Environmental stress and domoic acid production byPseudo-nitzschia: a physiological perspective

Natural Toxins Nat. Toxins 6: 127-135 (1998)

RESEARCH ARTICLE

Environmental Stress and Domoic Acid Production by Pseudo-nitzschia: a Physiological Perspective Youlian Pan,' * Stephen S. Bates2 and Allan D. (embelia' , Institute for Marine Biosciences, National Research Council of Canada, Halifax, Nova Scotia, Canada 2Fisheries and Oceans Canada, Gulf Fisheries Centre, Moncton, New Brunswick, Canada

ABSTRACT Production of domoic acid (DA) by the pennate diatom Pseudo-nitzschia multiseries is associated with physiological stress caused by silicate (Si) and/or phosphate (P) limitation. Such limitation may promote DA synthesis by (1) reducing primary metabolic activity, thus making available necessary precursors, high energy compounds, and cofactors, and (2) favoring the expression of genes involved in the biosynthesis of this toxin. In the case of Si and P-limitation, DNA synthesis and the progression through the cell division cycle are slowed, perhaps prolonging or arresting the cells in the stage of the division cycle which is most conducive to DA production. However, N-limitation results in an insufficient pool of cellular free N, which restricts synthesis of this nitrogenous toxin. A continuous supply of photophosphorylated high-energy intermediates (e.g., ATP and NADPH) is necessary for DA synthesis. In order to better understand the mechanism(s) of DA production, more studies are needed to elucidate: (1) the details of the biosynthetic pathway, (2) the regulation of enzymes involved in the pathway, (3) the relation between DA synthesis and the cell division cycle, (4) the cellular compartmentalization of DA biosynthesis, and (5) other environmental factors that may trigger DA production. Finally, these studies should be extended to include toxigenic Pseudo-nitzschia species other than P. multiseries, to confirm the commonality of these mechanisms. Copyright © 1998 John Wiley & Sons, Ltd.

Key words: domoic acid; Pseudo-nitzschia multiseries; neurotoxin; biosynthesis; environmental stress; silicon; phosphorus; nitrogen; diatom

INTRODUCTION

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Production of phycotoxins by diatoms was unknown until 1987, when amnesic shellfish poisoning (ASP) caused human illnesses and deaths in Atlantic Canada (Todd, 1993). The responsible neurotoxin, domoic acid (DA), was isolated and identified from extracts of toxic mussels (Wright et aI., 1989), which had fed upon blooms of the pennate diatom Pseudo-nitzschia muitiseries (Subba Rao et aI., 1988; Bates et aI., 1989) (previously known as Nitzschia pungens f. muitiseries, Pseudonitzschia pungens f. muitiseries and Pseudo-nitzschia pungens f. muitiseries [Hasle, 1995]). Subsequently, DA was detected in other members of the genus Pseudo-nitzschia at various locations around the world (Bates et al., 1998). The cosmopolitan distribution of toxigenic Pseudonitzschia species poses a threat to human health and to the fisheries and aquaculture industries. It is essential to understand the mechanisms of toxin production before the impact can be mitigated. Surprisingly, no studies have been carried out on the physiology or mechanisms of DA CCC 1056-9014/98/030127-09 $17.50 Copyright © 1998 John Wiley & Sons, Ltd.

production by the several species of rhodophycean macroalgae that were originally shown to produce DA (e.g. Takemoto and Daigo, 1958). Since the Canadian incident, environmental factors influencing DA production in pennate diatoms have been studied and documented. However, the mechanism of DA production particularly genetic regulation - is still far from clear. This review is not intended to cover all known aspects of the ecophysiology of Pseudo-nitzschia spp., some of which has been reviewed recently (Bates, 1998; Bates et al., 1998). Instead, it is to provide insights into the factors controlling DA production in Pseudo-nitzschia spp., especially those affected by environmental stress, and to suggest directions for further research on this topic.

*Correspondence to: Youlian Pan, Marine Biotoxins Program, NOA Johnson Rd, Charleston, SC 29412, USA. Received 30 October 1997; Accepted 15 May 1998

PAN ET AL.

128

FACTORS KNOWN TO AFFECT DOMOIC ACID PRODUCTION ;>.,

The production of domoic acid, a low molecular weight -amino acid, is circumstantially linked to external environmental stress, following the typical pattern for secondary metabolites synthesized by other protists, fungi and bacteria. In many organisms, secondary metabolite production is induced in response to environmental stress, such as temperature shock or nutrient deprivation, or it is associated with a change in life history status (e.g. cyst or spore formation). With reference to phycotoxins such as DA, the term 'toxin production' is frequently (mis)used to refer to the cell toxin quota (Qt), the amount of toxin per cell, rather than the net rate of cellular toxin accumulation, essentially the rate of toxin biosynthesis minus the loss terms (toxin catabolism, bio-transformation, leakage, excretion, etc.). Thus toxin production can be zero even in cells which have a high toxin content. Conversely, cell toxicity may be rather low even when toxin production is high, e.g. if cells are dividing rapidly. We use toxin production deliberately to refer to the rate processes.

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During early research on DA metabolism in Pseudonitzschia multiseries, substantial amounts of cellular DA were found only during stationary phase in batch culture (Bates et aI., 1989; Subba Rao et aI., 1990), and this was associated with a low concentration or depletion of macronutrients, e.g. inorganic silicate (Si) and phosphate (P). In a natural ecosystem subject to toxic Pseudonitzschia blooms, the peak in cellular DA concentration occurred about a week after the maximum concentration of P. multiseries cells in the water column (Smith et al., 1990). This suggested that DA production may be related to environmental stress, such as nutrient limitation, which resulted in the retardation of growth. These early findings led to the investigation of the effects of various nutrients that support cell growth in culture. In batch cultures of P. multiseries grown in medium 'f' (Ouillard and Ryther, 1962) and its modifications, the onset of stationary phase is usually associated with the depletion of Si (Bates et aI., 1989; Pan et al., 1991), rather than nitrogen (N)- or P-nutrients in the medium. This is a consequence of the high ratios ofN:Si (14.4) and P:Si (0.59), compared to the Redfield ratios of rv 1 and 0.07 (Redfield et al., 1963), respectively, which would describe nutritionally balanced growth. The highest production of DA occurs during stationary phase, although some is synthesized during late exponential phase when growth rate declines as a result of the depletion of Si in the medium (Figure 1(a)). Large amounts of DA are also produced in Si-limited Copyright © 1998 John Wiley & Sons, Ltd.

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continuous cultures, even while the cells are dividing, and the production rates are inversely related to growth rates (Pan et al., 1996b; Bates et al., 1996). As Silimitation (shown by reduced growth rates) becomes more severe in both batch and continuous cultures, DA production increases significantly; reintroduction of Si into the medium suspends DA production (Figure l(a)) during the subsequent increase in cellular Si (Pan et al., 1996a,b). There is thus a significant inverse correlation between the supply of Si and the production of DA (Bates et aI., 1991; Pan et aI., 1996a,b; Bates et al., 1996). In contrast, when germanium, a competitive inhibitor of Si-uptake, is introduced into the culture medium at the beginning of exponential growth to disable Si-uptake, neither DA production nor growth of the cell population occurs (Pan et al., unpublished). This suggests that the involvement of Si-limitation in DA production is indirect. In diatoms, Si is required not only for frustule formation, but also for other metabolic processes. For example, Si-limitation impedes the progression of the cell division cycle by interfering with the synthesis of DNA (Brzezinski, 1992; Bates and Richard, 1996). In the Nat. Toxins 6: 127-135 (1998)

STRESS AND DOMOIC ACID PRODUCTION BY PSEUDO-NITZSCHIA

centric diatom, Cylindrotheca fusiformis, DNA synthesis is 90 % inhibited by Si starvation but resumes immediately after Si re-supply (Darley and Volcani, 1969). Cessation ofDN1 synthesis by Si starvation is caused by a decrease in activity ofthe enzymes DNA polymerase(s) and thymilate (TMP)-kinase (Sullivan and Volcani, 1973), but not by a lack of energy or precursors. The DNA polymerases A and D are synthesized only in the presence of Si. However, at least 15 other proteins are formed only in the absence of Si, and their synthesis is repressed when Si is resupplied (Sullivan and Volcani, 1981). This suggests that Si affects the regulation of gene expression in diatoms. Okita and Volcani (1980) found an increase in polypeptide turnover in the absence of Si; four other previously undetected polypeptides, one of which may be DNA polymerase, are synthesized when Si is resupplied. This indicates that Si also participates in the synthesis of polypeptides (e.g. DNA polymerase), prior to DNA replication. In P. muitiseries, the inhibition of DNA polymerase by Si limitation may arrest cells at a particular phase in the cell division cycle that is conducive to DA production. Phosphorus plays a key role in the structure of cell membranes, in cell bioenergetics, and the synthesis of lipids (Siron et ai., 1989; Lombardi and Wangersky, 1991) and nucleotides (Cembella et ai., 1984). Increasingly, harmful algal blooms have been associated with conditions of P-limitation (e.g. Woodward and Owens, 1990; Graneli et ai., 1998). A positive correlation between P-limitation and high levels of cell toxin has been documented for the PSP toxin-producing dinoflagellate Aiexandrium tamarense (e.g., Anderson et ai., 1990), the haptophyte Chrysochromuiina poiyiepis (e.g., Edvardsen et ai., 1990), and the diatom P. multiseries (Bates et ai., 1991; Pan et ai., 1996c). In P. muitiseries, an elevated production of DA coincides with high levels of alkaline phosphatase activity (APA), an indicator of Plimitation, and of adenosine (tri-, di-, and mono-) phosphates (Pan et ai., 1996c). The relationship between DA production and APA is similar to that observed for the synthesis of antibiotics by bacteria under P-limitation, e.g. streptomycin by Escherichia coli, neomycin by Streptomyces fradiae, and ristomycin by Proactinomyces fructiferi var. ristomicini (Martin, 1977). The synthesis of both DA and these antibiotics is depressed by addition of inorganic P to the culture medium. In the case of P. muitiseries, P-limitation may be more closely implicated than Si-limitation in the inhibition of primary metabolism, thus having a greater effect on promoting DA synthesis. For example, the progression of the cell division cycle is impeded by the inability to form cell membranes and to synthesize DNA. However, by interfering with phosphorylation, P-limitation also inhibits the production of high-energy compounds, required for DA production (discussed below). Copyright © 1998 John Wiley & Sons, Ltd.

129

Unlike Si- and P-limitation, N-limitation is directly unfavorable for DA production by P. muitiseries. The availability of N in the growth medium is essential to DA production (Bates et ai., 1991) since it is a key structural element of DA. Nitrogen in the form of ammonium ion promotes DA production better than nitrate, when provided at equivalent concentrations of 220-440 11M (Bates et ai., 1993). Growth rates of P. muitiseries (Hillebrand and Sommer, 1996) and other phytoplankton (Dortch, 1990) on nitrate are usually equal to or exceed those on ammonium. In the synthesis of amino acids, including the pathway to DA, the reduction of nitrate to ammonium involves at least two enzymes and a series of electron transfers from the high energy compound, NADH; thus it is an energetically more expensive substrate than ammonium. However, high concentrations of ammonium tend to inhibit growth relative to the same concentration of nitrate. Therefore, the fact that ammonium supports higher DA production than nitrate can be interpreted as either a physiological stress imposed on diatom growth due to ammonium toxicity (Bates et ai., 1993; Hillebrand and Sommer, 1996) or the use of a more energetically favorable form of nitrogen for the synthesis of primary amino acids and DA. Addition of Tris, a common pH buffer, to P. muitiseries cultures substantially enhances DA production (Douglas et ai., 1993). The authors believe that Tris may act as an N-source because it is a primary amine and it may be more efficiently used than nitrate for amino acid (including DA) synthesis. Alternatively, Tris may play an indirect role by altering enzymatic reaction rates (Mahler, 1961), enhancing CO2 absorption (Ogata, 1966), or by complexing cations (McLachlan, 1963), including trace metals (Sunda and Guillard, 1976). Additionally, since the concentration of bicarbonate ions is sensitive to pH, buffered slightly alkaline systems will sustain the concentration of this ion and reduce the cost of carbon uptake. Lithium (Li) stimulates DA production even though it has no known nutritional role in the growth of P. muitiseries (Subba Rao et ai., 1998). In animal cells, Li alters the regulatory function of cyclic AMP (cAMP) (Peters et ai., 1992) and cAMP-induced phosphorylation (Brokaw, 1987), both of which may playa role in the synthesis of DA. The cyclic nucleotides, cGMP and in particular cAMP, have profound effects on the progress of mammalian cell division and on oxidative phosphorylation in general. In different systems, and depending on cell types, cAMP either promotes or blocks the progression of the cell division cycle in G1 phase (Depoortere et ai., 1996). In diatoms, the mechanism by which cAMP controls cell cycle progression is still unknown. Under Si starvation, cAMP levels in Cylindrotheca fusiformis increase steadily, nearly doubling (Borowitzka and Volcani, 1977). After adding Si, the cAMP level Nat. Toxins 6: 127-135 (1998)

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PAN ETAL.

increases drastically, but several hours later it decreases prior to initiating mitosis. Peak accumulation of cAMP substantially precedes that of DNA synthesis (Aline et al., 1984). Thus, Si-limitation and Li-addition, via their effect on cell cycle progression, may influence DA production in a similar way. As secondary messengers, cyclic nucleotides also directly or indirectly modulate ion channels. For example, cAMP regulates K+-selective channels in sea urchin sperm plasma membranes (Labarca et al., 1996). Uptake of Si and P requires the operation of a K+-Na+ pump. Changes in cAMP concentration will therefore change the rates of Si and P uptake and thus affect DA production.

Physical Factors

cultures (Pan, 1994; Laflamme and Bates, unpublished). However, there is still no evidence that bacteria are capable of DA production in the absence of P. multiseries (Bates, 1998). Bacteria from several different sources enhanced DA production in P. multiseries (Bates et al., 1995), suggesting that certain common bacterial compounds, either inorganic (e.g. bicarbonate ion) or organic, are stimulatory. For example, addition of gluconic acidl gluconolactone, produced by the bacterium Alteromonas sp. isolated from P. multiseries cultures, to axenic cultures of this diatom substantially increased DA production (Osada and Stewart, 1997). The association of P. multiseries with another bacterium, Moraxella sp., which is unable to produce gluconic acidlgluconolactone, did not substantially enhance DA production. Osada and Stewart (1997) suggest that the 'sequestering' ability of the bacterially produced gluconic acidlgluconolactone scavenges nutrients from the medium and thus increases the degree of nutrient limitation experienced by the diatom. Consequently, P. multiseries is stimulated to produce and release DA, which would chelate micronutrient cations, such as trace metals, rather than macronutrients anion, such as P04 -3 or Si0 3 -2. Alternatively, gluconic acidlgluconolactone may participate directly in the DA biosynthetic pathway. These compounds also may act as auxiliary carbon sources, by providing carbon skeletons involved in the pathway to DA.

Light is essential to DA production in diatoms (Bates et al., 1991; Whyte et al., 1995). A photon flux density of at least 100 ~mol photons m- 2 S-l is required to sustain DA production by P. multiseries (Bates, 1998). Production of DA in batch culture is suspended during prolonged darkness or after addition of the photosynthetic inhibitor DCMU (3-(3,4-dichlorophenyl)-1, 1-dimethylurea) (Bates et al., 1991), which blocks electron transfer between photosystems I and II, and hence impedes photophosphorylation. Light also influences the rate of nitrate reduction. In both algae (Dortch, 1982; Kumar et al., 1986; Corzo and Niell, 1994) and higher plants (Crete et al., 1997), nitrate reductase activity is generally much BIOSYNTHESIS OF DOMOIC ACID higher in the light. In addition, below the saturation threshold, light intensity is positively correlated with The DA molecule consists of a proline-like ring containcarbon assimilation in Pseudo-nitzschia multiseries (Pan ing an isoprenoid and a carboxymethyl side chain (Figure et al., 1991, 1996d). These findings show that light can 2). This entire molecule is derived from acetate (Douglas exert indirect effects on DA production by controlling the et al., 1992) via two separate precursor intermediates: a activity of photophosphorylation, nitrogen reduction, and glutamate derivative from the Krebs cycle (Laycock et carbon assimilation. al., 1989), and an isoprenoid structure likely derived from In contrast to the nutritional stresses discussed in the geranyl pyrophosphate, via acetyl CoA (Figure 2). previous section, physical stress due to low temperature Synthesis of geranyl pyrophosphate from acetyl CoA depresses DA production by P. multiseries (Lewis et al., requires 8 enzymes, 3 ATP and 2 NADPH molecules 1993), because of the depression of enzyme activity. (Luckner, 1984). Synthesis of a proline-like ring from 0(Nevertheless, P. seriata produces higher levels of DA at oxoglutarate also requires enzymes, plus substantial 4°C than at 15°C (Lundholm et al., 1994). This amounts of ATP and NADPH. Addition of proline to discrepancy is attributable to adaptive differences either axenic or non-axenic cultures of P. multiseries between cold water (i.e. P. seriata) versus cosmopolitan suppresses the stimulatory effect of gluconic acidl (i.e. P. multiseries) species (Bates, 1998). gluconolactone on DA production (Osada and Stewart, 1997). This suggests that an enzyme, which catalyzes the conversion of the glutamate to a suitable intermediate for Biological Factors condensation with the isoprenoid subunit to form DA, Domoic acid synthesis is significantly promoted by the may be subjected to a feedback modulation by proline. presence of extracellular bacteria, although they are not Addition of either glutamate or gluconic acidlglucoessential (Douglas and Bates, 1992). There is a significant nolactone enhances DA synthesis, slightly in the case of positive correlation between DA production and bacterial the former and significantly in the latter. When these two abundance after addition of glucose to batch cultures are added in combination, no stimulation is seen (Osada (Osada and Stewart, 1997) and to Si-limited chemostat and Stewart, 1997). This suggests that there may be an Copyright © 1998 John Wiley & Sons, Ltd.

Nat. Toxins 6: 127-135 (1998)

STRESS AND DOMOIC ACID PRODUCTION BY PSEUDO-NITZSCHIA

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al., 1992), before substantial DA is detected in the cells. There is, however, an insignificant quantity of DA produced by P. multiseries during the late exponential phase of batch culture when growth is declining. Pan et al. (l996a) characterize this as stage I DA production (Figure l(b)), which may correspond to the stage when the glutamate-like intermediate is derived either from the Krebs cycle or from free glutamate. During stage I, the number of bacteria is limited by the scarcity of organic substrates or by an increase of bactericidal compounds produced by actively growing algal cells (Fogg and Thake, 1987). When the culture reaches stationary phase, the bacterial number increases and DA synthesis is greatly enhanced (= stage II). This may correspond to a switch from the use of the Krebs derivative or free glutamate as a substrate for the synthesis of the glutamate-like intermediate to the use of a bacterially produced substrate or regulator, such as gluconic acidl gluconolactone (assuming that these compounds are metabolized). It is important to determine:

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alternate pathway other than via glutamate (Figure 2). Assuming that it can be taken up by P. multiseries cells, gluconic acidlgluconolactone produced by bacteria may act either as a precursor (unlikely because it is energetically unfavorable, although it may serve as a carbon skeleton for the glutamate-like compound) or as a regulator in an alternate pathway. Such a pathway could be more effective than the one via glutamate because elevated DA production is usually associated with a high abundance of bacteria, some of which produce gluconic acidlgluconolactone. Douglas et al. (1992) suggested that the isoprenoid subunit is synthesized prior to DA synthesis, i.e. during the exponential growth phase in batch culture. Therefore, the rate of DA synthesis would depend on the rate of synthesis of the glutamate-like subunit before its fusion with the isoprenoid subunit. The composition and absolute quantity of cellular free amino acids in microalgae changes as cells progress from exponential to stationary phase (e.g. Flynn, 1990). In P. multiseries, glutamate is a major component in the free amino acid pool during early stationary phase (Jackson et Copyright © 1998 John Wiley & Sons, Ltd.

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RELATIONSHIP TO PRIMARY METABOLIC PROCESSES

As discussed above, DA synthesis requires a substantial amount of biogenic energy and therefore competes with nutrient uptake and primary metabolism. For example, uptake of Si and P are energetically expensive metabolic processes with a substantial demand for ATP (Sullivan and Volcani, 1981). During periods of active nutrient uptake and growth, therefore, less ATP is available for DA synthesis (Pan et al., 1996b, c). Conversely, cellular levels of ATP increase in C. fusiformis under Silimitation (Coombs et al., 1967) and in P. multiseries under Si- and P- limitation (Pan et al., 1996b,c). This increase in ATP is attributable to its reduced demand as a result of a decline in primary metabolism; the ATP can then be used to support DA production. There is a significant inverse relationship between DA production and the growth rate of P. multiseries [Figure 1; also seen by Bates et al. (1996)]. Cellular DA begins to increase when growth slows as P. multiseries cells approach stationary phase. At stationary phase, carbon assimilation is substantially reduced (Pan et al., 1991), but on-going photophosphorylation maintains the production of ATP and NADPH, and thus DA production. Eventually DA production decreases or stops after cellular chlorophyll a concentration drops to a critical level «0.05 pg cell-I) during late stationary/senescence phase (Pan, unpublished). The increase in DA production Nat. Toxins 6: 127-135 (1998)

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can be reversed by the resupply of Si and the consequent nutrients such as vitamins and trace metals. These essential nutrients, when limiting, may (1) decrease resumption of growth (Figure l(a)). Accumulation of energy-rich intermediates and pre- primary metabolism, thereby making available necessary cursors during a period of active growth (e.g. during the precursor(s), high energy compounds and cofactors, and exponential phase) is important for subsequent DA (2) induce (or de-repress) gene expression for regulating production. Thus, a higher growth rate, accompanied by DA synthesis. In the case of Si-limitation, synthesis of a short exponential phase, results in a higher DA DNA and progression through the cell division cycle are production rate later in stationary phase (Pan et aI., slowed. However, N-limitation may inhibit DA synthesis 1996c). On a longer time scale - as an isolate of P. because of an insufficient pool of cellular free N, required muitiseries 'aged' over several years in culture - cellular to supply the ammonium moiety to DA. Synthesis of DA chlorophyll a and maximum growth rates declined, as did is not necessarily restricted to the cessation of cell the capacity for DA production (Pan, unpublished). This division, as the cells are also able to produce DA when decrease in DA production may be caused by decreases in they are dividing in chemostat culture under nutrientcell viability and cellular chlorophyll: the latter is limited conditions. A continuous supply of photoessential to photophosphorylation. The capacity to phosphorylated high-energy compounds (e.g., ATP, produce high levels of DA is regained after P. muitiseries NADPH) is necessary to enable DA synthesis. Noncells undergo sexual reproduction (auxosporulation) to nutrient ions, such as Li, may directly or indirectly affect recover their large initial cell size and viability (see DA synthesis through regulation of secondary messenDavidovich and Bates, 1998). Relationships among cell gers (cAMP) or regulators of gene expression linked to size, ability to undergo auxosporulation, and capacity to DA synthesis. produce DA are currently under investigation. In most diatoms, Si-limitation leads to an increase in SUGGESTIONS FOR FUTURE RESEARCH lipids and fatty acids, but this is not the case in P. muItiseries (Parrish et aI., 1991). This may be attributed During the past decade, considerable progress has been to a light limitation of triacylglycerol synthesis by self- made in understanding the physiology of Pseudoshading when cells reach a high abundance during nitzschia spp., allowing hypotheses to be developed. stationary phase (Parrish et al., 1991). Alternatively, What is lacking, however, is certain experimental data the activation of DA synthesis, which shares some of the that would permit all of this information to be gathered same precursors (e.g. acetyl CoA) with fatty acid into a paradigm that can describe the mechanism(s) biosynthesis, may have diverted the synthesis away from responsible for regulating DA production. In order to fatty acids. In contrast, in P-limited (based on the accomplish this, the following gaps in our knowledge composition of the culture medium) mass cultures of P. require further research. muItiseries, a positive correlation is evident between cellular DA and the content of certain fatty acids (Whyte • The biosynthetic pathway is not completely elucidated. et aI., 1995). This can occur if sufficient light is available Precursors for DA synthesis need to be more to support production of enough photosynthate and high specifically identified. For example, the role of energy compounds (e.g. ATP and NADPH) to supply gluconic acid/gluconolactone, which may act either both DA and fatty acid synthesis during the stationary as a nutrient scavenger, precursor or regulator, requires phase. study. More evidence is needed to confirm the hypothesis that two subunits are condensed from different pathways. The involvement of enzymes and CONCLUSIONS their kinetics merits investigation in order to better understand the roles of various environmental stresses. The pattern of DA synthesis is comparable to that of most other such secondary metabolites (e.g. drugs, antibiotics, Knowledge of the biosynthetic pathway will ultimately some other biotoxins, etc.) that are produced when lead to the identification of genes responsible for DA primary metabolism declines. Interestingly, this pattern biosynthesis. This will enable the production of differs substantially from that of certain other phycomolecular probes (cf. Scholin, 1998) to determine which forms and species of Pseudo-nitzschia have the toxins produced by dinoflagellates, such as the polyether toxins derived from polyketide biosynthesis (Wright and capacity to produce DA (Bates et al., 1998). Cembella, 1998) and the neurotoxic paralytic shellfish • Other environmental factors, such as bicarbonate ion concentration, trace metals and pH, affect the primary toxins (Cembella, 1998), which are produced constitumetabolism of microalgae, and will certainly affect the tively. The ability to synthesize DA is inversely related to production of DA. primary metabolic activity in P. muitiseries. Thus, DA production is enhanced when primary metabolism is • The detailed function of non-nutrient elements remains stressed by limitation of Si, P, and perhaps other essential unknown. For example, does Li ion change the Copyright © 1998 John Wiley & Sons, Ltd.

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regulatory function of cAMP in Pseudo-nitzschia spp., similar to that in animal cells? How does cAMP regulate DA synthesis? • Earlier studies suggested that Si-limitation indirectly enhance DA production via its influence on cell cycle progression, perhaps via its control of DNA synthesis; this must be confirmed. It is still unknown when DA is produced in the cell division cycle. Evidence of DA synthesis during certain phases of the cell cycle may enable a better understanding of DA biosynthesis with respect to enzyme activity and gene expression. • Cellular compartmentalization of secondary metabolites is well known in higher plants and microorganisms (Luckner, 1984). Many enzymes for the biosynthesis of secondary metabolites are located in plastids. For example, okadaic acid (OA), among the toxins responsible for diarrhetic shellfish poisoning (DSP), is found mostly associated with the chloroplasts of the epibenthic dinoflagellate Prorocentrum lima (Zhou and Fritz, 1994). Because DA is a small watersoluble molecule, it may be synthesized on the hydrophilic side of a membrane structure, perhaps within the chloroplasts. Precursors and enzymes for DA synthesis may be synthesized in the cytoplasm. Evidence is lacking to support these hypotheses. Studies on the localization of DA production will enable a better understanding of the biosynthetic pathway and a clearer picture of the effects of environmental stress. ACKNOWLEDGEM ENTS

We thank J.L.c. Wright, S. Johnson, T. Hu, and two anonymous reviewers for their constructive comments on the manuscript. This study is issued as NRC publication number NRCC 39774.

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