Interdisciplinary Minor in Global Sustainability, University of California, Irvine
Student papers, Spring 1998
Instructor: Peter A. Bowler
 
"The Cell from Hell"
by Boons Baythavong
 
Cyst Stage with  
Non-Tubular  
Spines
 
Abstract
 
"The cell from hell", as many scientists refer to this destructive microscopic dinoflagellate, has created a flurry of problems along the rivers and estuaries of North Carolina and stretching up towards the Chesapeake Bay. Not until 1991 was this mysterious murderer of millions of fish brought to the public’s attention and studied by Dr. JoAnn M. Burkholder. Pfiesteria piscicida possesses 24 different life stages and has the ability to transform to a variety of shapes. The predator kills fish and poses human health problems if its toxins are inhaled or contacted. It thrives on nutrients discharged into nearby rivers and streams, such as nitrates and phosphates. However, the government has future plans to put a halt to the explosion of Pfiesteria by reducing the amount of nutrient discharge into local rivers and streams. Also, new technology aids scientist to become more familiar with the newly discovered predator before it becomes out of control.
Introduction

In August 1997, approximately 30,000 fish were found floating with open sores covering there bodies in the Pocomoke River on the Chesapeake Bay (Pelley, 1998). Pfiesteria piscicida was the unknown culprit responsible for the deaths of the fish, until Dr. JoAnn Burkholder made the correlation between the dinoflagellate and the fish kills. Pfiesteria has a very complex life cycle, consisting of 24 different life stages and having the potential ability to transform into different shapes. Not only is it posing problems to fish, but it is also causing a variety of human health problems to those who come near its toxins. Nutrient discharge from nearby farms is nurturing the Pfiesteria and it continues to grow at an enormous rate. Thus, the government on both federal and state levels have established future plans to reduce the level of nutrient discharge responsible for the problem. Furthermore, as a government ally, new technology assists scientists to understand this unfamiliar creature.
 

Background
 

In the early 1990’s, an unknown source was responsible for killing hundreds of millions of fish in the coastal waters of North Carolina and extending as far north as the Chesapeake Bay area. Not until 1991 was this mysterious killer discovered and correlated to the mass deaths of fish on the east coast. This one-celled dinoflagellate, Pfiesteria piscicida (scientists call it "the cell from hell"), was responsible for wiping out fish and closing down fishing areas and shellfish beds. Dr. JoAnn M. Burkholder from the University of North Carolina State brilliantly correlated the discovery of the unknown dinoflagellate to the dead fish floating along the North Carolina’s rivers, streams, and coast (Broad, 1997).

Dr. Burkholder, a specialist in botanical limnology, arrived to North Carolina State in 1986. There, she was confronted with a problem that Dr. Edward Noga encountered while conducting studies of the causes of fish diseases. The unknown problem involved the mysterious deaths of all of his sample fish that were used for his studies. Different test methods were imposed to attempt to understand the cause of the problem, but all methods failed and still the problem could not be resolved. After long hours of frustration, Dr. Noga decided to give up on trying to resolve the problem. He cleaned out all his fish tanks and started the experiment over. However, the same problem reoccurred. At that time, the only possible prospects responsible for the killings were microscopic dinoflagellates discovered in the tanks (Barker, 1997).

Dr. Burkholder was approached with the problem. Dinoflagellates had been apart of Dr. Burkholder’s algal studies and she had attended many seminars featuring some of the leading researchers on toxic dinoflagellates. Thus, she was stumped with this unfamiliar dinoflagellate and could not identify it. Simultaneously, she became very fascinated with it and with Dr. Noga’s permission, she continued to study it. Dr. Burkholder began seeking expertise from different specialists of toxic dinoflagellates, but no one was familiar with this new specimen. As she progressed with the study of the dinoflagellate, from microscopic photographs she began noticing different shapes from the transformed dinoflagellate. At different stages of transformation, the dinoflagellate possessed the ability to emit a toxin to attack its prey. As a result, this dinoflagellate had been the culprit behind the unknown deaths of the fish in the lab. The problem still was not resolved because even after extensive cleaning of the fish tanks, the fish were still dying. Finally, Dr. Burkholder researched where the tilapia fish, used for the experiments, originated and the fish were traced back to the Pamilco River in North Carolina. At that time there has been a recent fish kill reported on that exact same river.

Life Stages

Pfiesteria piscicida was named after the scientist, Dr. Lois Pfiester, who was responsible for contributing information about the complex life cycles of dinoflagellates. Pfiesteria piscicida possesses the ability to transform its shape into multiple stages. In fact, its complex life cycle consists of 24 different encysted, amoeboid, and flagellated stages or forms. The cyst stages are the periods of dormancy and commonly occur along the muddy bottoms of North Carolina’s estuaries. However, the amoeboid stages are found in the water column and also along the muddy bottoms, but during this period they are feeding on other organisms by engulfing their prey. The dinoflagellate’s list of prey consists of bacteria, algae, small animals, and bits of fish tissue. Furthermore, the flagellated stages include vegetative or asexual cells, sexual cells or gametes, and motile sexual products or planozygotes, which feed more often by attaching to their prey’s cells and suctioning the prey’s contents (Burkholder, 1997).

Pfiesteria piscicida is normally a nontoxic predator, but becomes toxic only when it detects a substantial amount of an ephemeral substance that fish excrete within the surrounding environment. Pfiesteria usually does not become toxic with the presence of only a few fish. However, it needs a school of fish in order for the ephemeral substance to ignite encysted cells to become actively toxic. Similarly, the amoeboid and flagellated cells also become toxic with the presence of ephemeral substance. As a result, the small cells attack the surrounding fish by releasing a potent toxin to immobilize the fish and slowly destroys the skin. Often, open bleeding sores and hemorrhaging occurs. Finally, the Pfiesteria feeds on the epidermal tissue, blood, and other substances released from the sores. Once the fish are dead, flagellated stages transform into amoeboid stages and feed on the left over fish remains. On the other hand, if conditions become unfavorable, then the cells produce a protective outer covering, sink out of the water column into the muddy bottoms, and transform into dormant cyst stages. This whole process can occur in a matter of hours (Burkholder, 1997).

Burkholder recounts the situation from her lab experiments to show how the Pfiesteria transforms into different stages to attack its prey. The microscopic alga appears in a dormant state in the sediment at the bottom of the aquarium, encrusted in a scaly shell, when it is left alone. However, when fish are introduced into the water, a series of transformations occur, which turns the harmless cyst into a hungry predator. Exploding out of its shell, a swimming cell propels itself through the water by utilizing its two whiplike tails, or flagella. As it approaches the fish, it releases a powerful neurotoxin to initially stun its prey and eventually suffocating it by muscular paralysis. In addition, this toxin has a corrosive effect, which kills the fish skin and causes it to separate from the fish. Furthermore, the dinoflagellate changes into another form, by growing in size to accommodate the growth of a tonguelike absorption tube, or peduncle, which enables the dino to attach itself to the fish to feed at will. When the fish were removed form the aquarium, the dinoflagellate would retreat to the bottom the tank and transform back into the armored cyst stage (Barker, 1997). Again, the dino remains dormant until there is another opportunity to attack more fish.

Pfiesteria piscicida possesses incredible abilities to be able to survive in a vast array of conditions. It can thrive well in fresh water as well as salt water. Furthermore, it doesn’t even need either types of water to be able to survive. The dinoflagellate can survive without the all of the water (Barker, 1997). It has the ability to hibernate until the conditions are favorable. Once it senses that the environmental conditions are to its advantage, it transforms into a different stage to begin searching for food.

Pfiesteria piscicida can reproduce at the same time as it is feeding on its prey. Closely observed by Burkholder, she noticed that as the dinoflagellate was attacking its prey, sexual activity was occurring simultaneously. The cells simply divide into two, producing two gametes, and swim about fertilizing each other. As a result, they become larger forms of the original cell, which unless fish are present, fall to the bottom of the tank and become dormant (Barker, 1997). Therefore, as long as the dinoflagellates are feeding, sexual activity will occur. The rate of reproduction is dependent upon the source of food supply.

Source of Problem

Pfiesteria has the ability to survive in a wide range of conditions. When it can not detect the emphemeral substance from fish, it survives by feeding on bacteria, algae, and small animals. In turn, the algae thrives heavily on enriched nutrients such as nitrates and phosphates. Burkholder agrees: "We have established through field and lab research that [Pfiesteria] can be highly stimulated by both inorganic and organic nitrogen and phosphorus enrichments," she says, especially the nutrients in human sewage and swine effluent spills (Hileman, 1997). The nutrients are the source of food for the algae and therefore mass amounts of algal blooms occur. When the food supply of algae is very abundant, that results in a large amount of Pfiesteria growth (Burkholder, 1997). This functions as a food chain, but the problem is that the chain does not continue beyond the Pfiesteria. Currently, the unknown dino is not yet fully understood by scientists and predators of Pfiesteria have not yet been discovered.

The major contributors to the abundance of nutrients are hog and chicken farms, urban runoff sewage, and factory wastewater, which flow directly into the bays and estuaries. Similar to Burkholder’s correlations, the U.S. Environmental Protection Agency has reported a strong correlation between high nutrient levels, Pfiesteria, and algal blooms (Wiant, 1998). These contributors of nutrients are difficult to pinpoint where the sewage is actually coming from. Agricultural runoff is very difficult to control because it is unintentional and it occurs from rainwater draining towards the bays and estuaries. Similarly, urban runoff can not be controlled because it just gets washed away and it is too expensive to divert all of the urban or agricultural runoff through a sewage treatment facility. Already there is currently too much municipal sewage treated at sewage treatment facilities. However, there has to be something done about the amount of nutrients coming from these farms because Pfiesteria can potentially wipe out our future fishery industry and at the same time have catastrophic effects on the human health.

North Carolina is home to more than 3,500 large hog farms and more than 16 million pigs. It is considered to be the nation’s second largest pork-producing state behind Iowa. Mass amounts of untreated wastes from these hog farms that are suppose to be stored in lagoons usually leak out into the nearby rivers and estuaries. In 1995, five major lagoon spills discharged more than 30 million gallons of hog wastes into North Carolina’s rivers. As a result, that same year, approximately 10 million fish died in these waterways. A majority of the fish was reported with bleeding sores covering there bodies (Broad, 1997). This symptom can be traced back to Pfiesteria.

Effects on Humans

Not only does Pfiesteria piscicida have an impact on the health of fish, but it also causes complications on humans. Thirteen researchers who worked with diluted toxic cultures of Pfiesteria were subjected to serious adverse health by coming in contact with the water or by inhaling toxic aerosols from laboratory cultures. The symptoms from Pfiesteria include narcosis (a "drugged" effect), development of sores (in areas that directly contact water containing toxic cultures of Pfiesteria, and also on the chest and face), uniform reddening of the eyes, severe headaches, blurred vision, nausea/vomiting, sustained difficulty breathing (asthma-like effects), kidney and liver dysfunction, acute short-term memory loss, and severe cognitive impairment (equals serious difficulty in being able to read, remember one’s name, dial a telephone number, or do simple arithmetic beyond 1+2=3). For example, in January 1993, Burkholder began spending more time assisting with the actual experiments in the laboratory. She worked with beakers filled with extremely toxic batches of culture, holding the containers directly in front of her face to carefully pour the contents. At times, she recalled her eyes burning, so she raised a glove hand that was dripping with toxic water to rub them. As a result, she began to feel the narcotic effect take control along with breathing problems and severe stomach cramps. In addition, later she had problems teaching her class. She misspoke, her thoughts were confused, and she was unable to answer some very basic questions asked by her students. On her drive home, she remembered looking down at the speedometer and realized that she was going eighty-two miles per hour down streets whose speed limit was forty-five (Barker, 1997). Burkholder’s laboratory was shut down after she and a colleague fell ill. Her colleague’s reading ability deteriorated to the level of a 7-year-old. The effect temporarily lasted three months. Later that summer, the lab was reopened with state-of-the-art ventilation systems and isolation units to contain the organism (Pain, 1996). Many of these listed symptoms gradually disappear over time as long as the person is no long exposed to Pfiesteria. On the other hand, some of these effects have reoccurred in people after strenuous exercise from time to time as far up to six years after the initial exposure to Pfiesteria (Burkholder, 1997). Pfiesteria was also linked to fishers, water skiers, and those monitoring fish kills. They also complained of skin lesions and other health effects, such headaches and light-headedness. Consequently, Pfiesteria not only affects laboratory workers, but everyone who comes in contact with it outdoors.

Government’s response to problem

The government was forced to respond to attempt to resolve the problem due to the public health concern. As the outreaching news of the newly discovered Pfiesteria piscicida reached newspapers and journals, the White House, Congress, and the media became curious about the fascinating toxic algae. The curiosity led the government to launch a federal research project known as Ecology and Oceanography of Harmful Algal Blooms (ECOHAB). ECOHAB is targeted at attempting to understand the environment of toxic phytoplankton and benthic algae responsible for causing red tides and other complications, such as Pfiesteria, in our coastal waters. Harmful Algal Blooms have been a major concern in the past two decades because they are increasing at an enormous rate. The blooms have cost the United States approximately $1 billion in lost tourism, fishery closures, and fish and shellfish mortalities (Baker, 1998).

ECOHAB is supported by four different agencies: the National Science Foundation (NSF), the Environmental Protection Agency (EPA), the Office of Naval Research (ONR), and the National Oceanic and Atmospheric Administration (NOAA). In 1997, the agencies gave $3 million to ECOHAB to fund more research to understand questions as how fast the blooms grow, how they are distributed by currents, and how they move through the food web. In addition, each of the agencies has a specific interest in a particular field of these harmful algal blooms. The ONR is interested in learning about how the blooms might affect remote-sensing equipment and other military technology. However, the EPA’s interest is in the causes of the blooms. More specifically, they want to research Pfiesteria and many of the red and brown tides. It is assumed that one of the major causative factors is concerning the over discharge of nutrients and other pollutants into our coastal waters (Baker, 1998).

There has been action taken in attempt to reduce the amount of nutrient discharge by many different federal and state agencies. The United States Department of Agriculture (USDA), the EPA, the NOAA, and the Department of Interior are pushing for the reduction of nitrogen and phosphorous pollution in the Chesapeake Bay by 40%. In addition, North Carolina has taken action to control the farm nutrient runoff. In August 1997, Governor James B. Hunt Jr. signed a bill, entailing a two-year moratorium on new hog farms, a reduction on nutrient wastewater discharge, and new provisions for land use management. Also, Virginia Governor George Allen authorized $600,000 to be allocated for Pfiesteria research within the Virginia Department of Health (Hileman, 1997).

In September 1997, Maryland approved a $2 million emergency appropriation to aid Maryland farmers plant more crops to assist in absorbing unused crop nutrient, which result from the summer’s drought. Maryland and the USDA came together to expand the Conservation Reserve Program (CRP), by allowing 100,000 acres of environmentally-sensitive land near Maryland streams and rivers to be set aside and maintained to protect water quality. As Vice President Gore announced new federal and state efforts to protect the Chesapeake Bay, he stated, "This new partnership will further protect the water resources of Maryland and the Chesapeake Bay. This agreement means cleaner water, healthier fish, and a stronger environment for every family in Maryland. By protecting the lands adjacent to the tributaries of the Bay and by restoring wetlands, we can significantly reduce the amounts of nutrients, sediment, and pesticides that reach the waters of the Bay" (Gore, 1997). As a result, these future plans will have a dramatic effect on the prevention of nutrient discharge leaking into the rivers and streams. Restoration of wetlands will be a key factor because they have a great ability to absorb a lot of the accidental runoff of nutrients and restore the water quality. In addition, the protection of lands along the rivers will prevent development of hog farms, agriculture, or other facilities with the potential of discharging large amounts of nutrients. Also, the riparian areas along rivers will be able to reduce the amount of sediment and nutrients by as much as 90 percent if properly planted with protective vegetation (Gore, 1997).

New Technology

Pfiesteria piscicida and other dinoflagellates are very difficult to study and identify under microscopes, but biotechnology and remote sensing have made it possible for scientists to do the field research. Pfiesteria is very difficult to monitor due to their short duration and they take on a variety of formations. New molecular probes have allowed scientists the ability to view and identify these dinoflagellates. Parke Rublee, an associate professor of biology at the University of North Carolina, developed a DNA probe for Pfiesteria piscicida. This is a probe consisting of primer, which is a short piece of DNA unique to Pfiesteria piscicida and attached is a fluorescent dye. The primer binds to only the Pfiesteria cells. When this is looked at under a fluorescent microscope, the Pfiesteria cells fluoresce and can be separable from other species (Pelley, 1998).

Another potential technique to identify Pfiesteria piscicida is by the use of reporter gene assays. John Ramsdell, division chief of the NOAA Marine Biotoxins Program in Charleston, South Carolina, came together with Burkholder and colleagues to develop this new reporter gene assays. This technique works by isolating a mammalian gene that is only activated by the presence of toxins from Pfiesteria. They linked the mammalian gene to a gene for luciferase, which is an enzyme that gives fireflies their bright flashes of light. Furthermore, they inserted the enzyme into the mammalian gene and whenever the Pfiesteria’s toxins activate the mammalian gene, it turns on the luciferase. Therefore, bright flashes of light coming out of the mammalian gene alerts the scientists that Pfiesteria toxins are present (Pelley, 1998).

Remote sensing allows scientists to study algal blooms on a larger scale through the use of satellites. The process functions by tracking the water masses that are affected by the algal blooms with sea-surface temperature sensors. This process detects the reflectance of the chlorophyll and the algal blooms can easily be distinguished from the areas without any blooms (Pelley, 1998). This provides scientists clues in understanding how the blooms migrate along the coast. In addition, it gives the scientists ideas of the size of the blooms in a certain area. From there, the scientists can attempt to explain what is causing the bloom.

Conclusion

There would be enormous amounts of complications with the human health and the aquaculture ecosystem if the problem of Pfiesteria piscicida is not resolved now. If the detrimental effects from Pfiesteria’s toxins currently continue, our fish supply would be cut dramatically and our rivers and estuaries would be closed. In order to stop Pfiesteria from taking control of our rivers and estuaries, the discharge of nutrients must be cut back. Hog and chicken farms need to be built away from our nearby rivers to prevent this misfortune. In addition, we can not go on destroying our wetlands, which help in absorbing the nutrients and restore the quality of water. We need to look ahead into the future and confront our present problems now before they get out of hand. If we stop the problem now and we will not have to worry about the consequences and repercussions that will occur in the future. Consequently, the environment gives us hints to tell us that the pollution we are producing is detrimental to the ecosystem and eventually on human health. The death of millions of fish is a perfect example to show that we are dumping too much pollution into our waters. If our waters are too polluted for the fish, that means that they are too polluted for humans as well because we are not going to be able to use our rivers. Furthermore, the implications are still unknown about consuming the fish containing Pfiesteria’s toxins. Also, since the toxins are airborne, a swift wind can create problems for a whole city. As a result, the problem has to be stopped here.

The government’s response to the problem and the technology has come a long way since the discovery of Pfiesteria piscicida. The government has made reasonable future plans to achieve, which will indeed push us closer towards closing the war against Pfiesteria. The government realized the magnitude of the problem once the possibilities of Pfiesteria’s effects on the human health became clear. Technology has also worked well along aside the government just at the perfect time. Without the technology, our understanding of this mysterious dinoflagellate couldn’t be accomplished. The two are a dynamic duo at fighting against the dino. If government plans continue as predicted and technology continues to progress, then the victory over Pfiesteria piscicida looks promising.

Cited Literature:

Baker, B. 1998. BioScience 48: 12.

Barker, R. 1997. And the Waters Turn to Blood 1: 89-90.

Broad, W.J. 1997. National Wildlife 35: 10.

Burkholder, J.M. 1997. NCSU Aquatic Botany Laboratory Abstracts and Papers on Pfiesteria piscicida. www2.ncsu.edu/unity/lockers/project/aquatic_botany/p-abst.html: 5.

Burkholder, J.M. 1997. NCSU Aquatic Botany Laboratory Pfiesteria piscicida Homepage. www2.ncsu.edu/unity/lockers/project/aquatic_botany/pfiest.

Burkholder, J.M. 1997. NCSU Aquatic Botany Laboratory  Pfiesteria piscicida photographs.

www2.ncsu.edu/unity/lockers/project/aquatic_botany/algae.html

Gore, A. 1997. U.S. EPA Pfiesteria piscicida.
Www.epa.gov/OWOW/estuaries/pfiesteria/goreplan1.html: 1-2.

Hileman, B. 1997. Chemical and Engineering News 75: 15. 3-5.

Pain, S. 1996. New Scientist 149: 9.

Pelley, J. 1998. Environmental Science and Technology 32: 26-30.

Wiant, C.J. 1998. Journal of Environmental Health 60: 28.

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