Natural Products from the Sea: Can Seaweed Cure Cancer?

seaweed-cancer

Pharmaceuticals

For thousands of years, humans have depended on a “natural pharmacy” stocked with extracts from plants, animals, and microbes to treat a wide variety of diseases and medical problems. Research has resulted in the isolation and use of pure drugs and bioactive compounds. Today, 40 to 50 percent of drugs being used to treat human illness are derived from natural products, the majority of which are derived from terrestrial organisms (Bruckner 2002). We have come to depend on a range of medical treatments derived from natural products, such as antibiotics, pain killers, cancer therapies, and anti-inflammatory drugs, in our daily lives, and as a society we expect and value the availability of new drug therapies.

Current therapies and medicines for some serious diseases do not completely control the target disease or are available in limited supply. With the emergence of antibiotic resistance in bacteria and the spread of devastating diseases such as AIDS, there is an urgent need for new drugs. After several decades discovery and intensive searching among land-based organisms, novel natural products from terrestrial organisms with promising chemical and pharmaceutical properties are becoming harder to find.

Researchers are now turning with high expectations to marine organisms in their hunt for new medicines and other important bioactive compounds. The ocean is home to over 80 percent of all life forms on Earth. Thirty-four out of the 36 phyla of life forms are represented in the oceans; in contrast only 17 phyla are present in terrestrial habitats. The promise of a rich new source of useful pharmaceuticals and bioactive chemicals lies in our oceans.

Marine organisms have evolved a wide variety of toxins and novel chemical adaptations that help them compete successfully in demanding environments, such as a crowded coral reef, pounding surf, deep ocean sediments, thermal vents, and Arctic waters. Many sessile marine organisms, in particular invertebrates such as corals, mollusks, sponges, echinoderms, and bryozoans, have evolved chemical weapons to help them compete for limited resources, avoid predation, or deter overgrowth by competitors. Scientists anticipate that some of these compounds will provide useful drugs, especially antibiotics and anticancer agents, as well as agricultural products, and industrial enzymes.

Salinospora bacteria
Cultures of bacteria of the genus Salinospora.
Image Courtesy: National Science Foundation

Scientists are focusing their search for new marine bioproducts in habitats with high biodiversity and on marine microbial populations. Soil bacteria in the order Actinomycetes have already provided society with some of the most beneficial antibiotics and anticancer agents. Efforts to isolate marine actinomycetes from tropical ocean sediments have been extremely promising (Mincer et al. 2002). Members of Dr. William Fenical’s research team at the Scripps Institute of Oceanography have discovered at least 2,500 new species of marine bacteria, now called Salinospora, which are related to the land-based actinomycetes. More than 80 percent of these newly discovered bacteria demonstrate anticancer activity at very dilute concentrations. One compound, called Salinosporamide A, is a potent inhibitor of cancer growth, including human colon carcinoma, nonsmall cell lung cancer, and, most effectively, breast cancer (Feling et al. 2003). Salinosporamide A is currently in the process of completing Phase I clinical trials (National Institutes of Health 2009).

The use of marine organisms as pharmaceuticals and other useful products is not new. Eastern cultures have used marine organisms such as the sea horse, sea hares, and sponges in traditional medicines for centuries. Biotechnology opens new avenues for discovery. Several important drugs, including AZT (used to treat AIDS patients) and some anticancer drugs, have been isolated from sea organisms. Synthetic medicines such as ziconotide, a compound isolated from the venom of cone snails, are in clinical trials. Table 1 below lists marine-derived natural products in clinical and preclinical trials as of mid-2004.

Diatoms
Diatoms like the ones shown here are an example of a silicon-encased marine algae.
Image Courtesy: National Science Foundation 

Other natural products that can be purified from marine organisms are in use today as important nutritional supplements, industrial enzymes, cosmetics, sun screens, paint and plastic additives (as UV-blockers), natural pigments in fish food to add color to aquacultured species, and pesticides. For example, the carotenoids (orange-color pigments) astaxanthin, zeaxanthin, and lutein have high value and are being purified from microalgae and bacteria for use as anti-oxidants, anti-inflammatory agents, and as natural pigments for fish feed. Purification of these high-value carotenoid pigments is a growing marine biotechnology industry in Hawaii. Marine algae are also the source of two nutritional fatty acid supplements for infant formula. Some fatty acids are important for brain and eye development in fetuses and cardiovascular health in adults. These occur naturally in breast milk, but they have not been added to infant formula until recently.

Biodiversity

The ocean’s unexplored biodiversity is threatened by increased human use, climate change, environmental pollution, overharvesting, and increased collection as scientists search for new natural products. Scientists fear that some species producing the most beneficial compounds will become extinct before the full potential of these compounds can be realized. Finding a sufficiently large or renewable source of a potential pharmaceutical or bioactive compound is a major hurdle in the development of novel marine bioproducts. Many of the potentially important compounds will come from rare species with low biomass, slow reproductive rates, and a limited geographic distribution. Furthermore, because many of the invertebrate species that may be important sources of interesting compounds have little to no value in the fishing industry, countries have not developed management strategies to prevent overharvesting of these species.

Recent Advances

Recent advances in marine biotechnology and microbiology are helping scientists overcome many of the challenges involved in discovering useful bioactive compounds and bringing new marine natural products to market. Molecular genetic techniques, including the polymerase chain reaction and genomic sequencing, are being used to help researchers identify the genetic sequences involved in the production of natural products with interesting activities, as well as overcome some of the challenges of producing large enough quantities of an interesting compound.

The activities in this section of the curriculum are related to the search for biologically active compounds from marine organisms. Activities range from identifying products containing marine-derived compounds to testing for antibacterial properties of seaweed. Individual activities can be selected to complement classroom plans in biology, chemistry, or marine science classes, or the unit can be used as a whole.

Marine-derived natural products
Table 1. Update: Ziconotide was approved as a drug by the FDA on December 30, 2004.
Source: Data from David S. Newman, National Cancer Institute, Natural Products Division, Frederick, MD.

Bradley, David. 2003. Marine bugs make drugs. Reactive Reports Chemistry Web Magazine Issue #30. http://www.reactivereports.com/30/30_4.html. Accessed July 2009.

Bruckner, A.W. 2002. Life Saving Products from Coral Reefs. Issues in Science and Technology online. http://www.issues.org/18.3/p_bruckner.html. Accessed July 2009.

Feling, R.H., G. O. Buchanan, T. J. Mincer, C. A. Kauffman, P. R. Jensen, and W. Fenical. 2003. Salinosporamide A: a highly cytotoxic proteasome inhibitor from a novel microbial source, a marine bacterium of the new genus salinospora. Angewandte Chemie International Edition, English, 42(3):355–357.

Mincer, T. J., P. R. Jensen, C. A. Kauffman, and W. Fenical. 2002. Widespread and persistent populations of a major new marine Actinomycete taxon in ocean sediments. Applied and Environmental Microbiology. 68(10):5005–5011.

National Institutes of Health. 2005. The Nation’s Progress in Cancer Research Annual Report: Ocean is a Treasure Trove of Possible Anticancer Compounds. http://www.cancer.gov/nci-annual-report/1.html . Accessed July 2009.

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IIA 1. Shopping for Marine Natural Products

Background

Have you eaten your algae today? You probably have and didn’t even know it. Compounds derived from algae and many other marine organisms are in many of the foods we eat and the medications we take. No matter where you live, you have seen algae, whether it is seaweed in the oceans, green slime on the side of the aquarium, or the scum on the top of a pond. There are thousands of species of algae, many of which have been collected and eaten since prehistoric times. A variety of algal species also have properties that are beneficial to human health. Among them are Porphyra, familiar to us as nori, the wrapping used to make sushi, kelp, and Spirulina, touted for its health benefits and sold as a health supplement.

There is also a great potential to produce new pharmaceuticals from other marine organisms. Many marine organisms secrete compounds that help them survive in their environment. Some of these compounds have properties beneficial to humankind. There may be a wealth of pharmaceutical uses for marine organisms that we are not yet aware of.

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Focus Question

How many natural products from the sea can be found in supermarkets and drugstores?

notepad learning-objectives
Students will describe the various uses of marine organisms in food products and pharmaceuticals.
glass materials
Video Ocean Resources (see References and Further Reading section for reference)

Article “Drugs from the Sea” (see References and Further Reading section for reference)

Collection of foods and pharmaceutical products from a grocery store, health food store, or Asian food store that contain marine organisms

clock teaching-time
Two class periods
test-tubes procedure
1. Show the video Ocean Resources and have students read the article “Drugs from the Sea” (see the Resources and Further Reading section below for specific references).

2. Scavenger hunt: Students will compile a list of products that contain at least one ingredient from the ocean. If a visit to the grocery or health food store is not possible, students can do a Web search.

3. Possible words to look for include algin, alginic acid, carrageenan, spirulina, porphyra, and kelps.

4. Bring samples to class and allow students to read food-packaging labels to locate product words. Sample or even cook some of these products in class.

student-work
questionmark questions
1. Make a list of some of the products you found that contain marine-based substances. On the basis of your research, how prevalent are products developed from marine organisms in products you use?

2. What products do scientists hope to find by searching ocean organisms?

3. What clues do scientists use to locate these compounds?

teacher-key
answermark answers
1. Make a list of some of the products you found that contain marine-based substances. On the basis of your research, how prevalent are products developed from marine organisms in products you use?

Students’ results will vary, but some possible products include those which contain algins as emusifers (they prevent oils from separating from solids and keep food smooth and creamy), sushi nori, spirulina (a health supplement), beta carotene, carrageenan (derived from a red alga), and some omega-3 fatty acids (sometimes marketed as “krill oil”).

2. What products do scientists hope to find by searching ocean organisms?

New medicines, new products for various industrial uses, etc.

3. What clues do scientists use to locate these compounds?

They examine whether organisms have interesting defense mechanisms or unusual habits. Searching for the same genera as terrestrial microbes has proven fruitful.

computer references
Films for the Humanities and Sciences, P.O. Box 2053, Princeton, NJ 08543-2053.
Ocean Resources (23 minutes).

Mestel, Rosie. 1999. Drugs from the Sea. Discover Magazine Vol. 20
http://www.discover.com/issues/mar-99/. Accessed August 2005.

Washburn, Tracy. 1997. Medicinal Toxins. Science Notes, Summer 1997 http://scicom.ucsc.edu/SciNotes/9701/full/features/sponge/underwater.html. Accessed April 2005.

IIB 2. The Fight against Malaria: Promising New Drugs from the Sea

Background

mosquito
The Anopheles mosquito is a vector for malaria.
Photo Courtesy: WHO/TDR/Stammers

Malaria is an infectious disease caused by a sporozoan parasite that is transmitted through the bite of an infected Anopheles mosquito. Symptoms are chills and fever. Millions of people worldwide are affected by malaria and over a third of those infected will die from it. Many of its victims are young children. At one time malaria was more widespread than it is now. It is now confined mostly to Africa, Asia, and Latin America, where poverty and poor health care make controlling the disease difficult. In addition, many of the existing drugs for malaria are no longer effective because the parasites have grown resistant to the drugs. Drug companies are reluctant to fund research on cures for a disease that occurs mostly in countries unable to pay for treatment. Marine organisms have the potential to produce tremendous numbers of pharmaceuticals. Perhaps one of these drugs will be a new and inexpensive treatment for malaria.

Malaria is just one example of a disease that claims many lives in Third World countries and for which more research is needed. There are many other diseases, such as Chagas disease and river blindness, for which no medications and little research exist.

PDF file PDF file for this project

Focus Question

Why should finding a cure for malaria be a worldwide concern?

notebook
learning-objectives
Students will
• Describe the global concerns related to finding a cure for malaria

• Describe how researchers are searching for novel medicines in marine natural products

• Investigate the ethical issues involved in seeking a cure for malaria

glass
materials
Student handouts, such as the Introduction to the Natural Products (II. Unit Introduction) unit from this curriculum and other references listed below

Dr. William Gerwick’s PowerPoint presentation (II. Powerpoint Search for New Medicines from the Sea) on “Drug Discovery from Marine Organisms,” included in this curriculum

Internet access

clock
teaching-time
Two class periods or use as a strand throughout the unit.
testtube
procedure
1. Provide students with the article “A Death Every 30 Seconds” from Scientific American magazine (May 13, 2002). The book Fever Trail: In Search of a Cure for Malaria by Mark Honigsbaum also provides good background information on malaria. This book may be available in a library or for purchase on the Web.

2. Have students view the video Malaria, which can be downloaded from the 50 free Secrets of the Sequence video series developed by Virginia Commonwealth University. Use the accompanying study guide as a discussion tool. There is also an activity that accompanies this video that some teachers may find of interest but which is not needed to complete this activity.

3. Assign students to search the Web to locate research on the topic of antimalarial drugs. Students may also research Chagas disease or other infectious diseases affecting developing countries (see the background reading on Chagas disease (II.B.3) included on this disk).

4. Divide students into small groups to discuss the issue, “Should rich countries that are relatively unaffected by malaria help pay the cost of developing a new cure for the disease?”

5. Each group will produce a poster that includes the following:

a. A discussion of the issue
b. Pro position and reasons behind this position
c. Con position and reasons behind this position
d. Positives and negatives of each position
e. Group’s decision and justifications

6. Each group will present the poster to the rest of the class.

student-work
questionmark
questions
1. Why do you think there has been so little development of medications to cure diseases that afflict people in Third World countries?

2. What are some ways to support drug research for these diseases?

3. Do you think richer countries have an obligation to help find cures for diseases common in poorer countries? Why or why not?

teacher-key
answermark
answers
1. Why do you think there has been so little development of medications to cure diseases that afflict people in Third World countries?

Low incomes in these parts of the world cannot cover the cost of drug development, much less provide a profit for drug companies and their stock holders. Recouping these costs comes through the purchase of the medications.

2. What are some ways to support drug research for these diseases?

Development of not-for-profit companies and support through charitable institutions

3. Do you think richer countries have an obligation to help find cures for diseases common in poorer countries? Why or why not?

Student answers will vary. Evaluate the reasons for their answers and the supporting evidence they provide.

computer
references

Centers for Disease Control. Malaria facts. http://www.cdc.gov/malaria/about/facts.html. Accessed July 2010.

Editors, Scientific American. 2002. A Death Every 30 Seconds. Scientific American June 2002. http://www.scientificamerican.com/article.cfm?id=0005ADE5-04D6-1CDC-B4A8809EC588EEDF. Accessed July 2010.

Films for the Humanities and Sciences, P.O. Box 2053, Princeton, NJ 08543-2053. Ocean Resources (23 minutes).

Henkel, John. 1998. Drugs of the Deep. http://web.ebscohost.com/ehost/detail?vid=2&hid=111&sid=5ab40937-6c54-40ed-8b4f-24d197cd30fd%40sessionmgr111&bdata=JnNpdGU9ZWhvc3QtbGl2ZQ%3d%3d#db=awh&AN=186489. Accessed July 2010.

Honigsbaum, Mark. The Fever Trail: In Search of The Cure For Malaria. New York: Picador, 2003.

Institute for One World Health http://www.oneworldhealth.org/diseases/chagas.php. Accessed July 2010.

Malaria Journal http://www.malariajournal.com/start.asp. Accessed July 2010.

Mestel, Rosie. 1999. Drugs from the Sea. Discover Magazine Vol. 20. http://discovermagazine.com/1999/mar/cover. Accessed July 2010.

The Secrets of the Sequence video series. Virginia Commonwealth University. http://www.pubinfo.vcu.edu/secretsofthesequence/. Accessed July 2010.

IIB 3. Chagas Disease: A Case Study

Background

Chagas patient
This child in Panama is suffering from Chagas disease, manifested as an acute infection with swelling of the right eye.
Image Courtesy: Centers for Disease Control and Prevention, Dr. Mae Melvin

Millions of people around the world, a large percentage of whom are children under the age of five, die from malaria, sleeping sickness, and other persistent diseases. Much of the population of Third World countries lives on $1.00 a day, so purchase of medications to address these diseases is often beyond their economic ability. Making affordable new medicines available to Third World countries to control these diseases is a challenge facing drug companies and governments around the world.

The process of developing new medicines is lengthy and expensive. According to Dr. William Gerwick in the College of Pharmacy at Oregon State University, screening 15,000 new natural products may yield one effective therapy on the market. The process of bringing a new medicine to market may take 15 years. Drug companies must invest significant resources in identifying potential new drugs and ushering them through clinical trials. Many effective drugs are not available to people in Third World countries because they are too expensive.

Trypanosoma cruzi
Trypanosoma cruzi parasites.
Image Courtesy: Centers for Disease Control and Prevention

An example of this issue is Chagas disease, a life-threatening disease transmitted to humans by a triatomine insect containing the parasite Trypanosoma cruzi. The parasite is carried by a reduvidae beetle, an insect that infests mud, adobe, or thatch houses. The reduvid beetle bites its victims, and its feces infects the wound. Infection can also occur by rubbing the eyes with fingers carrying the beetle droppings. Chagas disease, found only in the Americas, is present in 19 countries in Central and South America. It is estimated that 10 to 12 million people are infected with T. cruzi and at least 50,000 people die each year. In the U.S., more than 100,000 people are infected with T. cruzi. Additionally, 100 million people in Latin America are believed to be at risk of infection. (Institute for OneWorld Health 2005). Infants and children often die from swelling of the brain associated with the disease. In other people the parasite will lie dormant for years only to emerge and lodge in the heart muscle, causing serious and irreversible damage to the heart. Chagas disease is the leading cause of heart disease in South America.

Because the only current treatment for Chagas is not very effective and has severe side effects, there is an overwhelming need for new effective, nontoxic, and inexpensive treatments. One promising new medicine to cure the disease has been discovered buried in the files of a drug company. Originally investigated for a treatment for osteoporosis, drug K-777 shows great promise to control the disease. An oral form is currently in preclinical studies to determine if it can enter human clinical trials. Since the people who are infected with this parasite are poor, there was little chance of K-777 ever being developed and tested as a Chagas treatment by a large for-profit pharmaceutical firm.

To address these problems, the nonprofit drug company Institute for OneWorld Health was founded in 2000, with the mission of developing “safe, effective, and affordable medicines for people afflicted with infectious diseases in the developing world.”

Financed by donations from private foundations, such as the Gates Foundation, the Institute for OneWorld Health is searching for cures for diseases such as malaria and Chagas.

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Focus Questions

notepad objectives
 glass  materials
 clock  teaching-time
 testtubes  procedure
   student-work
 questionmark

 questions

1. Millions of people die in undeveloped countries for lack of adequate medical care. Describe Chagas disease and how it affects its victims.

2. What circumstances deny poor people access to adequate care for Chagas and other diseases?

3. Why do you think there are not more organizations like OneWorld Health?

4. How would you fund organizations like OneWorld Health to bring new medicines to Third World countries? Brainstorm ideas. All serious ideas are welcome.

5. Research other Third World diseases that, like Chagas, kill millions every year for lack of adequate medications. Some examples include Vitamin A deficiency blindness, river blindness, elephantiasis, and malaria.

   teacher-key
 answermark

 answers

1. Millions of people die in undeveloped countries for lack of adequate medical care. Describe Chagas disease and how it affects its victims.

Infants and children often die from swelling of the brain associated with the disease. In other people the parasite will lie dormant for years only to emerge and lodge in the heart muscle, causing serious and irreversible damage to the heart. Chagas disease is the leading cause of heart disease in South America.

2. What circumstances deny poor people access to adequate care for Chagas and other diseases?

Lack of money to pay for treatment. Drug companies are unlikely to recoup the cost of research to develop new drugs and to make a profit for the company and its stock holders.

3. Why do you think there are not more organizations like OneWorld Health?

OneWorld Health is a charitable nonproft organization. The free enterprise system functions on for-profit companies.

4. How would you fund organizations like OneWorld Health to bring new medicines to Third World countries? Brainstorm ideas. All serious ideas are welcome.

Some examples include taxes on luxury items, donations from foundations interested in health issues, etc.

5. Research other Third World diseases that, like Chagas, kill millions every year for lack of adequate medications. Some examples include Vitamin A deficiency blindness, river blindness, elephantiasis, and malaria.

 computer

 references

Institute for OneWorld Health http://www.oneworldhealth.org/diseases/chagas.php. Accessed July 2010.

National Public Radio 2005. Nonprofit Drug Firm Targets Disease in Third World. http://www.npr.org/templates/story/story.php?storyId=4277956. Accessed July 2010.

IIB 4. Differentiating Cyanobacteria and Macroalgae

Background

Macrocystis
Macroalgae such as this Macrocystis are important habitat on temperate and northern reefs.
Photo courtesy: NOAA, OAR/National Undersea Research Program 

Cyanobacteria and macroalgae are not noticed as much as fish and invertebrates by people at the beach, unless they happen to be affected by the “swimmer’s itch” caused by a small clump of cyanobacteria caught inside their clothes, or have to walk along their favorite beach on the macroalgae strewn up by the surging waves. Current research in biomedicinals extracted from these marine flora serves to illuminate the importance of these underappreciated organisms and explains the value of biodiversity in the ocean. Explaining the organisms’ features also provides an entry point for teachers into classification and morphology.

Promising new extracts for cancer treatment are being isolated from the cyanobacterium Lyngbya majuscula (review Dr. William Gerwick’s PowerPoint presentation “Introduction to Drug Discovery from Marine Organisms”). Marine toxins and nutraceuticals are being extracted from cyanobacteria for use in the food and cosmetic industries (review Dr. Robert Bidigare’s PowerPoint presentation “Marine Natural Products and Human Health”).

The harvesting of these products is reminiscent of the harvesting of limu (the local term for freshwater and marine algae) by native Hawaiian populations for food and for medicinal and ceremonial purposes. The Hawaiians used the endemic brown alga, Sargassum echinocarpum, a number of ways: they applied it to cuts, ate it, and used it in ceremonial hula dances and in sessions of forgiveness where family misunderstandings were worked out (Abbott 1996). They augmented their diet of fish and poi (a paste made from taro root) with limu that contained appreciable amounts of vitamins and other minerals. In his 1970 publication "Seaweeds and Their Uses," Chapman estimated that about three ounces of some seaweeds provided more than the daily requirements of Vitamin A, riboflavin, and Vitamin B12, and about half the daily requirement of Vitamin C (Chapman 1970).

Some marine macroalgae grow on reef flats in the intertidal zone, where they are exposed to the harsh ultraviolet rays of the sun; other species grow in 100 m depths at the lowest reaches of the photic zone. They live in a fluid environment that places all organisms, their reproductive cells, and their exudates into contact with each other. It’s no wonder that a group of organisms exposed to such environmental conditions contains a vast array of chemicals to insure its members’ survival.

Classification & Taxon Characteristics

Whereas the limu used by the Hawaiians are eukaryotic organisms, Lyngbya is a prokaryotic genus of cyanobacteria, a taxon (classification group) now included within the domain or superkingdom bacteria.

The development of molecular tools allowed Carl Woese and his colleagues to probe the structure of nucleic acids, revealing significant differences among prokaryotic ribosomal RNA signature sequences. These investigations unveiled the two distinct prokaryotic lineages, one of which (Archaea) may have as much in common with the eukaryotes (organisms with nucleated cells) as with its other prokaryotic members (Campbell and Reece 2002). These studies have led to the designation of three super kingdoms—Bacteria, Archaebacteria, and Eukarya. In the former five-kingdom classification scheme, all prokaryotes were classified under the single kingdom Monera (see note 1, how to collect seaweeds).

The other macrophytic forms of marine flora are members of the superkingdom Eukarya. Within Eukarya, the macroalgae are still grouped under the Kingdom Protista. Ongoing research in ribonucleic acid and protein sequences of the cytoskeleton is clarifying the evolutionary relationships among different groups of Protista within the superkingdom Eukarya; however, the assignment of these groups to the kingdom or phylum level will be in flux for a while (Campbell and Reece 2002).

The marine algae are usually classified by color resulting from the presence of different pigments. Macroalgae that can be collected along seashores or by wading or snorkeling around the coastline are members of the Phaeophyta (brown algae), the Rhodophyta (red algae), and the Chlorophyta (green algae).

Phaeophyta Sargassum
Unidentified Phaophyta in the Gulf of Mexico.
Image Courtesy: National Oceanic and Atmospheric Administration
Pelagic Phaophyta in the genus Sargassum. The berry-like structures are gas bladders known as pneumatocysts, which provide buoyancy.
Image Courtesy: National Oceanic and Atmospheric Administration

The Phaeophyta (from the Greek phaios, meaning dusky, brown) are the largest and most complex of the algae. They are all multicellular and, in temperate waters, include 60 meter-long species of kelp that reach their length within a single season. In Hawaii, the brown algae are much smaller forms. The pigments present in the plastids of the browns include beta carotene, the xanthophylls—fucoxanthin and violaxanthin—and chlorophylls a and c. The cells of the Phaeophytes usually have one nucleus, often surrounded by vesicles called physodes. These physodes contain tannins, which seem to play a role in keeping the algae free of epiphytes, organisms that may live on their surface tissues. In their work with marine flora, Walters et al (1996) found that the larvae of two major biofouling organisms, the polychaete tubeworm Hydroides elegans and the bryozoan Bugula neritina, would not settle out in water conditionedby six species of brown algae (along with three greens, two reds, and the cyanobacterium Lyngbya majuscula). These browns with which no fouling was found were Sphacelaria tribuloides, Dictyota acutiloba and sandvicensis, Sargassum echinocarpum and polyphyllum, and Padina australis.

Also found in the browns are gel-forming substances called algins, which are used as thickeners for processed foods and other products. Minerals and iodine are also present in macroalgae, making them valuable additions to the human diet for soup stock and as additional ingredients in condiments. They also contain polysaccharides that are indigestible, causing diarrhea if eaten in excess.

Rhodophyta
An unidentified Rhodophyta algae in the Gulf of Mexico.
Image Courtesy: National Oceanic and Atmospheric Administration

Agar and carageenan are the gel-forming substance in the red algae or Rhodophyta (from Greek rhodos meaning red). The color of the reds is due to the accessory pigment in photosynthesis—phycoerythrin. Phycoerythrin is a member of the group of pigments called phycobilins, which are found in cyanobacteria as well. In shallow-water species of the Rhodophyta, the specimens may be more greenish because the chlorophyll is not fully masked by the phycoerythrin. The Rhodophytes may also contain a high concentration of phycoerythrin, so they appear almost black in water 260 meters deep. There the phycobilins and other accessory pigments can absorb the blue and green wavelengths of visible light that penetrate to that depth.

Most red algae are multicellular. In the tropics, red algae are the most abundant large algae (Campbell and Reece 2004). The plant body or thallus of many reds is filamentous and often finely branched or in lacy patterns. An example of this form is Porphyra, the seaweed that is dried in sheets as Japanese nori used to wrap sushi and rice balls. Among the reds that contained antifouling chemicals in the Walters et al study (1996) are Hypnea musciformis and Laurencia cartilaginea.

The third division of macroalgae is the Chlorophyta, the members of which are grass-green due to their chlorophyll-containing chloroplasts much like those in land plants. Cells of the Chlorophyta contain one too many chloroplasts or amyloplasts (starch-containing or starch-making plastids that are colorless). The pigments found in the greens are chlorophylls a and b, some carotenes, and xanthophylls. Included in the greens are microscopic unicellular members and multicellular forms, mostly smaller species. There are some freshwater members of the Chlorophyta that are pink or orange because their carotenoid pigments mask the chlorophylls they contain. These algae are cultivated in hypersaline lakes and sold as a source of beta-carotene.

The greens that have been found to have antifouling properties include Ulva fasciata and reticulata, and Halimeda discoidea (Walters et al 1996).

Chlorophyta Green grape algae Leafy green algae
Unidentified green algae in Saipan.
Image Courtesy: National Oceanic and Atmospheric Administration
Green grape algae (Caulerpa cavernosa) in the Gulf of Mexico.
Image Courtesy: National Oceanic and Atmospheric Administration
This leafy green algae (Anadyomene lacerata) is found in the Gulf of Mexico.
Image Courtesy: National Oceanic and Atmospheric Administration

PDF file PDF file for this project

Focus Question

How do cyanobacteria differ from macroalgae?

notepad

objectives

Students will

• Distinguish the differences in the cellular structure of macro-forms of cyanobacteria and macroalgae

• Begin to recognize different species of macroalgae

 glass

 materials

Field-collected samples of marine algae and cyanobacteria

Computer with access to Internet

One 8 1/2" x 11" drawing paper/student

Gerwick PowerPoint from this curriculum

Bidigare PowerPoint from this curriculum

Background reading from this section: “Differentiating Cyanobacteria and Macroalgae”

NOTE : L. majuscula is one of the few species of cyanobacteria that can easily be collected during kona weather (wind, waves and rain coming in from a southerly direction) in Hawaii. It is ripped from the reef substrates in deeper waters by the increased wave action and can be scooped up by hand from shallow waters all around the islands. The collection of other species of cyanobacteria is less predictable and occurs more serendipitously (Dr. Robert Bidigare, University of Hawaii, personal communication, 2004).

 clock

 teaching-time

Two class periods or homework

 testtubes

 procedure

1. Lecture on the characteristics of the two groups, emphasizing nucleus and pigment-containing structures.

2. Students will research the differences in cell structure between the two types of organisms—the cyanobacteria and the macroalgae—that are collectible by hand-gathering methods at the seashore.

3. Students will illustrate the cellular differences between one specific cyanobacterium and one macroalgae. They are to label the specific organelles and label the drawings with the scientific name of each of the two organisms and the group to which it belongs (Cyanobacteria, Rhodophyta, Phaeophyta, or Chlorophyta). Students may use the illustrations in Figures 6 and 7 as a guide.

4. Students will write a short essay (two paragraphs) emphasizing the differences between the two. References must be cited (Web sites or books).

5. Illustrations will be displayed around the room. To evaluate student work, check for nucleated algal and nonnucleated cyanobacterial cells; check for correct species and illustration and classification according to the resource materials.

   student-work
 questionmark

 questions

1. What are the main morphological differences between cyanobacteria and algae?

2. Why are cyanobacteria of particular interest to scientists?

3. For what kinds of things have alga been used?

   teacher-key
 answermark

 answers

1. What are the main morphological differences between cyanobacteria and algae?

Cyanobacteria are true bacteria (prokaryotes) and have no organized nucleus surrounded by a membrane and thin cell walls. Unlike other bacteria, they have chlorophyll and use the sun as an energy source. Algae have a true nucleus surrounded by a membrane, heavy cell walls, and chloroplasts that contain chlorophyll. They are classified according to the color pigments found in their tissues: green, brown, or red.


2. Why are cyanobacteria of particular interest to scientists?

Scientists believe cyanobacteria may be potential sources of compounds that have medicinal properties.

3. For what kinds of things have alga been used?

Some species have been valued as food. Alga have been used in ceremonies by indigenous people.

 computer

 references

NOTE: For Hawaii, two taxonomic treatises on these red, green, and brown algae are available to identify collected specimens—Marine Red Algae of the Hawaiian Islands (Abbott 1999) and Marine Green and Brown Algae of the Hawaiian Islands (Abbott & Huismann 2004). These books include taxonomic keys, photographs, drawings, and written descriptions accessible to individuals with a basic knowledge of scientific terms.

Abbott, Isabella A., and John M. Huismann. 2004. Marine Green & Brown Algae of the Hawaiian Islands. Bishop Museum Press, Honolulu.

Abbott, Isabella. 1999. Marine Red Algae from the Hawaiian Islands. Bishop Museum Press, Honolulu.

Abbott, Isabella. 1996 Limu, An Ethnobotanical Study of Some Hawaiian Seaweeds, Fourth Edition. National Tropical Botanical Garden, Lawai, Kauai, HI.

Campbell, Neil A., and Jane B. Reece. 2002. Biology, Sixth ed. Benjamin Cummings, San Francisco.

Chapman, V. J. 1970. Seaweeds and Their Uses. Second ed. Methuen & Co. Lt., London.

Walters, L. J., M. G. Hadfield, and C. M. Smith. 1996. Waterborne chemical compounds in tropical macroalgae: positive and negative cues for larval settlement. Marine Biology 126(3): 383–393.

IIB 5. Antibacterial Agents in Seaweeds

Background

Researchers studying marine cyanobacteria in order to find novel natural products are finding they contain an abundance of potent bioactive compounds with promising anticancer, antibiotic, and anti-inflammatory activity. Marine algae are among the largest producers of biomass in the marine environment. They also produce a wide variety of chemically active metabolites, potentially to protect themselves against other organisms. These active metabolites, also known as biogenic compounds, produced by several species of marine macro- and microalgae, have antibacterial, antialgal, antimacrofouling, and antifungal properties. Many of the metabolites are novel structures that represent unique biosynthetic pathways. Some of the most promising compounds function in target cells as tubulin polymerization inhibitors (compounds that inhibit the formation of cellular microtubules during the process of mitosis), actin polymerization inhibitors (compounds that inhibit development of actin filaments in cells), and neurotoxins.

Numerous promising compounds have been identified in the cyanobacterium Lyngbya majuscula (mermaid’s hair or fireweed). This filamentous cyanobacterium can be found in tropical and subtropical marine and estuarine environments around the world. Some strains cause swimmer’s itch (see William Gerwick’s PowerPoint presentation “Introduction to Drug Discovery from Marine Organisms").

The multistep process of drug discovery from marine algae and cyanobacteria involves years of research. The discovery process starts with the collection of samples followed by screening for biological activity. One of the strategies for natural product drug screening is a simple toxicity assay using cells such as cancer cell lines or microbial cultures, or indicator species such as brine shrimp. Extracts demonstrating significant activity in either of the assays are chosen for further purification and analysis.

In this experiment, students will explore the first steps in drug development by screening different seaweed samples for antimicrobial activity. Students will make a crude extract of seaweed or cyanobacteria and test for the presence of antibacterial properties against gram-positive and gram-negative bacteria. Students will compare the antibacterial effectiveness of their extract against known antibiotics and disinfectants.

PDF file PDF file for this project

Focus Question

How are the antibacterial properties of algal extracts determined?

notepad

objectives

Students will learn to extract compounds from algae and test them for antimicrobial activity.

 glass

 materials

Various species of fresh seaweed or marine cyanobacteria
Luria broth cultures of gram-positive and gram-negative bacterial species. Suggested species appropriate for high school level:
Gram-positive—Bacillus subtilis (25–30oC)
Bacillus megaterium (25–30oC)
Micrococcus luteus (25–30oC)
Staphylococcus epidermis (37oC)
Gram-negative—E. coli MM294 (or other K12 strain—37oC)
Serratia marcescens (25–30oC)
Pseudomonas aeruginosa (37oC)
Luria Broth or nutrient agar plates
Disinfectants in small beakers—bleach, Lysol, Pinesol, etc.
Antibiotic disks—ampicillin, penicillin G, tetracycline, streptomycin, erythromycin, etc.
(available from Difco at www.Difco.com Search for “BBL antibiotic susceptibility test disks”)
Extraction solvents—ethanol, methanol, hexane, acetone, etc.
Cold mortar and pestle
Scissors
1.5 ml tubes and microcentrifuge
Microfuge tube rack
20–100 μl Micropipettors and sterile tips
Forceps
Sterile Petri dishes
Beaker of 95% ethanol
Spreading rods
Bunsen burners and strikers
37oC and 30oC bacterial incubators
Beaker for waste/used tips
Permanent markers
Ice bucket with ice
Sterile 1/4-inch Schleicher and Schuell high-purity paper disks or Whatman filter paper (cut with a hole puncher)

 clock

 teaching-time

Three class periods:

Day 1—Extraction

Day 2—Antibacterial testing

Day 3—Recording results and class discussion

 testtubes

 procedure

Crude Extraction of Seaweed

1. Obtain 1 gram of fresh seaweed (* see notes below for collecting tips, including suggested species to collect) and remove all epiphytes and decayed areas. Rinse several times in distilled water and squeeze/pat dry.

2. Cut seaweed into small pieces with scissors or razor blade.

3. Using a cold mortar and pestle, thoroughly mash the chopped seaweed with 1 ml of solvent.

If needed, add more solvent, but try to limit the amount of solvent, because this will dilute your extract. Prepare different extracts using the solvents listed above.

4. Scrape the mashed seaweed/liquid into a labeled 1.5 ml microfuge tube and centrifuge for 5 minutes at maximum speed.

5. Carefully pipette out the supernatant to another labeled 1.5 ml tube.

6. (Optional) Filter sterilize the supernatant with a 0.2 µm cellulose acetate filter.

Preparation of Disks1.

Using sterile tips, pipette 20 µl of each extract onto two 1/4-inch filter paper disks in sterile Petri dishes and allow to dry (approximately 20–30 minutes). Note: if micropipettors are not available, use the following method. Place forceps in a beaker of 95% ethanol or isopropanol and tap off the excess alcohol when removing the forceps (or pass the forceps through a flame to burn off the alcohol). Pick up a sterile disk with the sterilized forceps and dip the disk into the extract. Be sure to touch the disk to the side of the extract tube and tap it to remove excess liquid, and then place the disk in a sterile Petri dish to dry.

2. Prepare negative control disks by pipetting 20 µl of the solvent in the extraction on two paper disks in sterile Petri dishes and allow to dry.

3. Prepare positive control disks of a store-bought disinfectant (Lysol, Pine-Sol, bleach, etc.) in the same manner.Antibacterial Testing

1. Obtain overnight Luria Broth (LB) suspension cultures of a gram-positive and gram-negative species.

2. Obtain 2 LB agar plates. With a permanent marker, label the bottom of the plate with your initials, bacterial species, seaweed extract, and date. Be sure to write along the edge of the plate in small letters so that your labels will not obscure your view.

3. Sterilize the spreading rod and spread 100 µl of bacterial suspension over the surface of each plate.

Dip spreader into the ethanol beaker and briefly pass it through a Bunsen burner flame to ignite the alcohol. Allow alcohol to burn away from the flame!

Lift lid of one plate only enough to allow spreading; do not place lid on the table.

Cool spreader by gently rubbing it on the surface of the agar away from the cell suspension.

Touch spreader to cell suspension, and gently drag it back and forth several times across the surface of the agar. Rotate plate on quarter turn, and repeat spreading motion. Be careful not to gouge the agar.

Replace plate lid. Return cell spreader to ethanol without flaming.

NOTE: if alcohol or spreading rods are not available, use sterile Q-tips to spread bacteria over the surface of the agar.

4. Using forceps, carefully pick up one of the extract disks and place it on the agar. See figure below. Press down on each disk to ensure close contact of the disk to the agar. Be sure to label the bottom of the plate.

5. Place the agar plates lid side down in the appropriate incubator

6. After 16 to 18 hours of incubation, measure the diameter of
millimeter. Measure from the edge of the disk to the edge of the
irregular, measure the zone at four different places (e.g., 12 o’clock,
o’clock) and calculate an average.

7. Create two data tables: one to record your observations and sketches showing the results of your antibacterial tests.

* Collecting tips:

Note 1: When collecting limu in Hawaii please be aware of the problems associated with invasive species. Place the limu in a bleach solution to kill the cells before discarding. This will help to prevent the species you’ve collected from spreading should any find their way back into the water.

Note 2: Collection suggestions for Hawaii: Limu kohu (Asparagopsis taxiformis) and limu alani (Dictyota sandvicensis) have produced positive results in this bioassay.

Collecting suggestion for species in Oregon: Try this assay with the brown seaweed rockweed (also known as bladder wrack, Fucus spp). Brown seaweeds are very common on the Oregon coast, and rockweed is present throughout the year rather than dying back at the end of summer as many seaweeds do. It contains relatively high amounts of iodine, reportedly from 0.03 to 1 percent, and is a good specimen to explore for antibacterial actions. The assay can also be performed with the green alga Enteromorpha intestinalis, the alga that looks like green Easter basket grass.

 

   student-work
 questionmark

 questions

1. What do the clear areas surrounding a disk indicate? What do differences in the width of the clear areas indicate?

2. What evidence do you have that the inhibition of the bacteria is due to the extracts on the disk and not the disks themselves?

3. Summarize the class data. Which seaweed extract was most effective against each of the bacterial species? What might account for the difference in effectiveness?

4. If a seaweed extract did not produce any inhibition zones, does that mean that the seaweed does not contain any antibacterial agents? Explain.

5. Compare the effectiveness of your seaweed extract to that of the commercially prepared antibiotics and disinfectants. How could you increase the effectiveness of your seaweed extract? Identify as many variables as you can that could affect the potency of your extract.

6. Which disinfectant (Lysol, bleach, Pine-Sol, etc) was most effective against the bacteria you tested?

7. Use a reference like the Merck Index to determine which chemicals are likely to be the active ingredients in the disinfectant you tested. Conduct an Internet search to see how these active ingredients affect bacteria. Include the URLs of the Web sites that provided you with information to answer this question.

8. Which antibiotic was most effective against the bacteria you tested? Conduct an Internet search to explain the mode of action of this antibiotic. Include the proper citations for your reference materials.

   teacher-key
 answermark

 answers

1. What do the clear areas surrounding a disk indicate?

Bacteria have been killed by the extract.

What do differences in the width of the clear areas indicate?

Greater antibacterial action by the extracts.

2. What evidence do you have that the inhibition of the bacteria is due to the extracts on the disk and not the disks themselves?

The control disks with disinfectants and sterile broth eliminate variables in the experiment.

3. Summarize the class data. Which seaweed extract was most effective against each of the bacterial species?

Answers will vary with individual results

What might account for the difference in effectiveness?

Stronger antibacterial action in different species of seaweeds.

4. If a seaweed extract did not produce any inhibition zones, does that mean that the seaweed does not contain any antibacterial agents? Explain.

No, it may kill species of bacteria not tested here.

5. Compare the effectiveness of your seaweed extract to that of the commercially prepared antibiotics and disinfectants. How could you increase the effectiveness of your seaweed extract?

Use a more concentrated sample from the seaweed.

Identify as many variables as you can that could affect the potency of your extract.

Any dilution of the extract, season the seaweed was harvested (i.e. at the end of summer the seaweeds would be declining)

6. Which disinfectant (Lysol, bleach, Pine-Sol, etc) was most effective against the bacteria you tested?

Answers will vary with individual results

7. Use a reference like the Merck Index to determine which chemicals are likely to be the active ingredients in the disinfectant you tested. Conduct an Internet search to see how these active ingredients affect bacteria. Include the URLs of the Web sites that provided you with information to answer this question.

8. Which antibiotic was most effective against the bacteria you tested?

Answers will vary with individual results

Conduct an Internet search to explain the mode of action of this antibiotic. Include the proper citations for your reference materials.

1. If antibacterial properties were discovered in one of the species of seaweed tested, test other species of the same genus to determine if the bioactive compounds are species specific.

2. Obtain the same species of seaweed from various locations and seasons to determine if habitat and season have any effect on the production of the bioactive compounds.

3. Traditional healers have often used seaweed as medicines. Research the types of seaweeds used and how the medicines were prepared by meeting with tribal elders, medicine men, kupuna, or Kahuna lapa’au.

4. Test the effect of traditional methods of preservations (e.g., salting of limu kohu and rolling the seaweed into balls) on the maintenance of antibacterial properties. Compare the antibacterial properties of preserved (salted or dried) seaweeds and fresh seaweeds.

 computer

 references

Bruckner A.W. 2002. Life Saving Products from Coral Reefs. Issues in Science and Technology online. http://bob.nap.edu/issues/18.3/p_bruckner.html. Accessed November 2005.

Gerwick, William, Department of Pharmacy, Oregon State University, July 27, 2004. “Introduction to Drug Discovery from Marine Organisms” (II.Resources.Gerwick) PowerPoint presentation at Marine Biotechnology Curriculum Workshop, Hawaii Institute of Marine Biology, Kaneohe, HI

IIC 6. Isolating Bacteria from Sponges

Background

Sponges
The variety of sponges seen here includes the yellow tube sponge (Aplysina fistularis), the purple vase sponge (Niphates digitalis), the red encrusting sponge (Spiratrella coccinea) and the gray rope sponge (Callyspongia).
Images Courtesy: National Oceanic and Atmospheric Administration

Sponges are sessile creatures that inhabit the ocean floor from rocky zones to the deep sea. They have no tissues or organs but rather are made up of fiberlike protein with hard, multipoint rods called spicules that give their body structure. They feed by pumping water through the cavities of their bodies with specialized cells equipped with little paddles or tails that beat to circulate the water. There are 10,000 different species of sponges worldwide, and they are now being explored as sources of compounds that may hold promise for new drugs to treat malaria and cancer. Sponges are unique animals, but it is the many types of bacteria found in their tissues that claim scientists’ attention. Many of the bacteria associated with sponges belong to the group Actinomycetes. This group includes the genus Streptomyces, which has been the source of a large percentage of the drugs we use. Current sources of these bacteria are terrestrial. The discovery that sponges also harbor these types of bacteria has stimulated a great effort to sample and identify sponge bacteria.

Collecting the sponges is the first step in the search for bacteria. Coral reefs are a rich source of sponge species, and the deep sea is a new frontier for exploration. Gathering the sponges may involve scuba diving or collecting deep-sea specimens with research submersibles. There are so many different species of sponges that finding those that harbor bacteria with bioactive properties can involve a lengthy search.

Once the sponges are collected, extracting and identifying their bacteria involves many steps and procedures. Crushing, grinding, and washing the sponge tissue releases the bacteria. The residue is diluted and then cultured on marine growth agar. Individual bacterial cells will reproduce on the agar medium, giving rise to colonies of identical cells. The isolated bacterial cells are stained and their biological activity can be tested. Bacterial samples of interest can be identified at the molecular level using the polymerase chain reaction followed by DNA sequencing (read IVB. Microbial Identification:Reading Genetic Name Tags with PCR and Sequencing (IVB24) in this curriculum).

PDF file PDF file for this project

Focus Question

How are sponges collected and processed to extract bacteria for biomedical research?

How are species of bacteria found in the tissues of sponges identified?

notepad

objectives

Students will
• Learn where sponges grow and the procedures and regulations governing their collection.

• Undertake the procedures and protocols used by scientists to extract and classify bacteria found in sponges.

• Gram stain bacteria and use the information for categorizing bacteria.

 glass

 materials

Student Worksheet (in this activity file)
IID 7. Create Your Own Product from the Sea (background section)(IID.7)
IVB. Microbial Identification: Reading Genetic Name Tags with PCR and Sequencing (IVB24) (Reading in this curriculum)

Bacterial Isolation

sponge samples

Ziploc bags

Sterile artificial seawater (ASW) Note: seawater salts can be purchased from pet stores that carry saltwater aquarium supplies and mixed according to directions. Make sure to sterilize the water (autoclave at 15 psi in a 2 L flask covered with foil for 30 minutes on a liquid sterilize cycle with 0 minutes of dry time).

Sterile 15 ml conical plastic tubes containing 9 ml of ASW

Sterile cutting boards and knives

Scalpels and blades

Sterile mortar and pestle

Micropipettes P1000 and P100 with sterile tips

MA2216 plates (prepared according to directions with Marine Bacterial Media from BD Biosciences Product Catalogue # 212185, see http://www.bd.com/ds/productCenter/212185.asp)

Gram Staining and Microscopy

Clean microscope slides

Crystal violet staining solution

Iodine

95% ethanol

Distilled water

Safranin

Microscope with 100x oil immersion lens or most powerful lens available to class

Freeze Cultures

DMSO

Sterile screw cap 1 or 2 ml tubes appropriate for freezing

-20°C or -80°C freezer

 clock

 teaching-time

1 to 3 days of teaching time, depending on growth of cultures.

Day 1—Collect samples, prepare dilutions, and plate

Day 2—Analyze colonies and Gram staining

 testtubes

 procedure

Isolation of Bacteria

1. Collect sponge samples from a field site. Sample the sponge underwater while wearing latex gloves and transfer sample to Ziploc bag.

2. Transfer to a second Ziplock bag and rinse briefly in sterile ASW to remove transient and loosely attached bacteria.

3. Place on cutting board and excise representative sections of approximately 1 cm3. Immediately freeze remaining material at -20°C.

4. Place cube of sponge in mortar; add 10 ml of sterile ASW and grind thoroughly with pestle for 2 to 3 minutes.

5. Decant sample into sterile 15 ml tube to give a 100 dilution.

6. Make a 10-fold dilution series to give dilutions of 10-1, 10-2, 10-3, 10-4, and 10-5.7. Plate out 100 µl of the 10-2, 10-3, 10-4, and 10-5 dilutions on MA2216 plates. Incubate at 30°C or ambient water temperature. Count colonies and pick isolates after 72 hours. Streak isolates on fresh MA 2216 plates and incubate overnight or until colonies form. Seal the plates and store at 4°C. If necessary, colonies may be transferred by streaking to a new plate to preserve the culture. Ideally, frozen stocks of individual strains would be prepared.

The process above uses mechanical methods (rinsing and grinding of the tissues) for dislodging bacteria from sponge tissues. The material is then diluted in dilutions of the order of 1 to 10, 1 to 100, 1 to 1000, etc., before gram staining. Without dilution, the bacteria cells may be so dense that it is difficult to work with them once they are freed from the sponge tissues.

Gram Stain and Light Microscopy

Although not definitive for species identification, analyzing bacteria samples at the macroscopic (colony) and microscopic (type) level provides useful data for students to strengthen their critical thinking skills. Comparison of colony morphologies such as color, shape, effect on agar, and comparison of bacterial characteristics elucidated from staining also allows students to study and appreciate marine microbial biodiversity if molecular equipment is not readily available.

1. Using a few cells from the colonies you mark, prepare smears on clean microscope slides.

2. Air dry and heat fix the smears.

3. Immerse each slide in a solution of crystal violet stain for 1 min, rinse with distilled water, and drain.

4. Immerse each slide in iodine for 1 minute, rinse, and blot dry.

5. Immerse in 95% ethanol for 15 s to destain then rinse with distilled water and drain.WP

6. Counterstain with safranin for 20 to 30 s, rinse, and blot dry. Examine under the microscope. Note the morphology of the bacteria: Are they rods, cocci, or other shapes? Are the bacteria stained with the Gram stain? Do the cells form filaments or clusters? How large are the cells?

Freeze Cultures (storing cultures for later study)

1. Choose the culture you find most interesting. Prepare an overnight culture (2 ml) of this strain in sterile liquid culture medium.

2. Label a sterile screw-cap tube with your name, date, and collection number for the culture you are freezing.

3. Add 0.2 ml of dimethyl sulfoxide (DMSO) to the tube and add 1 ml of an overnight liquid bacterial culture (save the remaining culture to prepare genomic DNA).

4. Shake gently to mix and freeze at -80°C. The cultures will remain viable indefinitely at -80°C.

5. To start a fresh culture from a frozen stock, scrape a small chunk of the frozen culture and transfer to a sterile tube containing 3 ml of broth. Do not allow the frozen culture to thaw. Return the frozen culture to the freezer immediately. If you do not have access to a -80°C freezer, prepare frozen cultures and store at -20°C.

   student-work
 questionmark

 questions

1. What are sponges?

2. Why are scientists interested in the bacteria associated with sponges?

3. Why might it be advantageous to a scientist to find that a natural product derived from sponges she is studying is actually being produced by a bacterium?

4. How do scientists identify bacteria at the molecular level?

5. Describe the results of your bacterial plating experiment. Where did you collect your samples? How many colonies did you isolate at each dilution?

6. Describe the morphology of the colony you chose for Gram staining. What were the results of the Gram staining?

   teacher-key
 answermark

 answers

1. What are sponges?

Sponges are sessile animals that inhabit the ocean floor from rocky zones to the deep sea. They have no tissues or organs but rather are made up of fiberlike protein with hard, multipoint rods called spicules that give their body structure. They feed by pumping water through the cavities of their bodies with specialized cells equipped with little paddles or tails that beat to circulate the water.

2. Why are scientists interested in the bacteria associated with sponges?

The bacteria may be producing interesting bioactive compounds that could be used to treat malaria or cancer.

3. Why might it be advantageous to a scientist to find that a natural product derived from sponges she is studying is actually being produced by a bacterium?

Sponges are difficult to collect and rear in aquaculture. To bring a drug to market, large, reliable quantities of the drug are needed for testing and production. Overharvesting of a sponge species for drug production could result in depletion or even extinction of the sponge species. Bacteria are easier to culture and grow in large quantities to produce enough of an interesting compound for testing.

4. How do scientists identify bacteria at the molecular level?

Scientists can use PCR and DNA sequencing to read an organism’s “genetic name tag.”

5. Describe the results of your bacterial plating experiment. Where did you collect your samples? How many colonies did you isolate at each dilution?

Answers will vary.

6. Describe the morphology of the colony you chose for Gram staining. What were the results of the Gram staining?

Answers will vary.

 computer

 references

Russell Hill Profile. http://www.umbi.umd.edu/comb/faculty-directory/hill/index.php.  Accessed July 2009.

IID 7. Create Your Own Product from the Sea

Background

How researchers select an ecosystem or class of organisms to test

Researchers in search of natural products don’t conduct that search randomly. By directing their search toward ecosystems or types of organisms most likely to contain useful compounds, they make their investigations more efficient and effective. Ecosystems with extreme environments are attractive to explore, because the organisms in those environments often contain unique compounds that enable them to survive. For example, organisms that live at very high temperatures contain enzymes that are able to function at high temperatures without denaturing. The Taq polymerase used for the polymerase chain reaction (PCR) was isolated from a high-temperature bacterium, Thermus aquaticus. The heat-resistant nature of the enzyme is critical to its ability to continue functioning through the many thermal cycles involved in PCR. These organisms that live under extreme conditions are called extremophiles.

Conus magus

Peptides from Conus magus venom may be used to treat pain, epilepsy, cardiovascular disease, and various neurological disorders
Image Courtesy: National Science Foundation

Ecosystems with high biodiversity are also prime sources for natural products, not only because each species may contain different compounds, but also because the competition among the species may have led them to evolve effective biochemical weapons and defenses. As species compete with each other, or as predators and prey evolve together, they develop new adaptations. For example, cone shells have toxins that allow them to paralyze their prey, thus enabling the rather slow-moving snail to catch fast-moving animals. The results of such evolutionary arms races may be unusual, complex compounds with unique properties. Because of their roles as interspecific weapons, these compounds are also likely to be biologically active. Returning to the example of the cone shells, because their toxins target nerve cells, these toxins are being researched as analgesics (pain-reducing drugs). As of December 30, 2004, ziconotide, a synthetic version of a toxin originally extracted from the cone snail Conus magus, was approved as a drug by the FDA.

In addition to concentrating their searches in ecosystems with high biodiversity or unique abiotic conditions, researchers may further focus on groups of organisms that are likely to have evolved compounds of interest (as researchers have focused on cone snails). For example, sponges may be a prime source of toxins or antibiotics because sponges are sessile (attached to the bottom and therefore not mobile), and yet they are not eaten or grown over. This suggests that the sponges must have some chemical means of repelling predators and competitors. Bacteria are also a likely source of bioactive compounds, partially because they have been around for much longer than any other group and therefore have had time to evolve many different chemicals and metabolic pathways, but also because they have been the source of so many important compounds.

When researchers wish to target specific species, the knowledge of indigenous people may be invaluable in further narrowing the search. Indigenous people have a rich understanding of the organisms in their area, and their medicinal practices can suggest excellent prospective sources of medicines.

How researchers isolate and test compounds

Once a species or group of species has been targeted for research, the search for compounds of interest begins in earnest. In some cases, the first step is determining how to culture the organisms. For example, approximately only 1 percent of the bacteria in the ocean have been cultured, so to grow and research the other 99 percent, new techniques for bacterial culturing had to be developed. In response to this need, the research firm Diversa has developed a high-throughput cultivation method based on the encapsulation of single cells in microcapsules. Researchers can also attempt to isolate and test individual genes rather than whole organisms. A sample of seawater might be filtered and the DNA extracted. Techniques can be used to amplify DNA sequences likely to be of interest and isolate segments that produce enzymes. The DNA segment can then be inserted into a host cell and the product of the enzyme thus produced can be analyzed.

Once a compound is identified it must be isolated from cells and tested for biological activity; such testing is known as screening. Isolating and screening a compound is usually done as part of a cycle that involves separating it into fractions. To accomplish this, the cells are placed in a solution, and the compounds are dissolved or suspended in that solution. The solution is then separated into different fractions, and those fractions are screened for their activity. The fractions that show the highest activity are then separated into even smaller fractions, and those smaller fractions are once again screened for activity. The process continues until a given fraction contains only one compound, and the compounds are individually screened for their activity to determine which ones show the highest activity.

Techniques for determining activity of the compounds include toxicity assays, ecologically oriented assays, and high throughput screening using either cell-based or cell-free assays.

In toxicity assays, samples of cells (for example, cancer cells or microbial cells) or indicator species such as brine shrimp are exposed to the compound. The toxicity of the compound is determined by whether the cells or indicator organisms survive.

Ecologically oriented assays test a compound’s ecological activity—how does it affect the behavior of target species? For example, if researchers were trying to find a product to discourage fish from feeding on a particular structure, they might impregnate strips of agar with potential compounds. The strips could then be placed in a fish tank, and the activity of a particular compound would be determined by seeing whether or not the fish ate it.

In cell-based high-throughput screening, cells are added to wells on a plate, different compounds are added to each well, and the effect of the compounds on the cells is measured. Sometimes cell-based screens use cells that are genetically engineered to possess the desired molecular targets or to contain a system for visualizing the effect of the compound. For example, researchers looking for a new anticancer drug targeted a compound called CEBP alpha transcription factor. Because CEBP alpha transcription factor may have an anti-leukemia effect, a drug that activates this factor would also have an anti-leukemia effect. The researchers engineered cells to produce luciferase when CEBP alpha transcription factor was activated. If a compound activated the factor, the cell would also produce luciferase, which in turn would cause a light-producing reaction. Whether a compound activated CEBP alpha transcription factor could then be measured by how much light it caused the cells to produce (Shoemaker et al.).

In cell-free high-throughput screening, instead of whole cells, proteins or other targets are added to the wells on the plate. The activity of the screened compounds can be measured in a variety of ways, including ELISA (enzyme-linked immunosorbant assay).

Producing natural products

Once a compound has been isolated, in order to become commercially viable and effective as a clinical treatment, it must be produced in large quantities. Various options exist, each with its pros and cons. These options include the following:

Culturing and harvesting the organism. Large numbers of the organism from which the compound was originally isolated are cultured (raised in a laboratory or other controlled environment), and the compound is extracted.

>Pros: This method takes advantage of the organism’s ability to produce the compound, which is particularly important in the case of very complex compounds. In the case of organisms found in other countries, it may also provide benefits to the local economy and the local citizens, who could be employed in the process.

>Cons: This processes usually requires a lot of space and extensive growing facilities (for example, many acres if the organism is a plant). Furthermore, it may not be known how to cultivate the organism. If wild populations are small, the processes of developing cultivation techniques not only may be very time consuming, but could potentially wipe out the very species researchers are trying to cultivate.

Inserting the gene into E. coli. The gene(s) that produce the compound or that produce the enzymes that synthesize the compound are inserted into E. coli bacteria. The engineered bacteria are then grown in vats, and the compound is isolated from the bacteria.

>Pros: E. coli can produce large quantities of the compound in a relatively small space and short time frame. Techniques for culturing E. coli are already well developed.

>Cons: Although not as controversial as genetically engineered food, this process still involves the somewhat controversial process of genetically engineering organisms. This process also requires more sophisticated technology than culturing and harvesting the original organism and may provide fewer employment opportunities for local people.

Synthesizing the compound. An industrial method for synthesizing the compound is developed.

>Pros: This process avoids all issues of culturing organisms and is likely to produce more uniform batches of the compound. This process is also the most familiar to and common among drug companies and thus has a well-developed infrastructure and wide knowledge base.

>Cons: Some compounds may be too complex to synthesize easily, or the synthetic pathway may be too expensive to be commercially viable. This is also the process least likely to benefit local people.

International agreements

The primary international agreement covering the search for natural products is the Rio Convention on Biological Diversity (CBD), adopted in 1992 and ratified by over 175 nations (the United States, however, has not signed the CBD). The Convention on Biological Diversity’s primary objectives are the conservation of biological diversity, the sustainable use of its components, and the fair and equitable sharing of benefits arising from the utilization of genetic resources. The CBD also affirmed the sovereign right of states to exploit their own resources.

The other main relevant international agreement is the World Trade Organization’s Agreement on Trade Related Aspects of Intellectual Property (TRIPS). Article 27(1) states that “patents shall be available for any inventions, whether products or processes, in all fields of technology, provided that they are new, involve an inventive step and are capable of industrial application.” Article 27(3)(b) states that member nations may exclude plants and animals (other than microorganisms) from patentability. There are many concerns over the granting of patents on living organisms, one of which is that such patents may interfere with the ability of indigenous people to practice traditional medicines if the medicines they use have been patented.

Arguments have also been advanced that the TRIPS and the CBD conflict with one another. A number of countries consider that there is an inherent conflict between the two agreements because one allows for private rights to be established over inventions based on genetic resources through patents and the other provides that countries have sovereign rights over their genetic resources. Proponents of this view argue that Article 27(3)(b) be amended to make the grant of patents contingent on the provision of a declaration of the origin of genetic resources, proof of prior informed consent where the genetic resources are the subject of traditional knowledge and evidence of fair and equitable benefit sharing, thereby ensuring compliance with CBD provisions. Other countries consider that no conflict exists as the agreements have different objectives and deal with different subject matter, and that patents can be granted for genetic material while complying with the CBD—its just a matter of how the agreements are implemented (Connelly-Stone 2003).

In this activity, students will use their creativity and their understanding of how natural products are developed to create a hypothetical natural product of their own. Students may be assigned a particular species, and then their job would be to determine what kind of natural product might be found in that species. Alternatively, students may be assigned a natural product, and then their job would be to determine what kind of species might contain that product.

PDF file PDF file for this project

Focus Questions

What is involved in finding a biological product and bringing that product to market?

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objectives

Students will
• Demonstrate the importance of biological diversity by illustrating the potential uses of compounds from different types of organisms

• Demonstrate their understanding of the relationship between an organism’s environment (including both abiotic features and competitive interactions) and the types of adaptive compounds that organism is likely to have

•Explain which biotechnology tools would be used to find, isolate, and produce a compound of interest

• Investigate the ethical issues involved in seeking biological products in other countries

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 materials

Student handouts

Internet access helpful

Poster board and other materials for creating presentations

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 teaching-time

One or two class periods

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 procedure

1.Assign each student either a biological product or an organism of interest (see Appendix A for tables of products and organisms).

2. Provide students with handouts describing the assignment.

3. Students conduct research and develop product presentations.

4. Students present products to the class.

5. Teacher conducts a class discussion examining
• the connections between an organism’s adaptations (to the environment and to competitors) and the kinds of compounds that might be found in them
• the kinds of information, infrastructure, and ethical and political considerations that may be necessary to decide how to produce a particular compound

6. Explore the ethical considerations involved in seeking natural products in other countries.

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 questions

1. After the 1992 Earth Summit in Rio de Janeiro, over 175 countries ratified the Convention on Biological Diversity. One of the main objectives of the Convention on Biological Diversity is the conservation of biological diversity. Using examples that you learned about during the natural product presentations, explain what biodiversity is and why it should be conserved. What are some unusual organisms on Earth, what special adaptations do those organisms have, and why might their unique chemistries be important to humans?

2. Another primary objective of the Convention on Biological Diversity is the sustainable use of the world’s biological diversity. When a natural product is isolated, companies that wish to develop and market that product must consider how to do so in a sustainable manner. How can genetically engineered bacteria be used to minimize the impact of mass production on wild populations of the source of a natural product? What are some benefits and drawbacks to this process?

3. The third main objective of the Convention on Biological Diversity is the fair and equitable sharing of the benefits from the use of genetic resources (including the unique gene products of organisms). Some of the naturally derived products developed by U.S. companies do come from the genetic resources found in the United States. For example, taxol, an important anticancer drug, was isolated from the Pacific yew tree (Taxus brevifolia). However, the search for new natural products is primarily taking place in countries other than the U.S.
Why is the search for new natural products focused on areas outside the U.S.?
What would you consider “fair and equitable sharing” of the benefits from any new natural products found in other countries by U.S. companies?

4. Once a promising organism has been identified, what steps are taken to identify whether it contains a compound of interest? What are the steps to isolate and study the compounds of interest?

5. Why are there laws governing the patenting of new products?

Sample Oral Presentation
Introducing ultraglue, the latest new product from the sea! Ultraglue is an all-natural, nontoxic, and extremely strong glue. Used by surgeons to glue together incisions, ultraglue is super safe, super strong, and waterproof. Ultraglue is made of byssus threads from the blue mussel. Blue mussels live in the intertidal zone, gluing themselves to rocky surfaces using their byssus threads. Since mussels must be able to withstand pounding surf, byssus is extremely strong. Being glued firmly to their rocks also makes the mussels harder for predators to eat. Since only the mussels with the strongest glue survive the harsh waves and are not eaten by predators, natural selection has led to the mussels developing an extraordinarily strong glue. And of course since the mussels live in water, their glue must be waterproof.
Blue mussels have a very wide distribution, being found all over the world in temperate and polar waters. In fact, blue mussels are a popular seafood in many areas. Because of this, blue mussels are cultivated in many areas, including Maine, Massachusetts, and Rhode Island. Since blue mussel cultivation is already a commercially viable process, we can simply tap into the current farming system. Because no one wants to eat byssus, we have developed a special process to remove the byssus from the mussels before they are shipped to stores. This will minimize the ecological impact of producing ultraglue, and will in fact make the existing cultivation of blue mussels more profitable and a more efficient use of our resources. We will be turning a wasted resource into a fantastic new product!

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 answers

1. After the 1992 Earth Summit in Rio de Janeiro, over 175 countries ratified the Convention on Biological Diversity. One of the main objectives of the Convention on Biological Diversity is the conservation of biological diversity. Using examples that you learned about during the natural product presentations, explain what biodiversity is and why it should be conserved. What are some unusual organisms on Earth, what special adaptations do those organisms have, and why might their unique chemistries be important to humans?

Biodiversity is the abundance and variety of plant and animal species that occur within ecosystems. Conservation efforts work to preserve habitats that support the diversity of life. The richness of life on earth took millennia to evolve and the loss of one species is a loss to all. We may be losing organisms that contain compounds that hold the cure for diseases or other uses that could enhance human life.

There are millions of organisms on earth, each unique in their adaptation to their particular environment. For example, organisms that thrive in the extremely high temperatures of deep ocean vents have attracted the attention of scientists. Bacteria from these environments are being investigated for a variety of applications from new medicines to heat-stable compounds. Other organisms that have evolved defense mechanisms are also of interest as they may have unique compounds.

2. Another primary objective of the Convention on Biological Diversity is the sustainable use of the world’s biological diversity. When a natural product is isolated, companies that wish to develop and market that product must consider how to do so in a sustainable manner. How can genetically engineered bacteria be used to minimize the impact of mass production on wild populations of the source of a natural product? What are some benefits and drawbacks to this process?

If a promising compound is identified but the organism from which it is extracted is rare or endangered, synthesis of the compound without harvesting the organism is desirable. It has been demonstrated that genes that code for the production of the compound can often be extracted and transferred to bacteria. The bacteria now have the capability to produce that compound and eliminate the need to harvest the original organism. Some people have concerns about genetically modifying organisms, and sometimes the compound cannot be generated if environmental conditions are not exactly right, and so this method does not work. It can also be time-consuming and expensive to identify the gene that produces the compound and determine how to insert it into the bacterial “host.”

3. The third main objective of the Convention on Biological Diversity is the fair and equitable sharing of the benefits from the use of genetic resources (including the unique gene products of organisms). Some of the naturally derived products developed by U.S. companies do come from the genetic resources found in the United States. For example, taxol, an important anticancer drug, was isolated from the Pacific yew tree (Taxus brevifolia). However, the search for new natural products is primarily taking place in countries other than the U.S.

Why is the search for new natural products focused on areas outside the U.S.?

Areas with high biodiversity, especially those that have not been thoroughly explored, are of particular interest to scientists searching for new compounds. These include tropical rain forests, coral reefs, and deep-sea habitats. These areas are all or mostly outside the boundaries of the U.S.

What would you consider “fair and equitable sharing” of the benefits from any new natural products found in other countries by U.S. companies?

If a natural product with useful compounds is identified in another country, then that country should share in the profits that the product will earn. A fair and equitable share would have to be worked out and agreed upon by the country marketing and earning the profit with the country of origin. Student answers will vary.

4. Once a promising organism has been identified, what steps are taken to identify whether it contains a compound of interest? What are the steps to isolate and study the compounds of interest?

First, scientists must learn how to either harvest or culture the organism, or both. A large number of the organism may be needed to extract useable quantities of the compound. Populations may not be large enough to support harvest, so the organism may be cultured instead. Extraction techniques must be perfected so that sufficient amounts of the compound in its pure form are available. Then tests must be run to determine if the compound acts the way the scientists have hypothesized it would. These tests might include toxicity assays, ecological assays, or cell-based high-throughput screening (in which cells are added to little wells and compounds are added to the cells in order to measure the compound’s effects). The next step might be to learn to synthesize the compound without the organism itself, often using genetically modified bacteria.

5. Why are there laws governing the patenting of new products?

These laws protect the intellectual property of those who have identified and conducted research to develop new products.

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 references

Connolly-Stone, Kim. 2003. Intellectual Property, Bioprospecting and Traditional Knowledge: Who Benefits? Paper to the Bioprospecting in New Zealand Seminar. http://www.med.govt.nz/ers/nat-res/bioprospecting/review/seminar-20030221/connolly-stone/paper/index.html. Accessed January 2005.

Dalton, Rex. 2004. Bioprospects less than golden. Nature 429: 598–600.

Maloney, Sarah. Date of publication unknown. Extremophiles: bioprospecting for antimicrobials. Mediscover: Infectious Diseases.

http://www.mediscover.net/Extremophiles.cfm. Accessed January 2005.

Rippin, Mike. May 2003. High throughput screening. Tessella Scientific Software Solutions. Issue V1.R1.M0. www.tessella.com/Literature/Supplements/PDF/hts.pdf. Accessed January 2005.

Shoemaker et al. Date of publication unknown. Application of High-Throughput, Molecular-Targeted Screening to Anticancer Drug Discovery.
http://www.bentham.org/sample-issues/ctmc2-3/shoemaker/shoemaker-ms.ht. Accessed January 2005.