York Times asked about the safety of tuna fish sandwiches in light of mercury contamination of tuna (NYT, 2/20/01, p. F2, C. Claiborne Ray). The advice was that a woman who could be pregnant should limit herself to not more than one tuna sandwich per week.

Why not eat safe, farm-raised catfish or tilapia? Is tuna the only source of fish protein people know about?

The best time to educate people is when they are young. This is the time both to build awareness and to train people to work in the now-fledgling industry. Aquaculture programs are being set up nationwide to teach teenagers the basis for aquatic technologies.

These industries will need workers at all levels. U.S. aquaculture currently employs about 180,000 people. With an expanding industry—and a more technology based one—these numbers will be increased. And the use of genomic technologies will require workers ranging from trained genomics experts, to water quality specialists, to engineers who design systems and technicians who run the systems and take care of the organisms.

[SLIDE 33]

These teenagersat a part-time aquaculture magnet school in Bridgeport, Connecticut, are working on a project to culture the rhodophyte, Chordus crispis, in a program sponsored by University of Connecticut.

[SLIDE 34]

They are typical of teenagers throughout the country who will be familiar with the techniques of aquaculture and ready for university training in the field.

The aquaculture industry itself must be environmentally responsible, and methods for environmental responsibility must be developed. Aquaculture efforts that pollute coastlines and cause changes in habitats are not acceptable.

[SLIDE 35]

And so in closing, I hope I’ve show that we have before us a great challenge – and greater opportunities. Marine habitats are being altered—by fishing, pollution, and human activities—yet we don’t understand the mechanisms that lead to environmental damage. Wild-caught seafoods are insufficient in numbers to meet the world demand. Aquaculture is essential to fill the gap.

Marine biotechnology, including aquaculture, is an emerging area of immense importance—but it has been the biotechnology industry’s stepchild and needs more attention. The U.S. has the technology to take the lead in the biotechnology of aquaculture, bringing tremendous benefit to our economy.