The sexual phase of the diatom <Emphasis Type="Italic">Pseudo-nitzschia multistriata</Emphasis>: cytological and time-lapse cinematography characterization

Pseudo-nitzschia is a thoroughly studied pennate diatom genus for ecological and biological reasons. Many species in this genus, including Pseudo-nitzschia multistriata, can produce domoic acid, a toxin responsible for amnesic shellfish poisoning. Physiological, phylogenetic and biological features of P. multistriata were studied extensively in the past. Life cycle stages, including the sexual phase, fundamental in diatoms to restore the maximum cell size and avoid miniaturization to death, have been well described for this species. P. multistriata is heterothallic; sexual reproduction is induced when strains of opposite mating type are mixed, and proceeds with cells producing two functionally anisogamous gametes each; however, detailed cytological information for this process is missing. By means of confocal laser scanning microscopy and nuclear staining, we followed the nuclear fate during meiosis, and using time-lapse cinematography, we timed every step of the sexual reproduction process from mate pairing to initial cell hatching. The present paper depicts cytological aspects during gametogenesis in P. multistriata, shedding light on the chloroplast behaviour during sexual reproduction, finely describing the timing of the sexual phases and providing reference data for further studies on the molecular control of this fundamental process.     

Sexual and vegetative phases in the planktonic diatom Pseudo-nitzschia multistriata

The planktonic diatom Pseudo-nitzschia multistriata (Takano) Takano is known to produce the toxin domoic acid and it is recorded during late summer and autumn in the Gulf of Naples (Tyrrhenian Sea, Italy). We describe the sexual cycle of this species and report information on the variability of growth and cell size reduction rates at different experimental conditions. We induced sexual reproduction by crossing monoclonal cultures of opposite mating type. P. multistriata has a heterothallic life cycle that follows the general pattern reported for other congeneric species. Sexual stages were detected in cultures with an average apical length between 55 and 39 μm. The size of the initial cells produced at the end of the sexual phase was comprised between 72 and 82 μm and the lower cell size detected in culture was 26 μm. Sexual reproduction was thus recorded within a size window corresponding to 39–71% of the maximum cell apical length. Both growth performances and cell size reduction rates depend on cell size. The largest cells showed slower growth rates and larger size reduction rates at each division, while the relationship was opposite for cells smaller then 60% of the maximum size.     

Domoic acid production by Pseudo-nitzschia australis and Pseudo-nitzschia calliantha isolated from North Chile

The morphology and toxicity of the four ubiquitous species belonging to the genus Pseudo-nitzschia found in mixed blooms of phytoplankton from northern Chilean waters were studied. The phytoplankton samples and cultures obtained were identified by scanning electron microscopy, revealing the presence of Pseudo-nitzschia australis, P. calliantha, P. pseudodelicatissima and P. subfraudulenta. This is the first report of P. calliantha in northern Chile. Toxin analyses using the LC–MS method confirmed the presence of domoic acid in P. australis and P. calliantha. Domoic acid was not detected in cultures of P. subfraudulenta. This study therefore confirms P. australis and P. calliantha as an unequivocal source of domoic acid in Chilean waters. P. australis is probably the most important producer of amnesic shellfish toxin in view of its domoic acid content. However, more research is needed to evaluate the potential for toxin production in P. pseudodelicatissima.     

Growth dynamics and domoic acid production of Pseudo-nitzschia sp. in response to oil and dispersant exposure

The diatom genus Pseudo-nitzschia is a common component of phytoplankton communities in the Gulf of Mexico and is potentially toxic as some species produce the potent neurotoxin domoic acid. The impact of oil and chemical dispersants on Pseudo-nitzschia spp. and domoic acid production have not yet been studied; preliminary findings from a mesocosm experiment suggest this genus may be particularly resilient. A toxicological study was conducted using a colony of Pseudo-nitzschia sp. isolated from a station off the coast of Louisiana in the Gulf of Mexico. The cultures were exposed to a water accommodated fraction (WAF) of oil and a diluted chemically enhanced WAF (DCEWAF) which was a mix of oil and dispersant (20:1). Exposure to WAF induced a lag phase but did not inhibit growth rates once in exponential growth. Cultures grown in DCEWAF did not experience a lag phase but had significantly lower growth rates than the Control and WAF cultures. The cellular quota of domoic acid was higher in cultures treated with DCEWAF and WAF relative to their control values, and half of the domoic acid had leaked out of the cells into the surrounding seawater in the DCEWAF cultures while all the domoic acid remained inside the cells in WAF-treated cultures. These results suggest that the presence of oil could lead to toxic blooms, but that the application of dispersant could decrease bioaccumulation of domoic acid through the food web.     

Temporal Changes in Population Structure of a Marine Planktonic Diatom

A prevailing question in phytoplankton research addresses changes of genetic diversity in the face of huge population sizes and apparently unlimited dispersal capabilities. We investigated population genetic structure of the pennate planktonic marine diatom Pseudo-nitzschia multistriata at the LTER station MareChiara in the Gulf of Naples (Italy) over four consecutive years and explored possible changes over seasons and from year to year. A total of 525 strains were genotyped using seven microsatellite markers, for a genotypic diversity of 75.05%, comparable to that found in other Pseudo-nitzschia species. Evidence from Bayesian clustering analysis (BA) identified two genetically distinct clusters, here interpreted as populations, and several strains that could not be assigned with ≥90% probability to either population, here interpreted as putative hybrids. Principal Component Analysis (PCA) recovered these two clusters in distinct clouds with most of the putative hybrids located in-between. Relative proportions of the two populations and the putative hybrids remained similar within years, but changed radically between 2008 and 2009 and between 2010 and 2011, when the 2008-population apparently became the dominant one again. Strains from the two populations are inter-fertile, and so is their offspring. Inclusion of genotypes of parental strains and their offspring shows that the majority of the latter could not be assigned to any of the two parental populations. Therefore, field strains classified by BA as the putative hybrids could be biological hybrids. We hypothesize that P. multistriata population dynamics in the Gulf of Naples follows a meta-population-like model, including establishment of populations by cell inocula at the beginning of each growth season and remixing and dispersal governed by moving and mildly turbulent water masses.     

Phylogeny of the Bacillariaceae with emphasis on the genus Pseudo-nitzschia (Bacillariophyceae) based on partial LSU rDNA

In order to elucidate the phylogeny and evolutionary history of the Bacillariaceae we conducted a phylogenetic analysis of 42 species (sequences were determined from more than two strains of many of the Pseudo-nitzschia species) based on the first 872 base pairs of nuclear-encoded large subunit (LSU) rDNA, which include some of the most variable domains. Four araphid genera were used as the outgroup in maximum likelihood, parsimony and distance analyses. The phylogenetic inferences revealed the Bacillariaceae as monophyletic (bootstrap support ≥90%). A clade comprising Pseudo-nitzschia, Fragilariopsis and Nitzschia americana (clade A) was supported by high bootstrap values (≥94%) and agreed with the morphological features revealed by electron microscopy. Data for 29 taxa indicate a subdivision of clade A, one clade comprising Pseudo-nitzschia species, a second clade consisting of Pseudo-nitzschia species and Nitzschia americana, and a third clade comprising Fragilariopsis species. Pseudo-nitzschia as presently defined is paraphyletic and emendation of the genus is probably needed. The analyses suggested that Nitzschia is not monophyletic, as expected from the great morphological diversity within the genus. A cluster characterized by possession of detailed ornamentation on the frustule is indicated. Eighteen taxa (16 within the Bacillariaceae) were tested for production of domoic acid, a neurotoxic amino acid. Only P. australis, P. multiseries and P. seriata produced domoic acid, and these clustered together in all analyses. Since Nitzschia navis-varingica also produces domoic acid, but is distantly related to the cluster comprising the Pseudo-nitzschia domoic acid producers, it is most parsimonious to suggest that the ability of species in the Bacillariaceae to produce domoic acid has evolved at least twice.     

Species of the planktonic diatom genus Pseudo-nitzschia of the Pacific coasts of Mexico

Species of the diatom genus Pseudo-nitzschia are common in the marine phytoplankton world-wide. Some species of this genus have been proved to be source of domoic acid (DA), a powerful toxin causing Amnesic Shellfish Poisoning (ASP) in humans and probably mass mortality in sea birds and mammals. Net plankton samples obtained during several cruises and seasons from the Pacific coasts of Mexico: western coasts of Baja California, Gulf of California, coasts of the tropical Pacific of Mexico (including the Gulf of Tehuantepec), were analyzed to study the species of the diatom genus Pseudo-nitzschia. Four species ( P. australis, a presumed toxic species, P. fraudulenta, P. lineola, P. pungens) and one former species of the genus, Nitzschia americana were recorded and studied by light and electron microscopy. The most common species was P. pungens, widely distributed along the Pacific coasts of Mexico. All other species appeared occasionally and in low relative abundances. The probable misidentification of P. australis as P. seriata is discussed, as well as the presence of another potentially toxic species, P. delicatissima, in the Gulf of California. No case of toxicity (ASP) has been fully documented and therefore related to toxic Pseudo-nitzschia species in the Gulf of California. This revised version was published online in July 2006 with corrections to the Cover Date.     

Species composition and toxicity of the genus Pseudo-nitzschia in Taiwan Strait,including P. chiniana sp. nov. and P. qiana sp. nov.

In a field survey in the Taiwan Strait during April 2016, the species composition and the domoic acid production of the diatom genus Pseudo-nitzschia were investigated. A total of 80 strains of Pseudo-nitzschia were established, and species identification was determined based on a combination of morphological and molecular data. Fourteen taxa were recognized, i.e., P. americana, P. brasiliana, P. calliantha, P. cuspidata, P. galaxiae, P. lundholmiae, P. multiseries, P. multistriata, P. pseudodelicatissima, P. pungens var. aveirensis, P. pungenus var. pungens and P. sabit, as well as two novel species P. chiniana C.X. Huang & Yang Li and P. qiana C.X. Huang & Yang Li. Morphologically, P. chiniana is characterized by striae comprising one or two rows of poroids, and valve ends that are normally dominated by two rows of poroids within each stria. Whereas P. qiana is unique by having a narrow valve width (1.3–1.5 μm) and sharply pointed valve ends. Both taxa constitute their own monophyletic lineage in the phylogenetic analyses inferred from LSU and ITS2 rDNA, and are well differentiated from other Pseudo-nitzschia species. Pseudo-nitzschia chiniana forms a group with P. abrensis and P. batesiana in LSU and ITS trees, whereas P. qiana is sister to P. lineola. When comparing ITS2 secondary structure, five CBCs and seven HCBCs are recognized between P. chiniana and P. abrensis, and four CBCs and ten HCBCs between P. chiniana and P. batesiana. Two CBCs and eight HCBCs are found between P. qiana with P. lineola. The ability of the strains to produce domoic acid was assessed, including a potential toxin induction by the presence of brine shrimps. Results revealed production of domoic acid in six strains belonging to three species. Without presence of brine shrimps, cellular DA (pDA) was detected in four P. multiseries strains (1.6 ± 0.3, 26.6 ± 2.7, 68.3 ± 4.2 and 56.9 ± 4.7 fg cell⁻¹, separately), one strain of P. pseudodelicatissima (0.8 ± 0.2 fg cell⁻¹) and one strain of P. lundholmiae (2.5 ± 0.4 fg cell⁻¹). In the presence of brine shrimps, pDA contents increased significantly (p < 0.05) in P. lundholmiae (strain MC4218) and P. multiseries (strain MC4177), from 2.5 ± 0.4 to 8.9 ± 0.7 and 1.6 ± 0.3 to 37.2 ± 2.5 fg cell⁻¹ respectively.     

Pseudo-nitzschia simulans sp. nov. (Bacillariophyceae), the first domoic acid producer from Chinese waters

The genus Pseudo-nitzschia has attracted attention because of production of the toxin, domoic acid (DA), causing Amnesic Shellfish Poisoning (ASP). Pseudo-nitzschia blooms occur frequently in Chinese coastal waters, and DA has been detected in several marine organisms, but so far no Pseudo-nitzschia strains from Chinese waters have been shown to produce DA. In this study, monoclonal Pseudo-nitzschia strains were established from Chinese coastal waters and examined using light microscopy, electron microscopy and molecular markers. Five strains, sharing distinct morphological and molecular features differentiating them from other Pseudo-nitzschia species, represent a new species, Pseudo-nitzschia simulans sp. nov. Morphologically, the taxon belongs to the P. pseudodelicatissima group, cells possessing a central nodule and each stria comprising one row of poroids. The new species is characterized by the poroid structure, which typically comprises two sectors, each sector located near opposite margins of the poroid. The production of DA was examined by liquid chromatography tandem mass spectrometry (LC–MS/MS) analyses of cells in stationary growth phase. Domoic acid was detected in one of the five strains, with concentrations around 1.05–1.54 fg cell⁻¹. This is the first toxigenic diatom species reported from Chinese waters.     

The influence of the Pseudo-nitzschia toxin,domoic acid,on microzooplankton grazing and growth: A field and laboratory assessment

We conducted field and laboratory experiments to determine whether the Pseudo-nitzschia-derived metabolite, domoic acid (DA), functions as a microzooplankton grazing suppressant. Using the seawater dilution technique in natural plankton communities along the Pacific Northwest coast, we found no significant relationship between dissolved DA and microzooplankton grazing rate on Pseudo-nitzschia spp. Dilution experiments amended with either 50 or 80 nM dissolved DA also showed no evidence that microzooplankton community grazing was affected by DA. The relationship between Pseudo-nitzschia spp. intracellular DA and microzooplankton grazing was less clear. On a subset of data where small Pseudo-nitzschia spp. cells dominated community composition, an apparent negative relationship between intracellular DA and microzooplankton grazing was observed. However, we provide evidence that this relationship is a microzooplankton response to Pseudo-nitzschia spp. growth rate, rather than cellular DA. In laboratory experiments, two diatom-consuming dinoflagellates, Protoperidinium excentricum and P. pellucidum, were fed single and mixed diets of a toxic and non-toxic Pseudo-nitzschia species and an optimal prey, Ditylum brightwellii. P. excentricum did not grow or ingest either the toxic or non-toxic Pseudo-nitzschia. However, P. pellucidum grew as well on the toxic Pseudo-nitzschia multiseries as it did on D. brightwellii, but did not grow on the non-toxic Pseudo-nitzschia pungens. Both dinoflagellates were capable of growing if Pseudo-nitzschia spp. diets were mixed with D. brightwellii. Addition of dissolved DA also had no negative effect on dinoflagellate growth when fed the optimal diatom diet. We conclude that domoic acid has no functional role in deterring microzooplankton grazing or growth rates. Further, our findings highlight the difficulty of defining the complex mechanisms that regulate predator and prey interactions within microplankton food webs.     

IDENTIFICATION OF CULTURED PSEUDO-NITZSCHIA (BACILLARIOPHYCEAE) USING SPECIES-SPECIFIC LSU rRNA-TARGETED FLUORESCENT PROBES1

Some, but not all, marine pennate diatoms of the genus Pseudo-nitzschia H. Peragallo are associated with the production of domoic acid, a naturally occurring amino acid responsible for amnesic shellfish poisoning. Distinguishing between potentially toxic and nontoxic representatives of this genus is time-consuming and difficult because it demands scanning electron microscopy of cleaned frustules. The objective of this work is to speed and ease identification of these organisms by using whole-cell (in situ) hybridization and species-specific large-subunit ribosomal RNA (LSU rRNA)-targeted oligonucleotide probes. Toward that end, cultures of P. australis Frenguelli, P. pungens (Grunow) Hasle, P. multiseries (Hasle) Hasle, P. fraudulenta (P. T. Cleve) Heiden, P. heimii Manguin, P. delicatissima (P. T. Cleve) Heiden, P. pseudo-delicatissima (Hasle) Hasle, and P. americana (Hasle) Fryxell were screened with a suite of 15 putative species-specific probes. Of those, a subset of eight probes was found that distinguished each species tested. In addition, Pseudo-nitzschia chloroplasts were labeled with a probe directed against a eubacterial-conserved sequence. Identification of new cultures based on their reactivity toward a set of probes agreed with species designations as defined by morphological criteria. Whole-cell hybridization is a rapid, simple, and cost-effective technique for discriminating among cultured Pseudo-nitzschia species.     

An assessment of Pseudo-nitzschia population dynamics and domoic acid production in coastal Louisiana

Over 1200 samples were collected from Louisiana estuarine and coastal shelf waters between 1989 and 2002, and analyzed to examine the population dynamics of Pseudo-nitzschia and to assess the potential threat posed by domoic acid (DA), a potent neurotoxin produced by some members within this toxigenic diatom genus. Results demonstrated that three species in this region (Pseudo-nitzschia multiseries, P. pseudodelicatissima complex, P. delicatissima) produce DA, and that particulate toxin levels were highest (up to 3.05 μg L⁻¹) during the spring bloom, while cellular concentrations were highest in the winter/early spring when P. multiseries was most abundant (up to 30 pg cell⁻¹). These particulate toxin levels are comparable to those seen in other regions (e.g., United States west coast) where DA poisoning events have occurred in the past. Pseudo-nitzschia were most abundant under dissolved inorganic nitrogen-replete conditions coupled with lower silicate and/or phosphate concentrations, and in the early spring months when temperatures were cooler. Pseudo-nitzschia were occasionally well-represented in the phytoplankton assemblage (≥10⁶ cells L⁻¹ in 14% of samples, over 50% of total phytoplankton in 5% of samples), indicating that planktivores (e.g., Gulf menhaden, Brevoortia patronus) may have little choice but to consume Pseudo-nitzschia cells, thereby providing potential vectors for DA transfer to higher trophic levels. By comparison, eastern oysters (Crassostrea virginica) present in estuarine waters may be more exposed to this toxin when Pseudo-nitzschia cells are part of a mixed assemblage, reducing selective grazing by these bivalves. C. virginica may thus represent the most effective vector for DA exposure in humans.     

我国沿海拟菱形藻属的2新记录种及其产毒特征分析

为澄清我国沿海拟菱形藻属(Pseudo-nitzschia)的物种多样性,并确认中国海域拟菱形藻属是否具有产生多莫酸(Domoicacid)的能力,采用毛细管显微操作技术从我国沿海水体中分离、纯化拟菱形藻细胞,建立了单克隆培养株系,并基于核糖体转录间隔区ITS1-5.8S-ITS2 序列构建了分子系统树。结果表明,结合在光学显微镜和透射电镜下观察的形态学特征和分子系统发育分析数据,鉴定到我国拟菱形藻属的2新记录种:银河拟菱形藻(P. galaxiae Lundholm & Moestrup)和微孔拟菱形藻(P. micropora Priisholm, Moestrup & Lundholm),对其形态学特征进行了详细描述,并与相似种类进行了比较研究。利用高效液相色谱(HPLC)技术对多莫酸特征进行了检测,结果表明培养株系并不产生多莫酸。这些为我国拟菱形藻属物种多样性和产毒特征研究提供了基础数据。     

Domoic acid production by Pseudo-nitzschia australis and Pseudo-nitzschia calliantha isolated from North Chile

The morphology and toxicity of the four ubiquitous species belonging to the genus Pseudo-nitzschia found in mixed blooms of phytoplankton from northern Chilean waters were studied. The phytoplankton samples and cultures obtained were identified by scanning electron microscopy, revealing the presence of Pseudo-nitzschia australis, P. calliantha, P. pseudodelicatissima and P. subfraudulenta. This is the first report of P. calliantha in northern Chile. Toxin analyses using the LC–MS method confirmed the presence of domoic acid in P. australis and P. calliantha. Domoic acid was not detected in cultures of P. subfraudulenta. This study therefore confirms P. australis and P. calliantha as an unequivocal source of domoic acid in Chilean waters. P. australis is probably the most important producer of amnesic shellfish toxin in view of its domoic acid content. However, more research is needed to evaluate the potential for toxin production in P. pseudodelicatissima.     

Development of a high-resolution molecular marker for tracking Pseudo-nitzschia pungens genetic diversity through comparative analysis of mitochondrial genomes

The harmful algal bloom (HAB) species Pseudo-nitzschia pungens is widely distributed in almost all continents. Accumulating evidence suggests that P. pungens has high genetic diversity and many strains can produce the toxin domoic acid (DA) that harms animals and humans. Nevertheless, different P. pungens strains cannot be distinguished using morphological features or using common molecular markers including 18S rDNA, 28S rDNA, ITS, cox1, and rbcL. As such, high-resolution molecular markers need to be developed to resolve P. pungens genetic diversity, facilitating accurate tracking of toxic P. pungens strains. We hypothesized that molecular markers with high resolution and high specificity can be designed through identifying regions with high genomic variations in the mitochondrial genome. Here, we describe the development of a new molecular marker Pseudo-nitzschia pungens mitochondrial 1 (ppmt1) with high resolution and high specificity through comparative analysis of mitochondrial genomes of nine P. pungens strains isolated from coastal regions of China. In conclusion, we have developed ppmt1 as a high-resolution and high-specificity molecular marker for tracking strains and genetic diversity of the HAB species P. pungens.

     

An autumn bloom of diatoms of the genus Pseudo-nitzschia H. Peragallo, 1900 in Amursky Bay, the Sea of Japan

A bloom of potentially toxic diatoms of the genus Pseudo-nitzschia was observed in October?CNovember 2005 in the northeastern part of Amursky Bay in the vicinity of Vladivostok (northeastern Sea of Japan) at a salinity of 31?C33.5?? and a water temperature of 6?C12°C. The most intense peak of Pseudo-nitzschia spp. cell density (with a mean value of 1428.9 thousand cells/liter), which was recorded in the second half of October after heavy rains, was mainly caused by the massive development of P. multistriata (67% of the total density) and P. calliantha (9%). A negative correlation was found between Pseudo-nitzschia spp. cell density and water salinity and NH4 concentration; a positive correlation was observed between diatom population density and water temperature.     

Nutrient ratios influence variability in Pseudo-nitzschia species diversity and particulate domoic acid production in the Bay of Seine (France)

The population dynamics of different Pseudo-nitzschia species, along with particulate domoic acid (pDA) concentrations, were studied from May 2012 to December 2013 in the Bay of Seine (English Channel, Normandy). While Pseudo-nitzschia spp. blooms occurred during the two years of study, Pseudo-nitzschia species diversity and particulate domoic acid concentrations varied greatly. In 2012, three different species were identified during the spring bloom (P. australis, P. pungens and P. fraudulenta) with high pDA concentrations (∼1400 ng l⁻¹) resulting in shellfish harvesting closures. In contrast, the 2013 spring was characterised by a P. delicatissima bloom without any toxic event. Above all, the results show that high pDA concentrations coincided with the presence of P. australis and with potential silicate limitation (Si:N < 1), while nitrate concentrations were still replete. The contrasting environmental conditions between 2012 and 2013 highlight different environmental controls that might favour the development of either P. delicatissima or P. australis. This study points to the key role of Pseudo-nitzschia diversity and cellular toxicity in the control of particulate domoic acid variations and highlights the fact that diversity and toxicity are influenced by nutrients, especially nutrient ratios.