Stephen S. Bates

Pseudo-nitzschia (Bacillariophyceae) species, domoic acid and amnesic shellfish poisoning: revisiting previous paradigms

Lelong A., Hégaret H., Soudant P. and Bates S.S. 2012. Pseudo-nitzschia (Bacillariophyceae) species, domoic acid and amnesic shellfish poisoning: revisiting previous paradigms. Phycologia 51: 168–216. DOI: 10.2216/11-37 Pseudo-nitzschia is a globally distributed diatom genus, some species of which produce domoic acid (DA), the neurotoxin that causes amnesic shellfish poisoning. This toxin killed at least three humans in 1987, launching numerous studies concerning the identification, distribution, ecology and physiology of Pseudo-nitzschia spp. Since previous reviews in 1998, knowledge has been gained about the fate of DA, including its accumulation by marine animals and its degradation by light and bacteria. Molecular techniques and more precise microscopy have enabled the description of new Pseudo-nitzschia species, 15 since 2002, including ones that are cryptic and pseudo-cryptic. An increasing number of the 37 identified species, including oceanic and coastal species, have been studied in laboratory culture. The sexual reproduction of 14 species has been documented. Fourteen species have now been shown to be toxigenic, although some strains are not always toxic under the testing conditions. The biotic and abiotic factors that modify DA production are reviewed, with a focus on how new discoveries have changed our original hypotheses about control mechanisms. Recent studies confirm that silicate and phosphate limitation trigger DA production. However, stress by low concentrations of iron or high concentrations of copper are newly discovered triggers, suggesting a trace-metal chelation role for DA. Organic sources of nitrogen (urea and glutamine), as well as changes in pH, CO2, salinity and bacterial concentration, also enhance DA production. Laboratory and field studies sometimes give divergent results for conditions that are conducive to toxin production. Gaps in knowledge include further information about the whole genome of Pseudo-nitzschia (including sexual stages), mechanisms of DA production and decline, presence or absence of a resting stage, heterotrophic ability, impact of viruses and fungi, and a more complete description of the ecological and physiological roles of DA.

SEXUAL REPRODUCTION IN THE PENNATE DIATOMS PSEUDO‐NITZSCHIA MULTISERIES AND P. PSEUDODELICATISSIMA (BACILLARIOPHYCEAE)

Clones of the domoic‐acid‐producing pennate diatom Pseudo‐nitzschia multiseries (Hasle) Hasle and of the potentially toxic P. pseudodelicatissima (Hasle) Hasle normally decrease in cell size in culture until they eventually die without undergoing sexual reproduction to regain the largest cell size. However, we induced sexual reproduction by mixing individual exponentially growing clonal cultures of the appropriate minimal cell size under the same conditions that are normal for vegetative growth. We observed pairing of parent cells (gametangiogamy); production of four morphologically isogamous, nonflagellated gametes per gametangial pair; rearrangement of the gametes and their fusion to form zygotes, revealing physiological anisogamy; enlargement of auxospores; and formation of long initial cells. Our observations of allogamous reproduction are consistent with those reported for other dioecious pennate diatoms. Clones of P. pseudodelicatissima from the Black Sea and from the CCMP culture collection failed to auxosporulate when mixed together, although they are the same species according to scanning electron microscopy. The range in apical length of P. multiseries was broader than that reported in the literature for field samples, necessitating a modification of the species description. Knowledge of the pattern and timing of sexual reproduction in Pseudo‐nitzschia spp. may provide insights into their bloom dynamics.

Growth and domoic acid production by Pseudo-nitzschia seriata (Bacillariophyceae) under phosphate and silicate limitation

Pseudo‐nitzschia seriata (Cleve) H. Peragallo isolated from Scottish west coast waters was studied in batch culture with phosphate (P) or silicate (Si) as the yield‐limiting nutrient at 15°C. This species produced the neurotoxin domoic acid (DA) when either nutrient was limiting but produced more when stressed by Si limitation during the stationary phase. Under P‐limiting conditions, exponential growth stopped after P was reduced to a low threshold concentration. Under Si‐limiting conditions, fast exponential growth was followed by a period of slower exponential growth, until Si became exhausted. A stationary phase was observed in the P‐limited but not the Si‐limited cultures, the latter showing a rapid decrease in cell density after the second exponential growth phase. Si‐limited cultures exhibited a further period of active metabolism (as indicated by increases in chl and carbon per cell) late in the experiment, presumably fueled by regenerated Si. DA production was low in exponential phase under both conditions. In P‐limited cultures, most DA was produced during the immediate postexponential phase, with little or no new DA produced during later cell senescence. In contrast, although a substantial amount of DA was produced during the slower exponential phase of the Si‐limited cultures, DA production was even greater near the end of the experiment, coincident with the period of chl synthesis and increase in carbon biomass. Comparison of the magnitude of toxin production in the two nutrient regimes indicated a greater threat of P. seriata‐generated amnesic shellfish poisoning events under Si rather than P nutrient limitation.

Environmental stress and domoic acid production by Pseudo-nitzschia: a physiological perspective.

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.

CRYPTIC AND PSEUDO‐CRYPTIC DIVERSITY IN DIATOMS—WITH DESCRIPTIONS OF PSEUDO‐NITZSCHIA HASLEANA SP. NOV. AND P. FRYXELLIANA SP. NOV.1

A high degree of pseudo‐cryptic diversity was reported in the well‐studied diatom genus Pseudo‐nitzschia. Studies off the coast of Washington State revealed the presence of hitherto undescribed diversity of Pseudo‐nitzschia. Forty‐one clonal strains, representing six different taxa of the P. pseudodelicatissima complex, were studied morphologically using LM and EM, and genetically using genes from three different cellular compartments: the nucleus (D1–D3 of the LSU of rDNA and internal transcribed spacers [ITSs] of rDNA), the mitochondria (cytochrome c oxidase 1), and the plastids (LSU of RUBISCO). Strains in culture at the same time were used in mating studies to study reproductive isolation of species, and selected strains were examined for the production of the neurotoxin domoic acid (DA). Two new species, P. hasleana sp. nov. and P. fryxelliana sp. nov., are described based on morphological and molecular data. In all phylogenetic analyses, P. hasleana appeared as sister taxa to a clade comprising P. calliantha and P. mannii, whereas the position of P. fryxelliana was more uncertain. In the phylogenies of ITS, P. fryxelliana appeared to be most closely related to P. cf. turgidula. Morphologically, P. hasleana differed from most other species of the complex because of a lower density of fibulae, whereas P. fryxelliana had fewer sectors in the poroids and a higher poroid density than most of the other species. P. hasleana did not produce detectable levels of DA; P. fryxelliana was unfortunately not tested. In P. cuspidata, production of DA in offspring cultures varied from higher than the parent cultures to undetectable.

Domoic acid production by Pseudo-nitzschia seriata (Bacillariophyceae) in Scottish waters

In 1999, a 49,000 km2 area in western Scottish waters was closed to shellfish harvesting due to the amnesic shellfish poisoning (ASP) toxin domoic acid (DA). The only previously confirmed DA producer identified had been Pseudo‐nitzschia australis Frenguelli. The toxin has appeared every year since and has led to more harvesting closures. We isolated and cultured two strains of Pseudo‐nitzschia seriata f. seriata (P. T. Cleve) H. Peragallo from western Scottish waters in 2001 and 2002. They were identified using TEM analysis of their morphological fine structure and sequencing of the internal transcribed spacer (ITS)1, 5.8S, ITS2, and partial large subunit (LSU) rDNA. The morphology of the Scottish P. seriata f. seriata strains differed slightly, for example, in the number of poroid rows, from descriptions in identification keys. Comparison of P. seriata sequences with those of two co‐occurring Pseudo‐nitzschia australis isolates showed an overall divergence of only 0.012. Sequence divergence between both species was highest in the ITS1 region (0.036). Combined morphological and genetic approaches are needed to identify closely related Pseudo‐nitzschia species. The P. seriata strains grew successfully at 15°C, suggesting that although seen as a psychrophilic species, it may also occur at higher water temperatures. All isolates produced DA in stationary phase (measured on day 25): 0.16–0.23 pg DA·cell−1 in P. seriata and 0.15–1.68 pg DA·cell−1 in P. australis. Our study is the first to identify P. seriata f. seriata as a DA producer in Scottish waters and indicates that at least it and P. australis can be responsible for ASP toxicity in that region.

CONFIRMATION OF DOMOIC ACID PRODUCTION BY PSEUDO‐NITZSCHIA AUSTRALIS (BACILLARIOPHYCEAE) ISOLATED FROM IRISH WATERS1

A nonaxenic isolate of the potentially toxic diatom Pseudo‐nitzschia australis (Frenguelli) from Irish waters was tested in two separate batch culture experiments. When grown under a low irradiance (∼12 μmol photons·m−2·s− 1 ; 16:8‐h light:dark cycle) for up to 40 days, the culture produced only trace amounts of the neurotoxin domoic acid (DA) during late stationary phase. Growth at a higher irradiance (∼115 μmol photons·m−2·s− 1 ; 12:12‐h light:dark cycle) resulted in DA production starting during late exponential phase and reaching a maximum concentration of 26 pg DA·cell− 1 during late stationary phase. Liquid chromatography coupled to mass spectrometry was used to confirm the identity of DA in the culture. Irradiance and photoperiod could be important factors that contribute directly or indirectly to the control of DA production in P. australis. This is the first record of a DA‐producing diatom in Irish waters, and results indicate P. australis may have been the source of DA that has recently contaminated shellfisheries in this area.

Microsatellite marker development and genetic variation in the toxic marine diatom Pseudo-nitzschia multiseries (Bacillariophyceae)

The genetic structure of phytoplankton populations is largely unknown. In this study we developed nine polymorphic microsatellite markers for the domoic acid–producing marine diatom Pseudo‐nitzschia multiseries (Hasle) Hasle. We then used them in the genotyping of 25 physiologically diverse field isolates and six of their descendants: 22 field isolates originated from eastern Canadian waters, two from European waters, and one from Russian waters. The numbers of alleles per locus ranged from three to seven and the observed heterozygosities from 0.39 to 0.70. A substantial degree of genetic variation was observed within the field isolates, with 23 different genotypes detected. The Russian isolate was the most genetically distinct, although there was also evidence of genetic differentiation at a more local scale. Mating experiments demonstrated that alleles were inherited in a Mendelian manner. Pseudo‐nitzschia multiseries primer pairs were tested on DNA from four congeners: P. calliantha Lundholm, Moestrup et Hasle; P. fraudulenta (P. T. Cleve) Hasle; P. pungens (Grunow ex P. T. Cleve) Hasle; and P. seriata (P. T. Cleve) H. Peragallo. Cross‐reactivity was only observed in P. pungens. Our results are a first step in understanding the genetic variation present at the Pseudo‐nitzschia“species” level and in determining the true biogeographic extent of Pseudo‐nitzschia species.

Feeding, egg production, and egg hatching success of the copepods Acartia tonsa and Temora longicornis on diets of the toxic diatom Pseudo-nitzschia multiseries and the non-toxic diatom Pseudo-nitzschia pungens

In 1987, there was an episode of shellfish poisoning in Canada with human fatalities caused by the diatom Pseudonitzschia multiseries, which produced the toxin domoic acid. In order to examine whether domoic acid in this diatom serves as a grazing deterrent for copepods, we compared feeding rates, egg production rates, egg hatching success and mortality of the calanoid copepods Acartia tonsa and Temora longicornis feeding on unialgal diets of the toxic diatom P. multiseries and the similarly-sized non-toxic diatom Pseudo-nitzschia pungens. Copepods were collected in summers of 1994, 1995 and 1996 from Shediac Bay, New Brunswick, Canada, near Prince Edward Island, the site of the 1987 episode of domoic acid shellfish poisoning. Rates of ingestion of the toxic versus the non-toxic diatom by A. tonsa and T. longicornis were similar, with only one significantly different pair of values obtained in 1994, for which A. tonsa had a higher mean rate of ingestion of the toxic than the non-toxic diatom. Thus, domoic acid did not appear to retard grazing. Analyses of copepods with high performance liquid chromatography (HPLC) revealed that copepods accumulated domoic acid when feeding on P. multiseries. Egg production rates of copepods when feeding on P. multiseries and P. pungens were very low, ranging from 0 to 2.79 eggs female−1 d−1. There did not appear to be differential egg production or egg hatching success on diets of the toxic and non-toxic diatoms. Mortality of females on the toxic diet was low, ranging from 0 to 20%, with a mean of 13%, and there was no apparent difference between mortality of copepods feeding on toxic versus non-toxic diatoms. Egg hatching success on both diets, although based on few eggs, ranged between 22% and 76%, with a mean percentage hatching of 45%. Diets of the non-toxic diatom plus natural seawater assemblages supplemented with dissolved domoic acid, revealed similar rates and percentages when compared to previous experiments. In summary, none of the variables measured indicated adverse effects on copepods feeding on the toxic compared to the non-toxic diatom.

Mass sexual reproduction in the toxigenic diatoms Pseudo-nitzschia australis and P. pungens (Bacillariophyceae) on the Washington Coast, USA.

Sexual reproduction is documented for the first time in field populations of the pennate diatoms Pseudo‐nitzschia australis Freng. and P. pungens (Grunow ex Cleve) Hasle (var. cingulata Villac and hybrids between var. cingulata and var. pungens). A bloom dominated by these species began on June 26, 2006, along Kalaloch Beach, Washington, USA, coincident with a drop in the Si(OH)4:NO3 ratio to below two. Multimodal size distributions were detected for both species, and synchronous auxosporulation occurred within the smallest size class during a 3‐week window. Auxospores and initial cells created a new class of large cells, and cells in the intermediate size classes increased in abundance during auxosporulation. Mating cells of both species were attached to colonies of surf‐zone diatoms. Paired gametangia, gametes, zygotes, auxospores, and large initial cells were found. Auxosporulation began first for P. pungens (June 30), apparently once a critical, high cell concentration was reached, followed by P. australis (July 5), when the total Pseudo‐nitzschia cell concentration reached 929,000 cells · L−1. Low frequencies of auxosporulation occurred throughout the bloom but increased 4‐fold for P. australis and 3‐fold for P. pungens when macronutrients were reduced to low levels on July 11. A 2‐year life cycle was estimated for P. australis and 3 years for P. pungens, both with annual auxosporulation. Domoic acid (DA) in razor clams reached a maximum of 38 μg DA · g−1 on July 18. A significant relationship existed between the percent of cells within the new size range and DA concentrations in razor clams on the same beach.