PSEUDO-NITZSCHIA SPECIES (BACILLARIOPHYCEAE) IN LOUISIANA COASTAL WATERS: MOLECULAR PROBE FIELD TRIALS, GENETIC VARIABILITY, AND DOMOIC ACID ANALYSES

An 18-month field survey of the Pseudo-nitzschia population present in Louisiana coastal waters was conducted comparing species abundance estimates by novel fluorescent molecular probes (16S large subunit rDNA oligonucleotide sequences) with traditional electron and differential-interference light microscopy. While the probe and microscopic analyses agreed on the presence or absence of four common Pseudo-nitzschia species ( P. multiseries (Hasle) Hasle, P. pseudodelicatissima (Hasle) Hasle, P. delicatissima (P.T. Cleve) Heiden, and P. pungens (Grunow) Hasle in 66% of the samples analyzed, the probes gave conflicting results with the microscopic methods in the remaining 34% of the samples. The majority of the discrepancies appear to be because of genetic variation within the Pseudo-nitzschia population, especially in P. pseudodelicatissima, indicating that the Monterey Bay Pseudo-nitzschia spp. may not be appropriate reference strains for distinguishing Louisiana Pseudo-nitzschia spp. Additionally, P. pseudodelicatissima has been associated with domoic acid (DA) activity in three field samples, at levels up to 22 times higher than the highest value given inother published reports of DA production by this species. The contemporaneous existence of multiple strains of P. pseudodelicatissima (toxic and non-toxic) presents new challenges to the study of the ecophysiology and population dynamics of this bloom-forming species.     

A procedure for large-scale purification of domoic acid from toxic blue mussels (Mytilus edulis)

Pure domoic acid is required for use in research to investigate the biological effects of this new shellfish toxin. It may also prove to be a useful tool in studies exploring the basis of Alzheimer's disease. In this paper we describe a procedure which is effective in obtaining adequate quantities of pure domoic acid from blue mussel (Mytilus edulis). The procedure involves tissue homogenization, treatment of homogenate with chloroform and methanol, and separation of different phases with the addition of water. The aqueous-methanolic phase (upper layer) contains water soluble components including domoic acid, the chloroform phase (lower layer) contains lipoid moieties, and the interphase contains denatured proteins. The aqueous phase containing domoic acid was removed, rotory evaporated to get rid of methanol, followed by ultrafiltration to remove high molecular weight contaminants. The filtrate was lyophilized, resuspended in 1 N HCl, centrifuged and the resulting clear solution subjected to column chromatography on C18 reversed phase silica gel. Fractions containing domoic acid were pooled, and lyophilized. A brownish dry powder contained pure domoic acid with 60–65% yield from the original tissue homogenate. Another 10–15% of domoic acid was mixed with its isomer, and can be further resolved to obtain an overall recovery of 75–80% of the starting material.     

DOMOIC ACID PRODUCTION IN NITZSCHIA SP. (BACILLARIOPHYCEAE) ISOLATED FROM A SHRIMP-CULTURE POND IN DO SON, VIETNAM

Domoic acid (DA), a neuroexcitatory amino acid, was detected in batch culture of the newly recognized species Nitzschia navis-varingica Lundholm et Moestrup . The production of DA by this diatom was confirmed by electrospray ionization mass spectrometry. The diatom was collected from a shrimp-culture pond in Do Son, Vietnam. The production of DA (1.7 pg·cell ^{− 1}) is within the levels reported for Pseudo-nitzschia multiseries (Hasle) Hasle. The DA production started during the late exponential growth phase and reached a maximum during the early stationary growth phase. Maximum DA levels in the axenic culture decreased to about half that of the nonaxenic culture (0.9 pg·cell ^{− 1} vs. 1.7 pg·cell ^{− 1}), suggesting that DA production by the new species is influenced by bacteria.     

Toxic events in the Northwest Pacific coastline of Mexico during 1992–1995: origin and impact

Previously considered as toxin-free, the Baja California Peninsula has witnessed several toxic algal blooms during the past three years. Apparently these ‘red-tide’ phenomena's outbreaks are not linked to any human related activity. This may just reflect better detection and training. Such events may be periodical and natural rather than induced. The most common types of marine toxins have been detected along the coast of the Peninsula and neighboring waters by mouse bioassay and chromatographic techniques. These are: Tetrodotoxin (TTX), Amnesic Shellfish Poison (ASP), Paralytic Shellfish Poisons (PSP), Diarrhetic Shellfish Poisons (DSP) and even Ciguatera (CFP), which are related to the presence of organisms of Prorocentrum sp. and Alexandrium sp. groups, and the diatom Pseudonitzschia sp. among others. There are also some indications about different kinds of TTX in the puffer fish of the region, and reasons to believe that we are facing a quite different pattern in toxic components, since PSP toxic potency (defined as the number of mouse units per gram(MU/g)of shellfish meat) is very high in spite of low dinoflagellates cell density registered. The ecological and social impact of the above has been considerable, with mass deaths of shellfish, seagulls, dolphins and turtles, and even some human casualties. The locally registered toxicity records: PSP found in one single fanshell reaches to 23 000 MU/100 g of tissue as determined by the mouse bioassay and, on a different event, two persons killed after ingesting puffer fish fillet. The largest reservoir of commercial marine organisms in Mexico is precisely the Northwest coast of the country and important plans for building large harbors and develop aquaculture areas are in progress. Therefore, a monitoring program is essential for an adequate management of such resources. Considering the large extension of the Peninsula (about1600 km)and, at this time, the lack of efficient communication means and scarce population, the implementation of such monitoring programs presents a big challenge. This revised version was published online in August 2006 with corrections to the Cover Date.     

The influence of light quality on the development of the brown algae Petalonia and Scytosiphon

Petalonia fascia (O. F. Müll.) Kuntze, P. zosterifolia (Reinke) Kuntze and Scytosiphon lomentaria (Lyngb.) J. Ag. have been cultured in white, blue and red light of equal quantum irradiance at 15°C. Hairs, knot-filaments and Ralfsia-like crusts were formed only in blue light, whereas the prostrate system of red-grown plants consisted entirely of sparingly-branched and mostly uniseriate filaments. The production of erect thalli from the prostrate system was controlled or stimulated by red light, but these erect thalli became fertile only in the presence of blue light. All three species exhibited all of these types of response, although specific differences in the degree of certain types of response were observed.     

FLOW CYTOMETRY AND MICROSCOPY OF GAMETOGENESIS IN NITZSCHIA PUNGENS,A TOXIC,BLOOM-FORMING,MARINE DIATOM1

The domoic acid-producing diatom Nitzschia pungens Grunow f. multiseries Hasle, which is responsible for amnesic shellfish poisonings in Prince Edward Island, Canada, underwent gametogenesis when senescent cells (i.e. in stationary growth phase for more than 290 days) were subcultured into fresh FE medium and light intensity was increased from 33 to 530 μE · m^?2· s^?1. The number of gametes produced varied with the salinity of the medium, with a maximum at 23.5‰. Cells in the exponential growth phase (0.8 div · d^?1) did not produce gametes, nor did senescent cells when transferred without change in light intensity. Anisogamous gametes, probably haploid, were isolated by combining conventional microscopy with flow cytometry. Zygotes resulting from syngamy yielded cigar-shaped naviculoid cells, morphologically different from parent cells (heteromorphism). These cells, with a division rate of 1.9 div · d^?1, could serve as a seed population and explain the origin and rapid progression of the toxic blooms of red-water proportions that have been a public health problem in Eastern Canada. Production of domoic acid by postexponential and moribund cells but not by gametes, zygotes, or immediately resulting cells, provides an insight into the dependence of toxicity on the developmental history of this diatom.     

The Probable Mechanism for Silicon Capture by Diatom Algae: Assimilation of Polycarbonic Acids with Diatoms—Is Endocytosis a Key Stage in Building of Siliceous Frustules?

Many organisms including unicellular (diatoms, radiolaria, and chrysophytes), higher plants (rice and horsetail) and animals (sponges) use silica as a main part of skeletons. The bioavailable form of silicon is silicic acid and the mechanism of silicic acid penetration into living cells is still an enigma. Macropinocytosis was assumed as a key stage of the silicon capture by diatoms but assimilation of monomeric silicic acid by this way requires enormous amounts of water to be passed through the cell. We hypothesized that silicon can be captured by diatoms via endocytosis in the form of partially condensed silicic acid (oligosilicates) whose formation on the diatom surface was supposed. Oligosilicates are negatively charged nanoparticles and similar to coils of poly(acrylic acid) (PAA). We have synthesized fluorescent tagged PAA as well as several neutral and positively charged polymers. Cultivation of the diatom Ulnaria ferefusiformis in the presence of these polymers showed that only PAA is able to penetrate into siliceous frustules. The presence of PAA in the frustules was confirmed with chromatography and PAA causes various aberrations of the valve morphology. Growth of U. ferefusiformis and two other diatoms in the presence of tri- and tetracarbonic fluorescent tagged acids points to the ability of diatoms to recognize substances that bear four acidic groups and to include them into siliceous frustules. Thus, partial condensation of silicic acid is a plausible first stage of silicon assimilation.     

A guide to the pronunciation of the scientific names for harmful algae

A historical review of scientific nomenclature and ofthe pronunciation of classical languages suggests thatthere is no objectively correct way to enunciate thetechnical terms applied to harmful algae. Any guideto pronunciation is always relative to some group ofspeakers; scientific nomenclature is an artificialconstruct without a population of normative speakers,living or dead, to whom the bewildered enunciator canhave reference. Thus a key to the pronunciation ofthe Latin and Greek scientific terms in alldisciplines, and a fortiori to the pronunciation ofthose terms applied to harmful algae, must be based onrules of common sense, mutual forbearance, and generalintelligibility. This article includes a guide topronouncing the names of harmful algae based on theseprinciples.     

Novel symptomatology and changing epidemiology of domoic acid toxicosis in California sea lions (Zalophus californianus): an increasing risk to marine mammal health

Harmful algal blooms are increasing worldwide, including those of Pseudo-nitzschia spp. producing domoic acid off the California coast. This neurotoxin was first shown to cause mortality of marine mammals in 1998. A decade of monitoring California sea lion (Zalophus californianus) health since then has indicated that changes in the symptomatology and epidemiology of domoic acid toxicosis in this species are associated with the increase in toxigenic blooms. Two separate clinical syndromes now exist: acute domoic acid toxicosis as has been previously documented, and a second novel neurological syndrome characterized by epilepsy described here associated with chronic consequences of previous sub-lethal exposure to the toxin. This study indicates that domoic acid causes chronic damage to California sea lions and that these health effects are increasing.     

Detection of the Dinozoans Pfiesteria piscicida and P. shumwayae: a review of detection methods and geographic distribution

Molecular methods, including conventional PCR, real-time PCR, denaturing gradient gel electrophoresis, fluorescent fragment detection PCR, and fluorescent in situ hybridization, have all been developed for use in identifying and studying the distribution of the toxic dinoflagellates Pfiesteria piscicida and P. shumwayae. Application of the methods has demonstrated a worldwide distribution of both species and provided insight into their environmental tolerance range and temporal changes in distribution. Genetic variability among geographic locations generally appears low in rDNA genes, and detection of the organisms in ballast water is consistent with rapid dispersal or high gene flow among populations, but additional sequence data are needed to verify this hypothesis. The rapid development and application of these tools serves as a model for study of other microbial taxa and provides a basis for future development of tools that can simultaneously detect multiple targets.     

贝类毒素监测的动物试验优化及替代方法

贝类毒素严重威胁水产品的质量,而且给人类健康带来潜在危害。由于其结构多样、作用机制复杂,贝类毒素监控计划通常使用动物方法,不仅成本高、定量难,而且不符合＂3R＂的要求。运用减轻动物痛苦和减少数量的优化方法,开发新的基于毒性作用机制和结构分析的细胞功能检测法、免疫学方法、化学分析法和生物传感器方法,这些动物试验替代方法的研究提高了贝类毒素监测的有效性,经过科学验证和认可后可用于监控目的 。