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HARMFUL ALGAL BLOOMS

Th e Role of in the Global Proliferation of Harmful Algal Blooms

NEW PERSPECTIVES AND NEW APPROACHES

BY PATRICIA M. GLIBERT, SYBIL SEITZINGER, CYNTHIA A. HEIL, JOANN M. BURKHOLDER, MATTHEW W. PARROW, LOUIS A. CODISPOTI, AND VINCE KELLY

Th is article has been published in Oceanography, Volume 18, Number 2, a quarterly journal of Th e Oceanography Society. Copyright 2005 by Th e Oceanography Society. All rights reserved. Reproduction of any portion of this article by photo- 198 Oceanography Vol.18, No.2, June 2005 copy machine, reposting, or other means without prior authorization of Th e Oceanography Society is strictly prohibited. Send all correspondence to: [email protected] or Th e Oceanography Society, PO Box 1931, Rockville, MD 20849-1931, USA. Eutrophication is now recognized to be one of the important factors contributing to habitat change and to the geographical and temporal expansion of some harmful species...

Cultural eutrophication, the of coastal waters by for example. Eutrophica- , is a result of population growth, food production tion is now recognized (, animal operations and ), and energy to be one of the impor- production and consumption, and is considered one of the tant factors contribut- largest pollution problems globally (Howarth et al., 2002). Pop- ing to habitat change ulation growth and food production result in major changes and to the geographical to the landscape, in turn, increasing discharges and and temporal expan- runoff from agriculture and populated lands. In addition to sion of some harmful algal population growth, eutrophication arises from the large in- bloom (HAB) species (Smayda, creases in the use of chemical that began in the 1950s 1990; Anderson et al., 2002). and which are projected to continue to escalate in the coming Some of the clearest examples of the relationship between decades (Smil, 2001). Both and are of HAB frequency and increases in total loadings to concern in eutrophication, but nitrogen has received far more coastal waters come from China. Since the 1970s, when escala- attention because it often limits in estuar- tion in use of chemical began in China, the number ies and coastal waters and because the global application of ni- of HAB outbreaks has increased over 20-fold, with blooms that trogen from synthetic fertilizers is far greater than that of phos- now are of greater geographic extent, more toxic, and more phorus (more information available at www.ut.ee/~olli/eutr/). prolonged (Anderson et al., 2002). Other examples of a positive Although cultural eutrophication is occurring globally, nu- relationship between nutrient loading and HAB outbreaks can trient export is not evenly distributed (Seitzinger et al., 2002a). be found throughout the world. In northern European waters, For example, inorganic nitrogen export to coastal waters is blooms of the mucus-forming HAB species Phaeocystis globosa estimated to be highest from European and Asian lands, al- have been shown to be directly related to the content though signifi cant discharge also occurs from the United States of riverine and coastal waters (Lancelot, 1995). In the United and other parts of the world. Furthermore, the rate of nutrient States, a relationship between increased nutrient loading from export to coastal waters has increased dramatically in recent the Mississippi to the Louisiana shelf and increased abun- years in some parts of the world. China, which consumed less dance of the toxic Pseudo-nitzschia pseudodelicatissima than 5 million tonnes of nitrogen fertilizer in the 1970s, now has been documented from examination of the diatom frus- consumes more than 20 million tonnes per year, representing tules preserved in cores (Parsons et al., 2002). 25 percent of global nitrogen fertilizer consumption (Figure 1) Indeed, strong positive relationships have been reported be- (more information available at www.fertilizer.org), leading to tween increasing human population and the intensity of some signifi cant increased nitrogen pollution of its coastal waters. HAB outbreaks. In , in the U.S. Pacifi c Northwest, Global increases in population growth and food production, where consistent shellfi sh monitoring has been carried out for and consequently eutrophication, have important impacts on decades, a strong positive relationship between the frequency of the environment, affecting the microbiota of adjacent waters, one algal , paralytic shellfi sh toxin (PST), and the growth

Oceanography Vol.18, No.2, June 2005 199 in human population has been reported One mechanism by which eutrophica- nutrient ratios, such as nitrogen:phos- (Trainer et al., 2003) (Figure 2). In this tion can shift coastal community struc- phorus (N:P) or nitrogen:silicon (N:Si) case, eutrophication was considered to ture toward the promotion of HAB spe- (e.g., Smayda, 1990; 1997), and some be one of the important factors contrib- cies is by changing the ratio of individual successes have been achieved in relating uting to the increased incidence of PST nutrients relative to other nutrients. shifts in these ratios to HAB outbreaks as intensive monitoring efforts have been Many studies have examined the rela- (Anderson et al., 2002). In Tolo Harbor, in place for many decades. tionships between HABs and inorganic , during the 1980s, the molar ratio of N:P decreased nearly 50 percent as both human population and sewage loading increased several-fold and, con- comitantly, the numbers of dinofl agel- late HAB outbreaks increased almost Contribution by global region to the consumption of the ten-fold (Lam and Ho, 1989). Altered world’s annual current use of 85 million tonnes of nitrogen nutrient ratios also have been correlated with shifts from diatom-dominated to others North fl agellate-dominated assemblages in America

China Latin Amer. some U.S., European, and Asian coastal waters. Even on short time scales, chang- W. ing nutrient ratios can be related to HAB outbreaks, as in Tunisian aquaculture East where toxic dinofl agellates de- Central South Asia Europe velop seasonally when N:P ratios change (Romdhane et al., 1998). While these examples demonstrate the relationship of several HAB species with Estimate of the increase in nitrogen both total nutrient loading or changes in fertilizer consumption through the Year 2030 specifi c nutrient ratios, many other dino- fl agellate species involved in HAB events have not appeared to follow these pat- terns, leading to much confusion about the role of eutrophication in their devel- 100 opment. One of the diffi culties in linking 80 nutrient loading to HABs is the multi-

60 2030 plicity of factors contributing to HAB species responses to nutrient loading. 40 2000 The inability to universally apply a single 20 Million Tonnes 1970 criterion, such as the N:P (or other nutri- 0 ent) ratio, to determine whether eutro- Asia and N & S W. Central N. America Europe Europe phication is stimulating HABs does not negate the utility of this approach. Rath- Figure 1. Th e use of nitrogen fertilizer varies considerably around the globe. Of the er, it underscores the interdependence of 85 million tonnes of nitrogen consumed annually, Asia uses about half. Th e greatest projected increases in global nitrogen fertilizer are also expected to be in Asia over environmental factors, physiological fac- the next 25 years. Data from the International Fertilizer Industry. tors, and trophic interactions in the out-

200 Oceanography Vol.18, No.2, June 2005 come of any species succession, as well as 6000 the importance of appropriately scaled temporal and spatial data (Glibert et al., 5000 this issue). The old paradigm (that HABs are stimulated by nutrient over-enrich- 4000 ment only if there is a direct and visibly evident increase in their ) is giv- 3000 ing way to an appreciation for the fact that nutrient enrichment can also indi- 2000 Average Decadal Average Maximum PST rectly stimulate HABs in more subtle but 1000 signifi cant ways over the long term—for example, by increasing that, in 0 turn, eliminates fi lter-feeding shellfi sh 01234 that would have consumed HABs but Population (Millions) that can no longer survive in the habitat Figure 2. An example of the relationship between population growth and HABs. (Burkholder, 2001). Th ese data are from Puget Sound, , USA, where continuous monitoring of paralytic shellfi sh poisoning has been ongoing since the mid 1950s. Plotted here INORGANIC NUTRIENTS ARE is the relationship between human population in the region for the past 40 years (data were derived from the U.S. census) and the average decadal maximum record- NOT THE ONLY NUTRIENT ed amount of paralytic shellfi sh (PST). Replotted from Trainer et al. (2003). SOURCE FOR MANY HABS Inputs of the major nutrients include both inorganic (ammonium, nitrate, nitrite, , silicate) and organic forms (dissolved organic and particulate matter). The magnitude and changing ganic material and manures is increasing duction and application statistics, it is composition of nutrients, both inorganic at a greater rate (Smil, 2001). Worldwide now estimated that represents more and organic, discharged to the coastal use of urea as a nitrogen fertilizer and than 50 percent of world nitrogen fertil- zone are beginning to be better under- feed additive has increased more than izer use, a dramatic global change in the stood and quantifi ed. For example, about 50 percent in the past decade (Glibert composition of nitrogen in one decade half of the dissolved nitrogen trans- et al., in press). Based on fertilizer pro- (Smil, 2001; Glibert et al., in press). The ported by to the coastal is in the form of dissolved organic nitrogen Patricia M. Glibert ([email protected]) is Professor, University of Maryland Center (DON) (Meybeck, 1982). Dissolved or- for Environmental , Horn Point Laboratory, Cambridge, MD, USA. Sybil Seitzinger ganic matter (DOM), the more inclusive is Visiting Professor, Rutgers University, Institute of Marine and Coastal , Rutgers/ term for organic material containing NOAA CMER Program, New Brunswick, NJ, USA. Cynthia A. Heil is Senior Research Scien- nutrients as well as carbon, is exported tist, and Wildlife Research Institute, Fish and Wildlife Conservation Commission, from all natural and anthropogenic land St. Petersburg, FL, USA. JoAnn M. Burkholder is Professor and Director, Center for Applied uses in watersheds, but the magnitude Aquatic , North Carolina State University, Raleigh, NC, USA. Matthew W. Parrow is and composition of this export is chang- Postdoctoral Research Associate, Center for Applied Aquatic Ecology, North Carolina State ing as land use changes. While fertilizer University, Raleigh, NC, USA. Louis A. Codispoti is Research Professor, University of Mary- use throughout the world is increasing land Center for Environmental Science, Horn Point Laboratory, Cambridge, MD, USA. Vince (Vitousek et al., 1997), the fraction of Kelly is Senior Research Assistant, University of Maryland Center for Environmental Science, total fertilizer that is composed of or- Horn Point Laboratory, Cambridge, MD, USA.

Oceanography Vol.18, No.2, June 2005 201 development of concentrated animal to HAB nutrition is also now beginning cies can consume (phagocytize) other feed operations in many countries, many to be recognized. Many HAB species rely living organisms or substances produced located near coastal areas, has also led to strictly upon for their by them, as illustrated in Figure 3. Many massive amounts of organic waste pro- energy and use inorganic nutrients (e.g., of the physiological and behavioral duced in relatively small areas of land nitrogen, phosphorus, carbon). Other mechanisms involved in these modes of that cannot absorb it over the long term species, however, use not only photosyn- nutrition are diffi cult to quantify, and (Mallin, 2000). thesis but also dissolved or particulate even more diffi cult to relate to species The mode of delivery of both inor- organic nutrients for some or all of their shifts or dominance. Nevertheless, the ganic and organic nutrients (especially nutrient demands (e.g., Granéli et al., list of HAB species with reported mixo- nitrogen) is not restricted to aquatic 1999; Stoecker, 1999). Organic nutrients trophic capability is long, and includes sources; atmospheric input is also im- have been shown to be important in species such as Chrysochromulina polyl- portant. In many estuarine and coastal the development of blooms of various epis, Dinophysis norvegica and D. acumi- waters the atmosphere may contribute HAB species, in particular cyanobacte- nata, Pfi esteria spp., and Prorocentrum up to 40 percent of the total inorganic ria and dinofl agellates (e.g., Paerl, 1988; minimum and Karlodinium micrum. Yet, plus organic nitrogen inputs (Howarth Glibert et al., 2001), and the impor- the extent to which they eat depends on et al., 2002), and local emissions near tance of this phenomenon is beginning other factors that regulate their nutri- large-animal operations and feedlots to be documented around the world. A tion. Prorocentrum minimum, for exam- may be even greater. It has been further recent study found that in ple, appears to become proportionately demonstrated that DON from atmo- the cyanobacterial fraction of the algal more mixotrophic when it becomes spheric deposition can stimulate coastal community was positively correlated inorganic nutrient-limited (Stoecker et production (Seitzinger with the fraction of nitrogen taken up al., 1997); however, it can take up some and Sanders, 1999), but much remains to as urea, and negatively correlated with organic nutrients, including humic sub- be understood in terms of the role of this the fraction of nitrogen taken up from stances, when supplied, regardless of nutrient source in HAB proliferation. the inorganic source, nitrate (Glibert et nutrient status (Granéli et al., 1985). As The chemical composition of DOM al., 2004). Other studies have shown that would be expected for most organisms exported from agricultural watersheds is some grow faster on urea that feed, feeding activity by Pfi esteria not known, but up to 50 percent can be than on other nitrogen sources (e.g., spp. decreases as the cells become en- taken up directly or indirectly by estua- Berman and Chava, 1999). gorged with prey. rine communities (Seitzinger In addition to direct uptake, many The complex mixture of organic et al., 2002b). The chemical composi- phytoplankton have other mechanisms nutrients in the environment can also tion, bioavailability, and effects of DOM to acquire nutrients. These phytoplank- have other stimulatory effects on HABs. on coastal plankton communities vary ton are referred to as mixotrophs (i.e., Organic nutrients may stimulate bacte- depending on the source of the DOM, species that “mix” their strategies for ria or other heterotrophs that, in turn, the plankton community composi- taking up nutrients). They use complex can stimulate the development of het- tion, and the season. We are beginning organic molecules for both nutrients erotrophic or mixotrophic HAB species to recognize the magnitude of export and energy, and their pathways for ob- by favorably altering the micro-environ- of DOM, but quantifying its variable taining these nutrient forms include ment. Thus, organic forms of nutrients composition and the extent to which it extracellular oxidation and hydrolysis may have many sources, from synthetic is contributing to eutrophication effects (Figure 3). Some phytoplankton, in- fertilizer, to natural terrestrial sources, within coastal environments is one of cluding an increasing number of HAB to release by other organisms. Organic the large challenges facing oceanogra- species, are now also recognized as able nutrients are, in turn, used by many phers today. to acquire their nutrients or energy via HAB species that have multiple acquisi- The signifi cance of organic nutrients ingestion: they eat! These spe- tion mechanisms.

202 Oceanography Vol.18, No.2, June 2005 STRAINS ARE NOT CREATED markedly in fundamental traits such as differences between strains of the toxi- EQUAL growth characteristics, , bloom- genic dinofl agellate Gymnodinium cat- Many generalizations about how HAB forming behavior, and responses to nutri- enatum in their requirement for the mi- species respond to nutrient enrichment ents and other environmental conditions. cronutrient, selenium. Strom and Bright have been based upon culture studies of Consideration of strain variations (2003) found that strains of the dimeth- only one or a few populations, or strains, within a given HAB species is fundamen- ylsulphide-producing haptophyte Emili- of a given species, cultured from a single tally important because many strains ania huxleyi differed in their growth cell isolated from the natural environ- can occur in the natural environment response when given inorganic (nitrate, ment. It is often assumed that the char- and because laboratory studies using ammonium) and organic (amino acids) acteristics of one strain maintained for cultured strains are heavily relied upon nitrogen sources. All four strains tested years under highly artifi cial laboratory to interpret, and to predict, how natural grew on nitrate and ammonium, but one conditions are representative of all strains populations respond to eutrophication. strain grew only on the inorganic nitro- of that species in the natural environ- Among many examples of signifi cant gen sources and, apparently, could not ment. Not so. Different strains within the differences among strains in response to utilize the organic nitrogen sources. The same species have been shown to differ nutrients, Doblin et al. (2000) described other three strains showed growth when

Urea Amino acids/proteins NO3 + NH 4 NO2 Urea + ? NO NH4 3 ? NO 2 Other NH + DON 4 Compounds? + Protein synthesis NH4 Figure 3. Schematic of the various pathways by which HABs may acquire their nitrogen. All phytoplankton can Particle nitrate, ammonium, or urea across cell membranes via passive dif- ingestion fusion or active transport. Some cells can transport large organic molecules across their cell membrane, and some cells have cell surface enzymes for breaking down larger organic molecules before transporting the nitrogen. Only a few cells have the ability to phagocytize other cells and particles. Although all possible pathways are shown, this is not intended to imply that all cells have all capabilities. Pathways that in- volve enzymatic reactions are indicated with a red circle. Pathways for which there is much uncertainty are indicated with a question mark.

Oceanography Vol.18, No.2, June 2005 203 given amino acids, but differed in the tant in maintaining dominance of one carbon or nitrogen, analyses of a few amino acids that supported growth— species versus stimulating competitor or individual compounds (e.g., urea, amino two of the strains grew on glutamine but consumer species. For example, whether acids, specifi c proteins), or compound not alanine, while the third strain grew nutrients are delivered in the spring with classes that account for a small percent on alanine but not glutamine. Such data freshwater runoff or in the summer (as is of total DOM (e.g., total carbohydrates, show the importance of including mul- the case in dry years) will impact down- total proteins)—ESI-MS permits exami- tiple strains in research to understand stream total accumulation, as graz- nation of a large suite of individual com- the nutritional ecology and physiology ers are more likely to be poised for rapid pounds in natural mixes of DOM. Ultra- of HAB species. algal consumption during the warmer fi ltration techniques have been used to months. In addition, it is important to characterize DOM by broad molecular PHYSIOLOGY IS COMPLICATED consider whether nutrients are delivered weight fraction, but such approaches do BY ENVIRONMENTAL in a pulsed manner, such as that which not allow characterization or identifi ca- CONDITIONS, TIMING, AND would follow heavy rainfall, or a more tion of individual compounds. ESI-MS TROPHIC INTERACTIONS continuous manner, such as may occur makes it possible to determine which The ultimate success of a given species from continuous sewage discharge. For compounds are used by organisms, and depends upon its ability to exploit the example, in the (Glibert which are not, and it also makes it pos- quantity and quality of available nu- et al., 2001) and the Neuse (both sible to follow rates of utilization of trients, the timing and intensity of the located on the U.S. East ) (Springer specifi c compounds. This technique nutrient supplies, and the interactions et al., 2005), blooms of Prorocentrum also provides molecular weight infor- with other environmental factors and minimum have occurred following ni- mation of ionizable compounds (in the competitor or consumer species. Further trogen delivery in heavy spring rains, form of a mass-to-charge ratio [m/z], complicating the interpretation of these but this species does not achieve dense which equals molecular weight for sin- effects is the response of individual spe- blooms during more dry years. gly charged compounds) and a measure cies that may also be separated in time of concentration as ion abundance. and space from the source of nutrient NEW TOOLS ARE YIELDING One such data set, in which changes in delivery, as nutrients are consumed, re- NEW INSIGHTS DOM were followed over a 14-day pe- cycled, and transformed. Attempts to re- The potential for advances in under- riod, demonstrates very different pat- late concentrations of nutrients to HAB standing the impacts of nutrient pollu- terns of use for different compounds. abundance may fail even in cases where tion on and relating eutrophication to Those with an m/z of 137 and 203 were certain nutrients are stimulatory during HABs and their proliferation is large. consumed by the natural plankton com- bloom development if these nutrients New and better tools are providing munity, while the compound with an are fully consumed by the time maxi- methodologies for addressing questions m/z of 253 was not used (Figure 4). As mum biomass is established. related to the complexity of the nutrient more researchers use this instrument, Many aspects of cellular physiology pool, the complexities of the time and compound libraries will be developed, also impact the affi nity of a cell for a par- space scales on which nutrients are deliv- allowing characterization of individual ticular form of nutrient and the immedi- ered, and the complexity of algal nutri- compounds and comparison among pat- ate fate of that nutrient once taken up. tion. Three of many examples are briefl y terns in use of specifi c compounds in Thus, a nutrient pulse may be assimilated highlighted here. comparable and by compa- by cells of the same species at different Electrospray ionization mass spec- rable species in different environments. rates depending on the strains pres- trometry (ESI-MS) is one such new tool. A classic dilemma in assessing whether ent, their nutritional history, and their In contrast to past methodologies for and to what extent nutrients are stimula- growth rates. The timing and intensity studying the DOM pool—which were tory to a given HAB is that the reporting of nutrient loading may also be impor- mostly limited to bulk analysis of total and sampling of bloom events typically

204 Oceanography Vol.18, No.2, June 2005 Figure 4. Example of use of ESI-MS to examine biological utilization of specifi c compounds in the complex mixture of dissolved organic matter (DOM) in aquatic environments. Th e mass-to- charge ratio (m/z) and ion abundance of individual DOM compounds in rainwater are shown in the top panel; the lower three panels show the change in concentration (relative units) due to microbial degradation over a 14-day experiment of three diff erent DOM compounds (m/z 137, 203 and 253) (after Spyres et al., 2003). In the case of compounds with m/z of 137 and 203, compounds were taken up in either linearly or exponentially with time, while in the case of m/z 253, there was no con- sumption over the time period of measurement.

occur after the HAB has developed. By ing the all-important antecedent condi- which concentrations decreased but re- that time, many nutrients are consumed, tions and to provide suffi cient resolution mained elevated for several days in com- leading to the potentially erroneous con- of the temporal scale. Deployments of parison to conditions prior to the pre- clusion that nutrients are unavailable and these monitors in the tributaries draining cipitation events. In the same study, max- therefore unimportant. Traditional ap- agricultural lands in the Chesapeake Bay imum nitrate + nitrite concentrations proaches for collecting information on watershed have shown trends in nutri- lagged behind rainfall events by several nutrient distributions, such as weekly or ent concentrations that vary with differ- days, suggesting either a groundwater bimonthly sampling, are also inadequate ent nutrient sources and that are heavily discharge following the rain or micro- to detect the short-lived nutrient pulses driven by rainfall events (Figure 5). In bial transformation of reduced nitrogen that often follow meteorological events. one such deployment, coincident with (e.g., ammonium and urea) following its Development of in situ nutrient moni- the rainfall events, phosphate “spikes” delivery. Fine-scale temporal resolution tors has provided a tool to begin resolv- were recorded, lasting only hours, after in nutrient concentrations also makes it

Oceanography Vol.18, No.2, June 2005 205 possible to calculate the N:P (in this case, nitrate + nitrite:phosphate) ratio of the dissolved inorganic nutrients (Figure 6). In this example, N:P ratios ranged from less than 1 to greater than 10 over a sev- eral-day time span. Most interesting is the fact that chlorophyll a concentrations increased several-fold after the short- term increase in nitrogen availability. While increases in nitrate + nitrite fol- lowing the rainfall drove the ratio higher, it was the draw-down of nitrogen that resulted in sharp declines in this ratio— and increases in chlorophyll a based on weekly sampling—following each short- term N spike. Use of these types of in situ instruments in conjunction with other remote methods for resolving additional Figure 5. Time series of nitrate + nitrite (upper panel), phosphate (middle panel), and environmental characteristics (Babin et rainfall and salinity (lower panel) in the Pocomoke River, one tributary of the Chesapeake Bay, in which in situ nutrient sensors were used for the dates indicted in 2001. Th e solid al., this issue) will yield the resolution black points are reference samples that were collected simultaneously by a small boat. In necessary to defi ne the ephemeral chang- the upper two panels, the solid green points are the values for chlorophyll a determined es in nutrients that can be signifi cant to from the same samples collected from the boat. Th e lower panel displays rainfall events (as measured at Snow Hill, Maryland, USA) and the fl uctuations in salinity. Data from L. phytoplankton cells. Codispoti and colleagues, University of Maryland Center for Environmental Science, Horn Flow cytometry, a methodology for Point Laboratory, Cambridge, Maryland, unpublished. rapidly measuring intrinsic or applied (stained) optical characteristics of in- dividual cells, has been used to study the occurrence, abundance, physiology, and diversity of aquatic for the past two decades. This technol- ogy has been increasingly applied to the study of HAB species, for example, to generally enumerate and specifi cally identify harmful algal cells in cultures and natural samples. More recently, fl ow cytometry also has been used as a tool for tracking changes in the complex structure of aquatic microbial com- munities (including , pathogenic bacteria, and harmful algae) as related to seasonal cycles, nutrients, or bloom

Figure 6. Ratio of (nitrate + nitrite): phosphate- and daily rainfall during the same in situ age. Such studies, including recent in situ nutrient instrument deployment shown in Figure 5. fl ow cytometric investigations of phy-

206 Oceanography Vol.18, No.2, June 2005 Figure 7. (A) Th in-section electron micrograph of a Pfi esteria piscicida fl agellated cell showing the large food vacuole in the upper portion of the cell (fv), a kleptochloroplast in the food vacuole (kch), and starch grains (st) apparently produced by the kleptochloroplast. Th e klepto- chloroplast had been ingested from a cryptomonad algal prey cell, and had been retained inside the food vacuole for at least 7 days. Scale bar = 5 µm (modifi ed from Lewitus et al., 1999). (B) Two cryptoperidiniopsoid dinofl agellates with food vacuoles containing ingested chloroplast ma- terial (red) from cryptomonad algal prey. Scale bar = 10 µm (modifi ed from Parrow and Burkholder, 2004).

toplankton assemblages, are providing ing photosynthesis to occur without the witus et al., 1999), while in other cases valuable insights about biological factors cellular expenditure of manufacturing they may not (Parrow and Burkholder, (e.g., infection, competition, ) the organelle. Other closely related spe- 2003). Differentiation between klepto- that infl uence the occurrence and distri- cies, such as some strains of cryptoperi- plastidy and simple digestion is prob- bution of HABs. diniopsoids, consume algal prey but lematic, but application of fl ow cytom- Recently, the cell sorting capacity of may not retain the chloroplasts. Cryp- etry is leading to a better understanding some fl ow cytometers has been applied, toperidiniopsoids that have eaten algal of these phenomena in different species as well, to assess modes of nutrition by prey are pigmented because of the algal and strains, and the possible metabolic HAB species. For example, one group of pigments consumed, whereas those that benefi t derived from such behavior. HABs that exhibits complex nutritional have not fed are colorless. Flow cytom- assimilation is Pfi esteria and the related etry has been used to sort live crypto- CONCLUSIONS pfi esteria-like species—small heterotro- peridiniopsoids after feeding trials with The complexities of nutrient sources, phic dinofl agellates that can kill fi nfi sh cryptomonad algae (Figure 7B), and to timing of delivery, nutritional mecha- and shellfi sh by a combination of toxin separate the pigmented cryptoperidin- nisms, complexities of the interactions production and feeding activity (Burk- iopsoid cells from the non-pigmented among members of the microbial com- holder et al., 2001). These dinofl agellates cells (Figure 8). The pigmented dinofl a- munity, and intrinsic differences in do not have their own chloroplasts, but gellates lost or digested the chloroplast physiology and behavior among strains can consume them from algal prey (Le- material in their food vacuoles within 48 within HAB species often make it dif- witus et al., 1999) (Figure 7A). The con- hours. Overall, these experiments show fi cult to differentiate the role of eutro- sumed “kleptochloroplasts”—or stolen that heterotrophic HAB species may phication in development of HABs from chloroplasts—may benefi t these cells for sometimes benefi t from photosynthesis other mechanisms that structure popula- short periods (hours to days) by allow- derived from ingested chloroplasts (Le- tions. Consideration of the full suite of

Oceanography Vol.18, No.2, June 2005 207 Figure 8. Flow cytometric histograms of side angle light scatter versus red autofl uorescence (indicating chlorophyll a, chla). Each data point denotes 1 cell. (A) Time = 0, a mixed culture containing algal prey cells and feeding dinofl agellates. Th e boxed region demon- strates the sorting parameters used to select dinofl agellates with red autofl uorescence of similar intensity to that of the algal prey. Th e dinofl agellates meeting these criteria were sorted and collected. (B) Th e sorted populations, reanalyzed under the same parameters 48 h later exhibited no detectable chla autofl uorescence and therefore clustered at the y-axis. AU = arbitrary units (from Parrow and Burkholder, 2003).

organic and inorganic nutrient forms, ECOHAB and MERHAB programs, Berman, T. and S. Chava. 1999. Algal growth the timing of inputs, and the range of Maryland and Grant, on organic compounds as nitrogen sources. Journal of Plankton Research 21:1423-1437. nutritional mechanisms, including mix- and NOAA South Florida Burkholder, J.M. 2001. Beyond algal blooms, otrophy and heterotrophy, will advance Research and Monitoring Program. We defi cits and fi sh kills: Chronic, understanding of when and where cer- wish to thank J. Alexander and C. Solo- long-term impacts of on tain species may dominate under specifi c mon for contributing to the work pre- aquatic ecosystems. Pp. in Waters in Peril, L. Bendell-Young and P. Gallaugher, eds. Klu- nutrient regimes. Impacts on the coastal sented here, and V. Trainer for sharing wer Academic Publisher, Norwell, Massachu- zone from the increasing use of synthetic graphics. This is Contribution No. 128 setts, USA. and manure fertilizers are projected to from the ECOHAB Program, NJSG-05- Burkholder, J.M., H.B. Glasgow, and N.J. Deam- worsen worldwide, especially in regions 586 from NOAA Sea Grant, and 3837 er-Melia. 2001. Overview and present status of the toxic Pfi esteria complex. Phycologia that are already sustaining increased pro- from the University of Maryland Center 40:186-214. liferations of HABs. for Environmental Science. Doblin, M.A., S.I. Blackburn, and G.M. Hal- legraeff. 2000. Intraspecifi c variation in the ACKNOWLEDGEMENTS REFERENCES selenium requirement of different geograph- ic strains of the toxic dinofl agellate Gymno- The ideas and research presented in this Anderson D., P.M. Glibert, and J.M. Burkholder. dinium catenatum. Journal of Plankton Re- 2002. Harmful algal blooms and eutrophi- paper were developed under several search 22:421-432. cation: Nutrient sources, composition, and Glibert, P.M., R. Magnien, M.W. Lomas, J. Alex- funding sources, including the NOAA consequences. 25:704-726.

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