Last updated 13 October 2000
Pfiesteria Interagency Coordination Workgroup
Glossary of Pfiesteria-Related Terms DISCLAIMER: This Glossary is intended to serve as a tool to improve communication between technical staff involved in Pfiesteria-related monitoring activities. It is a work-in-progress document and represents our best understanding of what is currently known about a complex subject. The majority of the information in this document results from work conducted in JoAnn Burkholder's laboratory. Additionally, federal, state and academic scientists reviewed and contributed to the information in the Glossary.Please contact one of the following individuals before any formal publication or reference:
Judy Kleindinst (508) 289-2745 jkleindinst@whoi.edu Joseph Macknis (410) 267-5748 macknis.joe@epamail.epa.gov JoAnn Burkholder (919) 515-2726 joann_burkholder@ncsu.edu PURPOSE: The purpose of this Glossary is to provide consistent and scientifically correct definitions of words and phrases used to describe events and activities related to toxic Pfiesteria piscicida and toxic Pfiesteria-like species. Some complex terms are defined at two levels. The first level provides a summary definition to improve the understanding of terms or phrases for a general non-technical reader. The second level provides additional or more detailed scientific information for the more technical reader.
Use of these terms will facilitate accurate and clear communication among scientists, managers, policy makers, the press, and the public. In this Glossary, several important terms are presented first because they are fundamental to understanding this issue. Other terms subsequently are listed in alphabetical order. New terms will be added to the glossary as needed.
Fundamental Terms
Pfiesteria piscicida
Level I - Pfiesteria piscicida is a microscopic organism called a dinoflagellate and is a natural part of the marine environment. It has been associated with fish lesions and fish kills in Maryland, Delaware and North Carolina. Most dinoflagellates are not harmful, and Pfiesteria is harmful only under certain conditions; in fact, some strains never produce toxins, and even the strains that can produce toxins are benign under most conditions and during most of the year.Level II - Pfiesteria piscicidais a one-celled organism called a dinoflagellate that is found in estuaries (brackish waters along the mixing zone where rivers meet the sea). Pfiesteria piscicida is a heterotrophic (animal-like) organism that feeds on a wide variety of prey including other one-celled organisms such as bacteria and algae, finfish and shellfish, and the remains of dead organisms that have settled to the bottom of estuaries. This dinoflagellate has many different life-cycle stages, including flagellated (with flagella or tail-like extensions), amoeboid (amorphous shape, without flagella) and encysted (dormant or inactive) stages or forms. These stages can have many different shapes (called morphs), and can vary greatly in size. Usually, the most commonly found forms of P. piscicida are colorless amoebae that remain in the bottom sediments (called a benthic habitat). Under laboratory conditions that include the presence of live fish, fish secretions, and fish excrement, P. piscicida can produce substances that are toxic to fish. When these triggers are present and detected in sufficient quantity, the stages ofP. piscicida that are toxic (especially toxic zoospores, which are asexual, motile flagellated cells) become abundant in the water column. The presence of low to moderate densities of toxic zoospores has been linked in both time and space with some fish kills, and with some fish disease events involving the presence of fish with lesions. The ability of populations, or strains, of P. piscicida to produce toxins varies widely. In fact, some strains never produce toxins, and even the strains that can produce toxins are benign under most conditions and during most of the year.
TPC (toxic Pfiesteria complex)
Level I - TPC (toxic Pfiesteria complex) refers to dinoflagellate species that have demonstrated toxicity through the production of bioactive compounds that cause erratic behavior, adverse health effects, or kill fish as evidenced in toxic bioassays (see toxic bioassays).
Level II - TPC (toxic Pfiesteria complex) refers to dinoflagellate species with strains that produce toxins when viewed under light microscopy in appropriate experimental conditions (as demonstrated by a positive test with fish bioassays [see below for description of the fish bioassay]. To be included in the TPC, a species must have all of these characteristics: 1) Strong attraction to live fish prey, 2) Ability to produce substances that are ichthyotoxic (i.e., cause erratic behavior, disease, or death in fish), and 3)Toxic activity (production of toxic substances by some strains within the species) that is stimulated by the presence of secretions, excretions, or the presence of (usually large numbers of) live fish. Note that confirmation of the species identity must be completed using scanning electron microscopy of suture-swollen or membrane stripped cells (SEM, see below). Other characteristics of secondary importance for membership in the TPC are a complex life cycle with many different stages; and inability to produce their own food (called heterotrophy). However, they sometimes consume algae that contain chloroplasts (small structures where photosynthesis occurs), and they can retain these structures (as kleptochloroplasts) in food vacuoles to help supplement their food resources. At present, there are two species in the TPC: Pfiesteria piscicida and Pfiesteria shumwayae. As additional species are verified to have all of the above characteristics, they will be added to the TPC.
TPC functional type
Level I - TPC functional type refers to the ability of different strains of these dinoflagellates to produce toxins. There are three functional types among strains of TPC species: 1) actively toxic strains producing substances which have caused stress, disease or death in fish; 2) nontoxic strains which are potentially toxic, but are not presently producing toxins; and 3) non-inducible ("never toxic") strains which are incapable of producing toxins in the presence of live fish or their fresh materials.
Level II - TPC functional type refers to the ability of different strains of these dinoflagellates to produce toxins. There are three functional types among strains of TPC species: 1) actively toxic strains are producing ichthyotoxic substances (confirmed by a positive result in fish bioassays [see below]) that have caused stress, disease, or death in fish; 2) nontoxic strains are potentially toxic — they are capable of producing toxins that cause fish stress, disease or death, but at present they are not producing toxins; and 3) non-inducible ("never-toxic") strains are incapable of producing ichthyotoxins in the presence of live fish or their fresh materials (see below).
Note that toxin strains of TPC species are difficult to maintain in culture. If denied access to live fish and fed other prey, they lose their ability to produce toxins within 6-8 months; and even if maintained in an actively toxic mode (given live fish several times daily), they generally lose their ability to produce toxins in laboratory cultures within a year or less. The phenomenon of lost ability to produce toxin over time in culture is an artifact of culture conditions - that is, the highly artificial conditions in culture aparently do not provide organic substances or other factors that allow toxic strains to retain the toxin-producing capability under field conditions (for example, between fish kill events). It is also important to note, as a second aspect of this issue, that many strains have been tested with fish immediately after field collection and have been found to be incapable of causing fish stress, disease or death. Thus, there likely are naturally occurring, non-inducible (benign) strains, in addition to the culture artifact problem of lost toxicity over time in culture.
The above distinctions among functional types of Pfiesteria are very important for consideration in efforts to understand the ecology of toxic Pfiesteria based on laboratory experiments. Non-inducible strains have been shown to have markedly different responses to environmental conditions (for example, nutrients, light, microbial prey, presence of herbivores, presence of fish) than strains that can be induced to make toxin in the presence of live fish. While certain types of molecular and comparative ecological information can be gained using non-inducible strains, these strains should not be used, or data from such strains extrapolated, in efforts to understand the ecology and behavior of toxic Pfiesteria.
Toxic Pfiesteria outbreak
Level I- Toxic Pfiesteria outbreak refers to a fish kill or fish disease event where an actively toxic TCP species has been collected, identified, and tested as actively toxic with fish bioassays [see below]. Toxic Pfiesteria outbreaks generally occur in mid-summer through mid-fall in quiet, poorly flushed brackish waters that are nutrient over-enriched. Most of the fish killed have been Atlantic menhaden, which are small fish that travel in large, dense schools and feed in poorly flushed estuaries with abundant phytoplankton.
Level II- Toxic Pfiesteria outbreak refers to a fish kill or fish disease event where an actively toxic TCP species has been collected, identified, and tested as actively toxic with fish bioassays [see below]. The TPC species must be present in water samples collected where the fish were diseased or dying, at high enough densities to harm or kill fish. Actively toxic TPC species can harm fish at cell densities of about 100 or more toxic zoospores/mL of water, and can kill fish at about 300 toxic zoospores/mL. Toxic Pfiesteria outbreaks generally occur in mid-summer through mid-fall in quiet, poorly flushed brackish waters that are nutrient over-enriched (with nitrogen and phosphorus pollution that encourages growth of algae and other organisms that are used as food by TPC species when fish are not readily available). Almost all of these outbreaks have been in North Carolina’s Albemarle-Pamlico Estuarine System (88 outbreaks in 1991-1998 with over a billion fish affected, especially in the Neuse and Pamlico Estuaries). Three small outbreaks have occurred on the lower eastern shore of Chesapeake Bay (ca. 30,000 fish affected, especially in the Pocomoke Estuary). Most of the fish killed have been Atlantic menhaden, which are small fish that travel in large, dense schools and feed in poorly flushed estuaries with abundant phytoplankton.
Other Terms
Algal bioassay
Level I - Algal bioassay is used to test water and sediment samples for the presence of dinoflagellates that consume the specific algae used in the test. Algal bioassays cannot be used to detect whether a strain of dinoflagellate can produce ichthyotoxins (see fish bioassay).
Level II - Algal bioassay is used to test water and sediment samples for the presence of dinoflagellates that consume the specific algae used in the test. This test usually detects benign species that, when subsequently tested with fish bioassays, do not grow or produce toxins when fish are present. Algal bioassays cannot be used to detect whether a strain of dinoflagellate can produce ichthyotoxins (see fish bioassay). Algal assays can stimulate growth of PLO dinoflagellates that can be isolated, cloned, and made available for comparison to TPC species, e.g., for genetic studies, ecological comparisons, etc. They can also be tested to determine whether they are toxic to fish; if so, they would be added to the TPC.
Background counts
Level I - background counts are counts of the number of Pfiesteria-like organisms (PLOs) in a given volume of water. They are conducted as part of baseline monitoring to determine the abundance and temporal and spatial distribution of PLOs in estuarine waters. Background counts provide no information about whether the organisms are capable of producing toxins.
Level II - background counts of Pfiesteria-like organisms (PLOs) are completed using a light microscope. These counts typically are done as part of water quality monitoring (in the absence of a fish kill or fish disease event, or outside of the area where such an event is occurring), just to see whether dinoflagellates that look like TPC species are present. These counts can sometimes provide information about the species present (especially autotrophic ‘lookalike’ species that are not members of the TPC). Background counts provide no information about whether the organisms are capable of producing toxins.
Biotoxin (Bioactive substance, in this case, a substance that harms fish; Toxin; Ichthyotoxin)
Level I - Biotoxin refers to a chemical produced by a living organism that adversely affects other organisms. In this Glossary, the terms are used to refer to substances that cause distress, disease, or death in fish. Once these toxins are chemically identified, assays can be developed to detect the toxins in water samples from fish kill/disease events. Such tests will represent a significant improvement over the more primitive fish bioassay approach (see fish bioassays), which is the only technique currently available to test for the presence of toxic TPC populations.
Level II - Biotoxin refers to a chemical produced by a living organism that adversely affects other organisms. Some of the biotoxins produced by the known TPC species to date (namely, Pfiesteria piscicida and species B) have been isolated, but none have yet been chemically identified or characterized. In this Glossary, the terms are used to refer to substances that cause distress, disease, or death in fish. Once these toxins are chemically identified, assays can be developed to detect the toxins in water samples from fish kill/disease events. Such tests will represent a significant improvement over the more primitive fish bioassay approach (see fish bioassays), which is the only technique currently available to test for the presence of toxic TPC populations. Note that the term ‘virulent,’ meaning toxic, has been used previously in reference to toxic or nontoxic (potentially toxic) strains of TPC species. However, because medical specialists use this term to indicate an infectious organism, and because TPC species are not known to be infectious, use of ‘virulent’ in reference to toxic strains of TPC species is discouraged.
Clonal culture of a TPC species begins with a single isolated cell. This cell is grown in appropriate conditions with prey such as bacteria, algae, or fish, to produce high densities of the organism. Thus, a clonal culture of a TPC species contains only that species and its prey.
Fish bioassay
Level I - fish bioassay is the only method currently available to determine if a sample contains TPC organisms that can produce bioactive compounds that cause erratic behavior, adverse health effects or kill fish. A valid fish bioassay requires that the sample be taken during an in-progress event in which fish are distressed or dying.
Level II — fish bioassay is the only method currently available to determine whether a sample contains strains of TPC species that can produce ichthyotoxins. The ichthyotoxins produced by TPC species have been linked to a suite of human health impacts. To protect laboratory workers from aerosolized toxins, the assay must be conducted in a specially designed biohazard III containment system with protective outerwear and respirators. Fish bioassays are conducted for two reasons: 1) In survey efforts, using water and/or sediment samples, to see whether potentially toxic TPC species are present in an area; or 2) In response to a fish kill or fish disease event, from water samples collected while/where fish are affected (behaving erratically, diseased or dying), to see whether actively toxic TPC species are present.
TPC species have a biochemical "memory" for recent stimulation by live fish, but they are sensitive to transport from the estuary to the lab and usually encyst (become dormant). If actively toxic when collected, they become active and produce a positive fish bioassay (i.e., cause disease or death of test fish) within 21 days (usually in 4-9 days). TPC species collected as part of surveys (i.e., not in response to reports of fish disease or fish kills) may also produce positive fish bioassays, but these tests take longer (8 to 10 weeks). If test fish show stress, illness, erratic behavior, or die, then water samples from the culture vessel used for the test are examined for the presence of Pfiesteria-like zoospores. The presence of potentially harmful cell densities of such cells (see toxic Pfiesteria outbreak) confirms this as a "positive fish bioassay," that is, with actively toxic TPC species present. Live fish are added to replace dead fish in the culture vessel for 1-2 more weeks. These fish serve as a food source and allow the TPCs to multiply, providing sufficient cell numbers for identification with scanning electron microscopy (see SEM). The culture is also examined for other organisms (bacteria, fungi, harmful algae, other harmful dinoflagellates) that can cause fish stress, illness, or death.
Thus, a "positive fish bioassay" can be used to confirm a toxic Pfiesteria outbreak and implicate TPC species as a cause of fish stress, illness, or death only when the following criteria are met: 1) Presumptive counts demonstrate potentially harmful PLO densities in field-preserved water samples that were collected when/where fish are diseased or dying; 2) Fresh (unpreserved) water samples collected at the same time are used for fish bioassay tests (or, for benthic species, water and surficial sediments); 3) The fish bioassays produce PLOs (see PLOs, below) in cell densities that, if actively toxic, are known to cause fish disease or death within 21 days, usually 4-9 days; and 4) Test fish become stressed, behave erratically, become ill, or die when such cell densities are present in the fish culture water. Also note that if other fish pathogens are detected, they are reported with the TPC species that are present as multiple causative agents in fish death/disease event. In such cases, the TPC dinoflagellate(s) species is isolated and re-examined separately for toxicity in an additional fish bioassay test.
Molecular probes
Level I - molecular probes are state-of-the-art diagnostic tools used to quickly determine the presence of Pfiesteria piscicida. They cannot be used to discern whether toxic (actively or potentially) populations of TPC species are present. They are still under development.
Level II — molecular probes refer to techniques that are used to detect the presence of the genetic material of TPC species. Three types of molecular probes for TPC species became available in 1998, and are currently in use by laboratories that are helping States to test for the presence of TPC populations in their waters. The first is called a hetero-duplex mobility assay (HMA), which amplifies target fragments of genetic material in polymerase chain reactions (PCR). The HMA method can determine whether cultures are monoclonal (that is, have only one dinoflagellate strain [from one species] present; see clonal cultures), or whether they contain multiple dinoflagellate species and strains. HMA probes are available for both Pfiesteria piscicida and Pfiesteria shumwayae. In another type of probe, target amplification by PCR has enabled screening of cultures and natural estuarine water samples to qualitatively detect the presence of P. piscicida. The third type of molecular probe is a fluorescent in situ hybridization (or FISH) ribosomal DNA probe, which allows visualization of whole P. piscicida cells and quantitative evaluation of P. piscicida cell densities in water samples.
These probes were developed by P. Rublee and D. Oldach using virulent clonal cultures of P. piscicida (from J. Burkholder’s laboratory; all three techniques) and Pfiesteria shumwayae (from J. Burkholder’s laboratory - HMA technique available thus far). The FISH probe has enabled researchers to discern P. piscicida from ‘lookalike’ species (for example, P. shumwayae, Gyrodinium galetheanum, ‘Cryptoperidiniopsis’ nov. gen.), and to estimate its abundance in water samples from the major fish kill and toxic Pfiesteria outbreak that occurred in North Carolina waters in 1998.
But note that these species-specific molecular probes cannot be used to discern whether toxic (actively or potentially) populations of TPC species are present. It is also important to remember that these probes are not "foolproof" or "guaranteed" — they offer promising techniques for more rapid detection of TPC populations, but there is always the possibility that species (toxic or nontoxic) which remain to be discovered will react with the probes, as well as the two known TPC species. Therefore, probe results need to be checked with SEM (see SEM) to verify TPC species identifications in clonal cultures and fish bioassays.
PCOs (Pfiesteria Complex Organisms)
Level I - PCOs (Pfiesteria Complex Organisms) is an incorrect acronym that should no longer be used. ‘Complex’ has a specific meaning referring to a group of species within a genus or to an assemblage of species with certain behavioral characteristics (for example, toxic activity, which is of critical importance when considering the Pfiesteria issue). The recommended term to describe dinoflagellates that have similar structural but not necessarily behavioral characteristics as toxic Pfiesteria piscicida is Pfiesteria like organisms (see PLOs -Pfiesteria like organisms).
Level II - (PCOs, Pfiesteria Complex Organisms) is an incorrect acronym that should no longer be used. ‘Complex’ has a specific meaning referring to a group of species within a genus or to an assemblage of species with certain behavioral characteristics (for example, toxic activity, which is of critical importance when considering the Pfiesteria issue). Examples of the correct use of the term ‘complex’ are the toxic Alexandrium complex, which refers to the species within the genus that produce saxitoxins and their derivatives; and the toxic Pseudo-nitzschia complex, which refers to the species within the genus Pseudo-nitzschia that produce the toxin, domoic acid. Currently the term PCOs is mis-applied to dinoflagellate species that simply look like Pfiesteria piscicida under a light microscope, but have never been known to produce toxins. The recommended phrase to describe dinoflagellates that are similar in appearance but not necessarily similar to Pfiesteria piscicida in ability to produce toxin and other important behavioral characteristics is ‘Pfiesteria-like organisms’ (see PLOs).
PLOs (Pfiesteria-like organisms)
Level I - PLOs (Pfiesteria-like organisms) is a term used to describe dinoflagellates that look similar to Pfiesteria piscicida as determined by high power conventional light microscopy. They may have similar structural but not necessarily behavioral characteristics as toxic Pfiesteria piscicida. The number of PLOs in a given volume of water may be determined as part of presumptive counts (see presumptive counts) or background counts (see background counts).
Level II - PLOs (Pfiesteria-like organisms) refer to zoospores of dinoflagellate species that look similar to Pfiesteria piscicida zoospores and motile zygotes under the light microscope (400x or higher magnification). Cell concentrations of PLOs are used in both background counts (see background counts) and presumptive counts (see presumptive counts). Note that in efforts with light microscopy to discern the presence/absence of TPC dinoflagellates, ‘Pfiesteria-like’ zoospores can be eliminated as truly "Pfiesteria-like" if they have chloroplasts freely distributed in the cytoplasm of both the upper and lower part of the cell. Thus, organisms such as Gyrodinium galatheanum and Gyrodinium estuariale may resemble TPC zoospores in their shape, but these organisms which photosynthesize on their own (with their own chloroplasts, rather than kleptochloroplasts that may be retained by TPC species) can easily be ruled out as TPC zoospores using light microscopy.
Presumptive counts
Level I - presumptive counts are counts of the number of PLOs in a given volume of water and conducted as the first step in determining whether or not TPC was involved in a fish distress event. Note: Presumptive counts alone cannot provide information about whether the organisms were actively toxic. Fish bioassays (see fish bioassays) and SEM (see SEM) identification are both needed to confirm TPC as a factor in causing adverse fish health, fish disease or fish kills.
Level II — presumptive counts are conducted as the first step in assessing whether TPC species were involved in fish kills or fish disease events. Water samples from the in-progress event are collected, immediately preserved (with acid Lugol’s iodine fixative), and examined with a light microscope to see if Pfiesteria-like zoospores had been at high enough densities (see toxic Pfiesteria outbreak) to harm or kill fish if the organisms were actively toxic TPC species. But NOTE: Presumptive counts cannot provide information about whether the organisms were actively toxic. Presumptive counts are best used: 1) To determine whether potentially harmful cell densities of dinoflagellates that might be TPC species are present (see toxic Pfiesteria outbreak) and, thus, could have caused the fish kill/disease event; and 2) to eliminate the TPC from further consideration as causative agents of the fish disease or fish kill event, if such cell densities are absent.
SEM (scanning electron microscopy)
Level I — SEM (scanning electron microscope) allows high magnification of specially prepared cells to confirm that toxic Pfiesteria complex (see TPC) was a causative factor in an event in which fish exhibited erratic behavior, adverse health effects, or were killed. It is the final test, proceeded by presumptive counts and fish bioassays needed to confirm implication of TPCs as the causative agent in a fish distress event.
Level II - SEM (scanning electron microscope), the current ‘gold standard’ for identification of TPC species, allows much higher magnification (ca. 1500-5000x or higher) than light microscopy. SEM of specially prepared ‘Pfiesteria-like’ zoospores (with outer membranes stripped from the cells, or with cell sutures swollen) is used to confirm the identity of TPC species from positive fish bioassays. SEM cannot be done on most field samples because the ‘Pfiesteria -like’ dinoflagellates (PLOs that may include members of the TPC) are usually present in low number, relative to other plankton that make it very hard to find the relatively few ‘Pfiesteria -like’ cells. Since SEM is an expensive procedure, the cells need to be easily located to minimize the expense. Moreover, many ‘Pfiesteria -like’ zoospores (preferably 100 or more from each sample) must be rotated to observe minute features on the top, bottom, front and back views of each cell before a species identification can be confirmed. Such effort requires at least two days for one sample, even when ‘Pfiesteria -like’ zoospores are abundant.
Please note that, while the focus of this Glossary is the toxic Pfiesteria complex, certain other harmful algal species (here, including dinoflagellates) can cause fish stress, disease, and death. For example, the photosynthetic dinoflagellate Gymnodinium breve is highly toxic to fish in Florida waters; Gyrodinium galatheanum has been linked to cultured fish death; and many hetero-trophic dinoflagellate parasites (some with Pfiesteria-like appearance in their zoospore stages) can cause fish disease and death; and toxic chrysophytes such as certain Chatonella and Heterosigma species have also been known to cause fish death. Much more needs to be learned about interactions between harmful algae and fish kills/disease, and it is important to 'be on the lookout' for other harmful algae, as well as TPC species, in evaluating causative factors involved impaired health of estuarine and cultured fish populations.