Critical Needs in Harmful Algal Bloom Research

JoAnn M. Burkholder

In this presentation, I shall describe areas in which progress is critically needed in the field of harmful algal research. All previous speakers have discussed what they have depicted as “black holes” in basic understanding of the topics they addressed. Harmful algal bloom research is surely another area that could be similarly cast.

“Harmful algae” refers to algae that are undesirable to humans because (1) they produce toxins that impair the health of humans and desirable fish and wildlife; (2) they parasitize desirable organisms in the food web, such as commercially valuable finfish and shellfish; (3) they become too abundant and overgrow desirable habitat for fish such as seagrass meadows, so that the beneficial plants cannot receive enough light to survive; and/or (4) they become too abundant and then, at night, use most or all of the oxygen in the water for their respiration, so that fish and other desirable organisms suffocate or become seriously physiologically stressed. Harmful algae include prokaryotic blue-green algae or cyanobacteria. More recently, the term has been used to include organisms that are not really algae —for example, certain nontoxic animal—like dinoflagellates, which cause fish disease (e.g., Amyloodinium ocellatum); and certain toxic animal-like dinoflagellates (e.g., the toxic Pfiesteria complex), which do not have their own chloroplasts for photosynthesis but which resemble plant-like dinoflagellates in appearance and certain other general features (Burkholder 1998; Lewitus and others 1999). Here, reluctantly, the cur-

Department of Botany, North Carolina State University, Raleigh, NC



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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP Critical Needs in Harmful Algal Bloom Research JoAnn M. Burkholder In this presentation, I shall describe areas in which progress is critically needed in the field of harmful algal research. All previous speakers have discussed what they have depicted as “black holes” in basic understanding of the topics they addressed. Harmful algal bloom research is surely another area that could be similarly cast. “Harmful algae” refers to algae that are undesirable to humans because (1) they produce toxins that impair the health of humans and desirable fish and wildlife; (2) they parasitize desirable organisms in the food web, such as commercially valuable finfish and shellfish; (3) they become too abundant and overgrow desirable habitat for fish such as seagrass meadows, so that the beneficial plants cannot receive enough light to survive; and/or (4) they become too abundant and then, at night, use most or all of the oxygen in the water for their respiration, so that fish and other desirable organisms suffocate or become seriously physiologically stressed. Harmful algae include prokaryotic blue-green algae or cyanobacteria. More recently, the term has been used to include organisms that are not really algae —for example, certain nontoxic animal—like dinoflagellates, which cause fish disease (e.g., Amyloodinium ocellatum); and certain toxic animal-like dinoflagellates (e.g., the toxic Pfiesteria complex), which do not have their own chloroplasts for photosynthesis but which resemble plant-like dinoflagellates in appearance and certain other general features (Burkholder 1998; Lewitus and others 1999). Here, reluctantly, the cur- Department of Botany, North Carolina State University, Raleigh, NC

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP rent general misuse of the term algae will be followed through inclusion of heterotrophic dinoflagellates under the broad umbrella of harmful algae, although they more correctly should be considered as animal-like protozoans. Harmful algal blooms have received a great deal of attention, but remarkably little is known about them. This discussion first addresses remote sensing techniques for detecting harmful algae, as requested, and then focuses mostly on critical research needs regarding toxic algal species, as opposed to other types of harmful species that cause oxygen deprivation or other undesirable conditions but do not produce toxins. ADVANCED TECHNIQUES FOR DETECTING HARMFUL ALGAL BLOOMS Various remote sensing techniques are available for detecting certain harmful algal blooms, but their value is limited. Remote sensing has helped scientists to track several types of established surface blooms formed by organisms such as certain cyanobacteria, chrysophytes, and dinoflagellates. For example, the toxic dinoflagellate, Gymnodinium breve, has been forming blooms in Florida waters for more than 100 years, and it also once bloomed in North Carolina's coastal waters during 1987 (Landsberg and Steidinger 1998; Steidinger and others 1998). In the latter case, it was determined retrospectively that this bloom originated from G. breve cells that were transported northward with the Gulf Stream (Steidinger and others 1998). An extremely unusual set of weather conditions allowed small eddies from the Gulf Stream to drift to North Carolina shores basically intact during early autumn. The G. breve inoculum increased enough to contaminate shellfish that concentrated them by filter feeding, thus requiring widespread shellfish harvest closures throughout most of the next winter season. That event caused about $26 million of damage to North Carolina; some fishermen never recovered from the losses they sustained. The analysis tracking G. breve northward from Florida (through sea surface temperature patterns) was retrospective. That is, the origin of G. breve, once detected in North Carolina waters, was determined belatedly from remote sensing records of temperature patterns from the Gulf Stream. Dense blooms of other toxic algae have also been tracked retrospectively with remote sensing (e.g., Pelaez 1987). However, in general, very little is known about how to prevent blooms, or even to track blooms as they begin to develop. Remote sensing techniques, which would permit design of improved early warning systems for mitigation efforts, are not available to enable detection of initial phases of these blooms. From the perspective of setting early warning systems in motion or mitigating

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP impacts, it would be much more desirable to detect the initiation phase of harmful algal blooms than blooms that are fully developed. In surveillance, remote sensing techniques are useful in tracking surface water temperatures, salinity, current direction, turbidity, and chlorophyll; however, these techniques have not yet been of use to advance general understanding of factors that control how blooms develop and then dissipate. Remote sensing techniques can sometimes be of value in tracking fully or moderately developed blooms, especially if the harmful species that form them are photosynthetic with plant-like pigments, or if scientists are certain that the bloom distribution closely follows certain temperature patterns or other environmental conditions that can be reliably tracked (e.g., Franks 1995; Johannessen and others 1989). However, in most estuarine and coastal waters, within practical constraints the currently available remote sensing techniques basically can track only blooms approaching 10 µg or more of chlorophyll/L (Kirk 1994). Thus, such techniques can detect moderate to dense blooms of photosynthetic harmful microalgae and development of undesirable macroalgal growth (e.g., Enteromorpha or Ulva species in sewage-enriched estuaries). In contrast, some toxic dinoflagellates do not have chlorophyll; and those with chlorophyll sometimes occur in very low cell densities (with chlorophyll a much less than 10 µg/L) that, nonetheless, are sufficient to cause shellfish to become too contaminated with toxins to be safe for human consumption (Falconer 1993a). Remote sensing would not be adequate to track these organisms; nor, in many situations, can the available techniques distinguish between harmful species and other co-occurring benign species with similar pigments. Remote sensing techniques, then, are of use primarily to track environmental conditions that may be associated with harmful algae. CRITICAL RESEARCH NEEDS The four most critical research needs are research-quality cultures, life cycles, toxin identification and detection, and detection of toxic strains. Brief discussions are provided for each. Research-Quality Cultures From most of the species that have been rigorously tested—from diverse groups including toxic cyanobacteria, chrysophytes, diatoms, and dinoflagellates—it has been established that within a toxic species there is actually a range in toxicity (e.g., Anderson 1991; Bates and others 1998; Burkholder and Glasgow 1997; Edvardsen and Paasche 1998; Gentien and

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP Arzul 1990; Gorham and Carmichael 1988; Skulberg and others 1993; Sperr and Doucette 1996). Some strains within a population of a “toxic species” can be benign, that is, unable to produce toxin or producing negligible/ undetectable toxin. Moreover, many toxic strains lose toxin-producing capability when maintained in culture for more than several weeks to months, apparently as an artifact of the (highly artificial) culture conditions (e.g., Bates and others 1998; Edvardsen and Paasche 1998; EPA 1999). As they shift from toxic strains to strains that show no detectable ability to produce toxin further, these strains also undergo fundamental changes in physiological and behavioral characteristics. The danger inherent in misuse of noninducible or “permanently non-toxic” cultures (cultures in which toxicity can no longer be induced; EPA 1999), ostensibly to gain insights about toxic strains of harmful algae, is illustrated by the following example. The toxic dinoflagellate Pfiesteria piscicida is a complex animal-like organism, as mentioned (Burkholder and Glasgow 1997). Its response to nutrient enrichment depends on its previous history of feeding, rather than following a typical growth curve with concentration of nutrient added. Pfiesteria is stimulated to produce toxin by the presence of live fish (hence the name of the first known species, piscicida, meaning “fish killer” as reported by Steidinger and others 1996; also see Burkholder and Glasgow 1997, Burkholder and others 1992, Fairey and others 1999). However, this organism extends retention of kleptochloroplasts from algal prey, is attracted to light in plant-like behavior, and shows minimal attraction to fish after it becomes non-inducible (i.e., unable to stress or kill fish in repeated bioassays) over several months in culture with live fish. These are profound changes. Biohazard III containment systems are required to culture toxic Pfiesteria with fish to protect laboratory workers from its aerosolized neurotoxins that fish-killing cultures apparently emit (Glasgow and others 1995). To avoid use of (expensive) biohazard III facilities, Pfiesteria can be cultured with algal prey in a temporarily nontoxic mode. However, if toxic strains are cultured for several weeks on algal prey without live fish, most lose their ability to produce toxin. Some strains have been tested repeatedly over 4 years in various culture conditions, and their loss of toxin-producing capability appears to be permanently noninducible, given the present state of knowledge about Pfiesteria species (WHOI 2000). Recently, millions of dollars have been directed to federal agencies to address the toxic Pfiesteria issue (Epstein 1998). In the past 2 years, however, much funding has been spent for research on noninducible strains, fed for months to years on algal prey without live fish, which have been supplied to the scientific community at large by a federally funded, national phytoplankton center in the northeastern United States that specializes in growth of plant-like algae. In repeated tests by independent

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP laboratories, these noninducible strains of Pfiesteria have proven to be incapable of causing fish stress, disease, or death. Their increased reliance on chloroplasts retained from algal prey makes them more plant-like than toxic strains (Lewitus and others 1999), and they respond to indirect stimulation by nutrient enrichment (mediated through algal prey) more strongly than toxic strains under certain conditions—data with important ramifications if used as planned by state and federal agencies as a basis for setting levels of nutrient reductions to discourage Pfiesteria growth (e.g., State of Maryland 1998). The poultry industry in the Chesapeake Bay region has opposed recent state and federal efforts to reduce nutrient loading from this industry to the Bay. Toxic Pfiesteria has been shown to be stimulated directly by nutrient enrichment, thus indicating an enrichment "connection" (Burkholder and Glasgow 1997). However, use of noninducible Pfiesteria strains would biasstudies in favor of finding stronger indirect stimulation of Pfiesteria by inorganic nutrients, mediated through the abundance of algal prey that respond directly to the nutrients. This information would not be well received by the poultry industry or others whom government agencies have attempted to move toward strengthened (and costly) nutrient controls, in part by invoking the Pfiesteria/nutrient linkage. Thus, the compromised validity of research findings about the behavior, ecology, and physiology of toxic Pfiesteria that were erroneously based on use of permanently nontoxic strains could be compounded by serious socioeconomic ramifications that would be avoidable if the importance of toxic versus permanently benign strains is considered in the research design. Similarly, other findings about the behavior and ecology of toxic Pfiesteria, based on strains that are incapable of producing toxin, would be questionable. The case of Pfiesteria provides but one illustration of a serious problem that is affecting the general field of harmful algal research. The previously mentioned phytoplankton culture center is endorsed by a consortium of federal agencies and commercially supplies cultures of many toxic algal species to the scientific community at large. The cultures commonly are contaminated with other algal species (e.g., Oldach and others 2000). Some of the cultures have been maintained for many years and are not checked or are infrequently checked to determine whether the strains are still capable of toxin production. Yet, the scientific community relies heavily on these cultures for use in research to further understanding about the behavior, physiology, and ecology of toxic algae. To avoid compromise of the validity of scientific insights about the physiology, behavior, and ecology of toxic algae, cultures of all “toxic algae” commercially provided for use by the general scientific community should be tested frequently to verify toxic activity. Laboratories that

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP are relied on to commercially supply cultures for research on toxic algae should be required to obtain fresh field isolates as often as is necessary to ensure that fundamental traits (especially the ability of the strain to sustain toxic activity) are maintained in the cultures. Alternatively, arrangements should be made with a laboratory that specializes in production of clonal cultures with demonstrated toxicity to check cultures of the national phytoplankton center frequently to ensure that they still are capable of toxic activity. The issue of quality control/quality assurance is of major importance in this context. Laboratories that state expertise (e.g., in grant proposals and letters of intent) in providing clonal cultures, techniques, toxin, or other products/services in harmful algal research should be required to provide supporting evidence of expertise in the form of peer-reviewed international science publications on the specific subject, or demonstration of cross-confirmation of their data by a second laboratory with such expertise, or both. Although this stipulation of quality control/quality assurance may seem obvious, it unfortunately is not being required by many federal grant programs in harmful algal bloom research (e.g., indicated in correspondence from ECOHAB-funded scientists expressing concern about the culture quality issue, to the NOAA Coastal Ocean Program, July 1999). Toward the goal of advancing knowledge about toxic strains of algal species—the strains that are germane from the perspective of public concern—assured availability of research-quality cultures is of critical importance and it needs to be more rigorously addressed by the consortium of federal agencies involved. Life Cycles Many of the problems inherent in developing techniques to track harmful algae, beyond established blooms of certain photosynthetic species, are grounded in lack of basic information about their biology. The reality is that scientists do not understand the various forms or stages that many of these species can assume (Burkholder 1998; Table 1). The life cycles are poorly characterized and poorly understood. If the range of forms is not known for many of these taxa, then it is difficult to identify or track them, especially with certain techniques in light microscopy that remain in wide use. For example, of the approximate total of ca. 60 toxic dinoflagellate species (Burkholder 1998), most of the life cycles are incompletely known (Table 2). Many dinoflagellates have animal-like traits (Schnepf and Elbr ächter 1992). About half of the described species are heterotrophs, without their own chloroplasts, and about half are plantlike with chloroplasts. Moreover, many of the plantlike species have well-developed

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP TABLE 1. Toxic Dinoflagellates with Characterized or Partially Characterized Sexual Life Cyclesa Species Gametes Sexual induction Sexual cyst * Alexandrium catenella Heterothallic, isogamous Low N or P + * Alexandrium tamarense Heterothallic?; isogamous, anisogamous Low N or P, increased temperature + Amphidinium carterae Homothallic, isogamous Increased salinity, photoperiod? + Amphidinium operculatum Homothallic?; isogamous (anisogamous) Increased salinity Not known Coolia monotis Homothallic, isogamous Increased nutrients? + Dinophysis acuta Anisogamous? Factors not known Not known Gambierdiscus toxicus Isogamous Factors not known Not known * Gonyaulax monilata Heterothallic?, isogamous Low N + Gymnodinium breve Homothallic or heterothallic, isogamous Factors not known + Gymnodinium catenatum Heterothallic, isogamous Low N and P + Pfiesteria piscicida Heterothallic, anisogamous Dying fish, organic P + Pfiesteria shumwayae sp. nov. Heterothallic?, anisogamous Dying fish, organic P + Prorocentrum lima Homothallic?, isogamous Organic P + Prorocentrum micans Isogamous, anisogamous High nutrients?, temperature shock + Pyrodinium bahamense Heterothallic?, isogamous Factors not known Hypnozygote a Sexual reproduction has been suspected or confirmed in 15 of the ca. 60 toxic dinoflagellates known. Asterisks (*) indicate the 3 species for which the life cycle has been reported as completely characterized. Species are indicated as homothallic (with gametes of the same mating type) or heterothallic (having gametes from different [+,−] types). Question marks (?) indicate observations that are suspected but have not been verified. Flagellated gametes area of similar (isogamous) or different (anisogamous) size; in the case of Prorocentrum micans, gamete protoplasts unite through a mucilaginous tube (conjugation). N indicates nitrogen; P indicates phosphorus. Hypnozygotes are resting-stage products of sexual reproduction. Adapted from Walker and Steidinger 1979; Nakajima and others 1981; Steidinger 1983; Faust 1992, 1993; MacKenzie 1992; Anderson and others 1998 and references therein.

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP TABLE 2. Harmful Estuarine and Coastal Marine Microalgae That Have Been Linked to Anthropogenic Nutrient Enrichment Harmful species Link to cultural eutrophication Chattonella antiqua Bloomed under cumulative high loading of poorly treated sewage and other wastes, coinciding with human population growth (Japan; fish kills, toxic). Chrysochromulina polylepis Toxic outbreaks followed change in nutrient supply ratios from cumulative increased nutrient loading (Europe; fish kills, toxic). Gymnodinium mikimotol Bloomed under cumulative high loading of poorly treated sewage and other wastes, coinciding with human population growth (Japan, as G. nagasakiense; fish kills, PSP). Gonyaulax polygramma Bloomed under cumulative high loading of poorly treated sewage and other wastes, coinciding with human population growth (Japan; fish kills from oxygen depletion). Nocriluca scintillans Bloomed under cumulative high loading of poorly treated sewage and other wastes, coinciding with human population growth (Japan; fish kills from oxygen depletion). Nodularia spumigena Blooms followed change in nutrient supply ratios from cumulative increased nutrient loading by sewage, agricultural wastes (Baltic Sea; estuary in Australia). Toxic Pfiesteria complex (P. piscicida, P. shumwayae sp. nov.) Most kills [with highest cell densities] have occurred in P- and N-enriched estuaries (e.g., near phosphate mining, sewage inputs, or animal waste operations); between kill events, can prey upon flagellated algae that are stimulated by inorganic nutrients; bloomed 1 wk after a major swine effluent lagoon rupture (with extremely high phosphorus and ammonium) into an estuary, in a location where high abundance of these dinoflagellates previously had not been documented; highly correlated with phytoplankton biomass in other eutrophic estuaries (mid-Atlantic and southeastern United States; fish kills, epizootics). Phaeocystis spp. Bloomed following cumulative high loading of poorly treated sewage (Europe; fish—Phaeocystis pouchetii); blooms were correlated with altered N/P ratios from cumulative increased nutrient loading (P. pouchetii); bloomed 1 week after a major swine effluent lagoon rupture into a eutrophic estuary (Phaeocystis globosa, along with Pfiesteria piscicida; southeastern United States).

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP Protocentrium minimum Bloomed under cumulative high loading of poorly treated sewage and other wastes, coinciding with human population growth (Japan; fish kills, toxic); blooms coincide with cumulative high loading of N from sewage, agricultural runoff, atmospheric loading, etc. (southeastern United States). Prymnesium parvum Toxic outbreaks usually have occurred under eutrophic conditions (fish kills). Toxic Pseudo-nitzschia complex species Have occurred with sewage and other wastes (Canada; ASP); consistent seasonal blooms in the Mississippi and Atchafalyu River plume areas, associated with hypereutrophic conditions and in Prince Edward Island, Canada, following anthropogenic nutrient loading and drought).† † Note that many of the known harmful estuarine and marine microalgae and heterotrophic or animal-like dinoflagellates also have been shown to be stimulated by N and/or P enrichment in culture, which is expected since they are photosynthetic. Also note that blooms of the toxic Pseudo-nitzschia complex have not been associated with cultural eutrophication in the northwestern United States. Adapted from Burkholder 1998 and references therein. heterotrophic capabilities (Hansen 1998). Some of these species have proven difficult or not yet possible to grow or maintain in culture, probably because unknown organic substances needed for growth are not available in laboratory conditions. Similarly, some toxic chrysophytes are known to have amoeboid stages that, thus far, have not been successfully maintained in culture (e.g., Estep and McIntyre 1989). As additional examples, other harmful (but not toxic) dinoflagellates include certain species that parasitize finfish, shellfish, zooplankton, and benign algae (Cachon and Cachon 1987). The life cycles of most parasitic dinoflagellates are completely unknown (Cachon and Cachon 1987; Pfiester and Popovský 1978). Many of the species remain to be described from one to two stages that can be recognized to date. Successful culture requires the prey, which complicates cloning procedures, especially if the prey are larger organisms (e.g., fish) with a suite of associated contaminating microorganisms and if fresh prey must continually be supplied to the parasites, so that prey sterilization becomes impractical. The environmental requirements of many of the free-living life cycle stages of most parasitic species are poorly understood. Additional information is needed for development of suitable culture media. Thus, for some harmful algae, culture media needed for successful growth and maintenance of the various life stages have not yet been developed. Obviously, such limitations translate into severe restrictions

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP in what scientists are presently able to learn about these organisms under experimental laboratory conditions. A related problem merits mention. Historically, research on toxic algae was conducted using defined culture media that had been developed to grow photosynthetic species, that is, strict autotrophs or auxotrophs. Unfortunately, such media narrowly constrained these species so that their heterotrophic behavior was missed (e.g., Jacobson and Anderson 1996). The type and abundance of available food sources have been shown to strongly influence the stages or forms that are present in the (few) toxic dinoflagellates that have been rigorously examined with an array of potential food sources (e.g., Burkholder and Glasgow 1997). Thus, in restricting the nutritional mode of these organisms, various stages in their life cycles may not have manifested (Popovsk ý and Pfiester 1990). Characterization of the life cycles of many harmful algal species is a critical research need. This information is of fundamental importance to enable scientists to determine ecological controls on bloom dynamics (e.g., nutrient enrichment; Table 3) and to design improved techniques for tracking both planktonic and benthic stages of these organisms. Techniques that may be especially useful in addressing this critical need include the following: Low-pressure (high-vacuum) scanning electron microscopy enables live samples to be viewed so that in-progress transformations can be observed at high resolution. Additional gene probes and other markers discern various stages (e.g., green fluorescent probes). Fluorescently labeled molecular probes are useful for discerning species of interest among many other species and assorted “debris” in field samples (e.g., Scholin 1998a). Gene-specific and other toxin probes are critically needed for many harmful species (also see below). Such probes would be of value in verifying toxic stages within the life cycles of harmful species that, in turn, would enable determination of the range of stages that are most important to detect and track. The various probes require greater speed in application, more automation, and more amenability to field use than current techniques. A note of caution is warranted: Although molecular probes used for species identifications are often considered as species-specific, the possibility remains that the probes will cross-react with closely related species that have not yet been tested (Gallagher 1998). Thus, use of probe technology for establishing species identifications should be cross-confirmed with scanning electron microscopy of morphological traits whenever possible, at least on a “spot-check” basis.

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP TABLE 3. Status of General Knowledge about Toxic Algaea Feature Toxic dinoflagellates Toxic chrysophytes Toxic blue-greens (cyanobacteria) Life cycles ~ 60 species, many incomplete or unknown; complex life cycle in 2 spp. (Pfiesteria); but heterotrophic food sources (aide in detecting complex life cycles) not yet tested for most species ~ 15 species; 1 of 3 Pseudo-nitzschia spp. known; 9 chrysophyte spp. poorly or incompletely known (complex life cycles, amoeboid stages in some spp.); 2 of 8 raphidophyte spp. known ~ 40 species, with simplistic (prokaryotic) life cycles known or suspected in most Toxins > 50 known; 21 saxitoxins + derivatives characterized; 9 brevetoxins characterized; 4 ciguateratoxins well characterized (many poorly known), 4 DSP toxins characterized; ≥ 2 Pfiesteria toxins partially characterized In diatoms, domoic acid + isomers characterized; in chrysophytes, several galactolipids, octadecapentaenoic acid, prymnesins, and other uncharacterized toxins; in raphidophytes, hemolytic substances (e.g., poly-unsaturated fatty acids), superoxide radicals mostly poorly characterized; other uncharacterized toxins > 70 known including > 50 microcystins, 3 anatoxins, 16 saxitoxins, 5 opsins, 2 cylindrosperm opsins, 1 nodularin, 1 ciguatera-like toxin Purified toxins [availability] Major saxitoxins, brevetoxins commercially available; okadaic acid + several other DSP toxins and derivatives available Domoic acid commercially-available Major microcystins, anatoxins, saxitoxins, and nodularin commercially available Toxin assays (for rapid field or laboratory use) Saxitoxins, brevetoxins as groups (major forms); more limited ciguatoxin assays Domoic acid commercially available Major microcystins saxitoxins as groups; nodularin a Diatoms are considered here within division Chrysophyta as class Bacillariophyceae. Adapted from Falconer 1993a; Hallegraeff and others 1995; Burkholder 1998 and references therein; and Chorus and Bartram 1999 and references therein.

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP laboratory data. Unfortunately, there have been no medical or epidemiological studies to determine whether such impacts are occurring in human populations chronically exposed to okadaic acid. In fact, such studies have not been conducted for most algal toxin exposures. A second general problem in assessing range of impacts is that, as mentioned, many algae toxins remain only partially characterized, without available purified standards or detection assays (Fairey and others 1999; Falconer 1993a; Hallegraeff and others 1995). In addition to the critical need to fully characterize more of the toxins from harmful algae, assays are also greatly needed to enable rapid, routine, reliable detection of these toxins in potable water supplies, natural waters, seafood, and aquaculture facilities. A probe for domoic acid, for example, together with species-specific molecular probes to verify the presence of the toxic algae that produce it, was valuable in relating the recent sea lion disease and die-off in California to diatoms in the toxic Pseudo-nitzschia complex (Scholin and others 2000). Although assays for the better known toxins—saxitoxins, brevetoxins, okadaic acid and certain other (DSP) toxins, and certain ciguateratoxins—are commercially available (e.g., Hallegraeff and others 1995), they are limited in ability to reliably detect more than a few of the toxins that are targeted. In other words, the commercially available assays for saxitoxins cannot be used to detect all of the major saxitoxins; those available for DSP toxins fail to detect all of the major DSP toxins; and so forth. Other limitations in quantification or specificity have led state and federal agencies involved in seafood safety issues to forego relying on these assays in favor of the traditionally used (but less sensitive) mouse bioassay (Cembella and others 1995). Concerted research to develop improved rapid, reliable assays for detecting algal toxins is critically needed to advance understanding about impacts from harmful algae on human health and natural resources. Scientists must also strengthen insights about how these toxins can be harnessed more effectively for beneficial medicinal use. For example, Pfiesteria toxins have been reported to cause profound learning disabilities manifested as short-term memory loss in mostly reversible impacts (Glasgow and others 1995; Grattan and others 1998), with indication of involvement of the hippocampus as a target region of the central nervous system. They also have been experimentally shown to cause severe learning disabilities in small mammal studies (rats; Levin and others 1999). These toxins could be of value in research to advance understanding of human memory function, but they are, as yet, incompletely characterized (Fairey and others 1999). Their chemical structures (identity) must be obtained before modes of action in affecting human health can be determined with certainty.

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP Detection of Toxic Strains Although various techniques are in hand (e.g., immunoassays, enzyme assays, neuroreceptor binding assays, cytotoxicity assays) to detect toxic strains in laboratory cultures, the available technology is much more limited for use in detecting and tracking toxic strains within mixed field populations. Molecular probes, where available, can detect the species but cannot discern toxic status. Assays for rapid detection of certain toxins have been developed, as mentioned —although with limitations (Table 3). These assays enable detection of the presence of toxin in waters, shellfish tissues, and other materials that are directly sampled (Hallegraeff and others 1995). However, in practical use the available assays do not make it possible to discern between toxic and nontoxic strains in natural phytoplankton or benthic algal samples. If the toxic members within a population could be tracked in field conditions, insights about environmental controls on the toxic strains, which are of primary interest in natural resource and health issues, could be strengthened. Scientists could better understand how a range of organisms across aquatic food webs is exposed. Impacts of exposure could be more accurately tracked through seafood (Falconer 1993a) and through food webs (e.g., Shumway 1995), and improved diagnostics for human health effects could be designed. Thus, additional techniques to enable detection and tracking of toxic strains in field populations are needed. PRESENT STATUS OF CONTROL AND PREDICTION Basic research on strategies to control harmful algal blooms has been limited, but such research remains in primitive status (Boesch and others 1997). Many harmful algae are detected in a reactive rather than proactive response mode, in part because of the sporadic occurrence of these species, and scientists are currently faced with the problem of attempting to develop control strategies for species that they basically know very little about—at least, for many harmful algae. Each of the three standard types of control strategies—physical or mechanical, chemical, and biological—have been considered for controlling harmful algal blooms. In general, they have not worked well except in limited situations. For example, physical mixing of the water to disrupt the density-based stratification has been used to minimize noxious freshwater blue-green algal (cyanobacterial) growth in ponds and small lakes, and sometimes in aquaculture facilities (Ross and Lembi 1999). As another example, death of striped bass was averted in coastal aquaculture facilities in initial stages of toxic Pfiesteria outbreaks by rapidly replacing the water (and thereby removing the toxic dinoflagellates) in brackish ponds where the

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP fish were being grown. In most larger lakes, estuaries, and coastal marine waters, such techniques generally have not been feasible. There are a few notable exceptions; for example, salmon pens simply have been moved away from waters containing harmful diatom or toxic flagellated chrysophyte blooms (Boesch and others 1997). In the realm of chemical controls, poisons such as copper sulfate have been applied for many years in small lakes, and in some aquaculture facilities under limited circumstances, to control noxious freshwater cyanobacteria (Ross and Lembi 1999). Bleach pellets have been added to the bottom-accumulated sediments in drained coastal ponds and aquaria to eliminate cysts of Pfiesteria, before addition of cultured striped bass and flounder. In most larger lakes, estuaries, and coastal marine waters, such physical and chemical techniques generally have not been feasible. As an additional problem, although many kinds of chemical poisons can be used to kill harmful algae, they are not species-specific. Thus, their use is usually impractical because many beneficial, cooccurring species would also be destroyed (Taylor and Pollingher 1987). Another type of chemical control, reduction of nutrient pollution (although more difficult to accomplish for socioeconomic reasons) has proven highly successful in minimizing the growth of certain harmful algal species. The best success incidents have been documented for toxic freshwater cyanobacteria in which phosphorus reduction to lakes (varying in size from small lakes to Lake Erie) has significantly reduced growth of the undesirable algae (Wetzel 1983). In small ponds, the same effect sometimes has been accomplished by adding nitrogen fertilizer to increase the N:P ratio and encourage growth of desirable green algae (with high nitrogen optima) rather than noxious cyanobacteria with high phosphorus requirements (Ross and Lembi 1999). In certain poorly flushed estuaries and marine coastal embayments, some harmful algal species have been linked to stimulation by nutrient pollution (Table 2). As a result, long-term strategies targeting nutrient reductions are under consideration or, in the case of the Chesapeake Bay and Pfiesteria, are being imposed (State of Maryland 1998). Biocontrol may be the most promising of control strategies, but it remains the least understood. Certain cyanophages are under consideration for control of noxious cyanobacteria under limited conditions (e.g., certain cyanobacteria strains in small ponds; Lembi and others 1988). A virus with potential for reducing blooms of brown-tide organisms has been discovered (Milligan and Cosper 1994). A dinoflagellate from the Pacific Northwest was found to attack a certain harmful dinoflagellate species under culture conditions, but the latter species occurs in the southeastern United States (Taylor 1987). The beneficial versus detrimental effects of attempts to introduce the dinoflagellate from the Northwest to

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP the Southeast, and the feasibility of being able to achieve success in controlling the southeastern species through this effort, are unknown. Recently there has been much discussion about the use of clay additions to control harmful algal species, a practice that, although primitive, has been used with success in certain aquaculture operations in the Orient (Anderson 1997; Pérez and Martin 1999). Clay particles adhere to the mucilage of certain algal species (e.g., montmorillonite clays adsorb cyanobacteria; reviewed in Burkholder 1992). The organisms coflocculate or settle out with the clay, and some species of algae can be killed in this way. Major concerns in using such control techniques are impairment of shellfish feeding, clogging of zooplankton apparati, and clogging of finfish gills. Aside from attempts to minimize these potentially serious impacts, the degree of success of such an approach depends on the type of clay used and the algal species in question. Some harmful algal species such as cyanobacteria are susceptible to reduction by clay additions, whereas others are not. Noxious freshwater species of Anabaena (including toxic Anabaena circinalis and A. flos-aquae) were highly susceptible to sedimentation and subsequent death with certain clays such as montmorillonite (Avnimelech and others 1982; Burkholder and Cuker 1991). However, addition of another clay common to the area, kaolinite, proved beneficial to these organisms, which grew well after settling out (Burkholder and Cuker 1991). The algae apparently benefited, as well, from high phosphorus supplies that were adsorbed to the clay. In contrast, Yu and others (1995) reported that dinoflagellate species Prorocentrum minimum (sometimes toxic to shellfish; see review by Landsberg 1996) and nontoxic Noctiluca scintillans were more adversely affected by coagulation with kaolinite than with montmorillonite. Some “naked” (unarmored) dinoflagellate species lacking protection from thick cell wall-like coverings appear to be especially vulnerable to cofloccuation with clays (e.g., various Gymnodinium and Gyrodinium species), and if they adsorb directly to the clay particles, they are destroyed (Burkholder 1992). However, dinoflagellates have a remarkable ability to rapidly form temporary cysts (Taylor 1987). The naked species rapidly excrete copious mucilage that surrounds the cells. At the same time, the organisms take up nutrients that were adsorbed to the clay particles. In this way, some species actually appear to derive benefit from the clay (Burkholder 1992). If there is a small area on the outer thick, mucilaginous cell covering that is left uncovered by the clay, then once the water column is cleared, the dinoflagellate protoplast emerges through that area. Armored dinoflagellates use their outer covering of cellulose plates with membranes as a protective barrier, and “molt” the outer covering with adsorbed clay once the water column is clear. Thus, many algae can survive clay-loading events and apparently can actually benefit from nu-

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP trients adsorbed to the clay particles. Such mechanisms for survival and benefit are logical especially in estuaries and certain turbid coastal environments. Clay applications can work under limited conditions in certain circumstances but may cause other problems inasmuch as they can promote potentially serious detriment to desirable aquatic life such as sensitive filter-feeding shellfish species (e.g., Howell and Shelton 1970). In discouraging but realistic writing, Boesch and others (1997) made the following statement in a publication that was cosponsored by NOAA and the National Fish and Wildlife Foundation: “It is premature to conclude whether control strategies are feasible, applicable or advisable because there is insufficient information to judge effectiveness and weigh benefits against costs.” Thus, control is in primitive status within the realm of harmful algal blooms. Prediction obviously is in similar status, given scientists' fundamental lack of knowledge about many harmful algal species and the current limited technologies for recognizing and tracking toxic strains. Some known conducive environmental conditions can be tracked; however, many of the factors that influence these blooms, especially the nutritional ecology of the algal species and controlling biological interactions, are not known. The exception of progress in prediction is cyanobacteria blooms in freshwater ecosystems (Wetzel 1983). Nonetheless, the degree of toxicity of these bloom formers is difficult to predict. Toxicity can be highly variable from strain to strain within the same cyanobacteria bloom (Gorham and Carmichael 1988), also true of toxic prymnesiophytes, toxic diatoms, and some toxic dinoflagellates as mentioned. Moreover, the environmental signals that trigger toxicity are unknown for nearly all toxic species. SUMMARY The most important basic challenges in the field of harmful algal bloom research are, first, to fundamentally ensure that research-quality cultures are available for use by the scientific community at large, which presently is not the case, especially for toxic algal species and despite the intent of federal consortium agencies engaged in supporting such effort. Second, the life cycles of many of these species need to be characterized so that they can be recognized and tracked in various stages. Armed with that information, scientists will be able to determine much more about their occurrence and behaviors. Third, more of the toxins and toxin derivatives from these organisms need to be identified so that assays can be developed to track the toxins through the food web, to improve diagnostics for human health exposure and animal exposure, and to determine modes of action. Effective medical treatment will remain beyond reach

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP until the modes of action (i.e., the metabolic pathways in which these toxins function) are understood—information that can be obtained with certainty only after the toxins are identified. These data would also enable scientists engaged in medical research to more effectively harness the potential beneficial uses of the toxins. Fourth, improved techniques are needed for detecting toxic strains among field populations of these organisms, which typically have benign (non-toxin-producing) as well as toxic strains that are physiologically and behaviorally distinct. Until scientists know much more about the life cycles and toxins of these organisms, harmful algae will remain in the realm of the enigmatic, difficult for the public to understand. Without the fundamental information that can be provided only through the critically needed research that was identified here, the panic that is fostered by lack of understanding (e.g., “economic halo effects” as described in Epstein 1998) will continue to occur—along with all of the hardship that such panic creates wherever people depend heavily on the affected freshwater, estuarine, and marine resources for economic sustainability. REFERENCES Anderson DM. 1991 Toxin variability in Alexandrium. In: Granél E, Sundstrom B, Edler L, Anderson DM, eds. Toxic Marine Phytoplankton. New York: Elseview. p 41-51. Anderson DM. 1997 Turning back the harmful red tide. Nature 388:513-514. Anderson DM, Cembella AD, Hallegraeff GM, eds. 1998 Physiological Ecology of Harmful Algal Blooms. NATO ASI Series G. Ecological Sciences. Vol 41. New York: Springer-Verlag. Aune T, Yndestad M. 1993 Diarrhetic shellfish poisoning. In: Falconer IR, ed., Algal Toxins in Seafood and Drinking Water. New York: Academic Press. p 87-104. Avnimelech Y, Troeger BW, Reed LW. 1982 Mutual flocculation of algae and clay: Evidence and implications. Science 216:63-65. Bagnis R. 1993 Ciguatera fish poisoning. In: Falconer IR, ed. Algal Toxins in Seafood and Drinking Water. New York: Academic Press. p 105-115. Bates SS, Garrison DL, Horner RA. 1998 Bloom dynamics and ecophysiology of domoic acid-producing Pseudo-nitzschia species. In: Anderson DM, Cembella AD, Hallegraeff AD, eds. Physiological Ecology of Harmful Algal Blooms. NATO ASI Series G. Ecological Sciences. Vol. 41. New York: Springer-Verlag. p 267-292. Boesch DF, Anderson DM, Horner RA, Shumway SE, Tester PA, Whitledge TE. 1997 Harmful Algal Blooms in Coastal Waters: Options for Prevention, Control and Mitigation. Washington, DC: US Department of Commerce and US Department of the Interior. Burkholder JM. 1992 Phytoplankton and episodic suspended sediment loading: Phosphate partitioning and mechanisms for survival. Limnol Oceanogr 37:974-988.

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP Steidinger KA, Burkholder JM, Glasgow HB Jr, Hobbs CW, Truby E, Garrett J, Noga EJ, Smith SA. 1996 Pfiesteria piscicida gen. et sp. nov. (Pfiesteriaceae, fam. nov.), a new toxic dinoflagellate genus and species with a complex life cycle and behavior. J Phycol 32:157-164. State of Maryland. 1998 Water Quality Improvement Act of 1998. Annapolis, MD: Maryland General Assembly. Taylor FJR. 1987 Dinoflagellate morphology. In: Taylor FJR, ed. The Biology of Dinoflagellates. Botanical Monographs. Vol 21. Boston: Blackwell Scientific Publications. p 24-92. Taylor FJR, Pollingher U. 1987 Ecology of dinoflagellates. A. General and marine ecosystems. In: Taylor FJR, ed. The Biology of Dinoflagellates. Botanical Monographs. Vol 21. Boston: Blackwell Scientific Publications. p 398-502. Walker LM, Steidinger KA. 1979 Sexual reproduction in the toxic dinoflagellate Gonyaulax monilata. J Phycol 15: 312-315. Wetzel RG. 1983 Limnology. Philadelphia: Saunders. WHOI [Woods Hole Oceanographic Institute]. 2000 Glossary of Pfiesteria-Related Terms. Pfiesteria Interagency Coordination Work Group, chaired by J. Macknis, US EPA, Baltimore, MD. Available at <http://www.redtide.whoi.edu/pfiesteria/documents/glossary.html>. Yu Z, Zou J, Ma X. 1995 Application clays for removal of red tide organisms. III. The coagulation of kaolin on red tide organisms. Chinese J Oceanol Limnol 13: 62-70.