ULTRASTRUCTURE OF THE RED TIDE DINOFLAGELLATE GYMNODINIUM BREVE. I. GENERAL DESCRIPTION1,2,3

Gymnodimium breve Davis, an unarmored marine dinoflagellate has a cell covering (theca) composed of four membranes. The inner two membranes represent a vesicular layer and in tangential section, the theca appears composed of polygonal areas. Unusual threat ridges are located in the cingular region between the epi- and hypocone. This osmotically sensitive species is extremely vesiculate with dispersed areas of cytoplasm containing typical eukaryotic organelles as well as other organelles found only in dinoflagellates. The non-vesiculated cytoplasm is continuous in serial sections. The chloroplasts can contain either quasi-radial or parallel lamellae typically consisting of three thylakoids each. The pyrenoid is multiple-stalked and lacks a starch cap. The dinophycean pusule is simple and similar to those found in several unarmored marine species. The nucleus is typically dinophycean but the chromosomes appear to lack nonfibrillar material.     

PFIESTERIA PISCICIDA GEN. ET SP. NOV. (PFIESTERIACEAE FAM. NOV.), A NEW TOXIC DINOFLAGELLATE WITH A COMPLEX LIFE CYCLE AND BEHAVIOR1

The newly described toxic dinoflagellate Pfiesteria piscicida is a polymorphic and multiphasic species with flagellated, amoeboid, and cyst stages. The species is structurally a heterotroph; however, the flagellated stages can have cleptochloroplasts in large food vacuoles and can temporarily function as mixotrophs. The flagellated stage has a typical mesokaryotic nucleus, and the theca is composed of four membranes, two of which are vesicular and contain thin plates arranged in a Kofoidian series of Po, cp, X, 4′, 1a, 5″, 6c, 4s, 5″′, and 2″″. The plate tabulation is unlike that of any other armored dinoflagellate. Nodules often demark the suture lines underneath the outer membrane, but fixation protocols can influence the detection of plates. Amoeboid benthic stages can be filose to lobose, are thecate, and have a reticulate or spiculate appearance. Amoeboid stages have a eukaryotic nuclear profile and are phagocytic. Cyst stages include a small spherical stage with a honeycomb, reticulate surface and possibly another stage that is elongate and oval to spherical with chrysophyte-like scales that can have long bracts. The species is placed in a new family, Pfiesteriaceae, and the order Dinamoebales is emended.     

Kryptoperidinium foliaceum blooms in South Carolina: a multi-analytical approach to identification

Observations following the discovery of Kryptoperidinium foliaceum blooms in South Carolina (SC), USA, suggest that a multi-analytical approach, using a standard, minimal set of criteria, should be adopted for determining dinoflagellate species identity and taxonomic placement. A combination of morphological, molecular, and biochemical analyses were used to determine the identity of this “red tide” dinoflagellate, first documented in SC waters in the spring of 1998. Results from thecal plate tabulations (based on scanning electron and epifluorescence microscopy), gene sequence data, species-specific PCR probe assays, and microalgal pigment profiles were analyzed and compared to reference cultures of K. foliaceum. Comparative data showed marked inconsistencies among the K. foliaceum reference culture isolates. In addition, the SC bloom isolate was shown to be mononucleate, contrary to previous reports for K. foliaceum, suggesting a more transient endosymbiotic association than previously considered.     

SEXUAL REPRODUCTION IN THE TOXIC DINOFLAGELLATE GONYAULAX MONILATA1

The sexual cycle of Gonyaulax monilata Howell was observed in stationary cultures and in nitrogen-deficient medium. The armored, isogamous gametes fuse in a characteristic manner with cingula at oblique angles. Nuclear fusion lags slightly behind cytoplasmic fusion. The zygote enlarges for several days. The dark, double-flagellated planozygote encysts within 1–3 wk. Early hypnozygotes are round to ovoid and contain lipid and one or two large golden-yellow globules. As the hypnozygote matures, the globules become smaller and the cytoplasm darkens and pulls from the wall. All cysts examined contained only one nucleus. A very dark, uninucleate post-hypnozygotic cell escapes through an archeopyle and within 24 h divides into daughter cells which divide in 24–48 h forming a small chain. The production of thick walled zygates in culture implies that such resting stages in marine sediments could serve as a source stock for blooms. This species causes toxic red tides and the existence of benthic “seed beds” consisting of hypnozygotes is now plausible.     

DESCRIPTION OF A NEW GENUS OF PFIESTERIA‐LIKE DINOFLAGELLATE,LUCIELLA GEN. NOV. (DINOPHYCEAE), INCLUDING TWO NEW SPECIES: LUCIELLA MASANENSIS SP. NOV. AND LUCIELLA ATLANTIS SP. NOV.1

A new genus of Pfiesteria‐like heterotrophic dinoflagellate, Luciella gen. nov., and two new species, Luciella masanensis sp. nov. and Luciella atlantis sp. nov., are described. These species commonly occur with other small (<20 μm) heterotrophic and mixotrophic dinoflagellates in estuaries from Florida to Maryland and the southern coast of Korea, suggesting a possible global distribution. An SEM analysis indicates that members of the genus Luciella have the enhanced Kofoidian plate formula of Po, cp, X, 4′, 2a, 6″, 6c, PC, 5+s, 5?, 0p, and 2″″. The two four‐sided anterior intercalary plates are diamond shaped. The genus Luciella differs from the other genera in the Pfiesteriaceae by a least one plate in the plate tabulation and in the configuration of the two anterior intercalary plates. An SSU rDNA phylogenetic analysis confirmed the genus as monophyletic and distinct from the other genera in the Pfiesteriaceae. The morphology of Luciella masanensis closely resembles Pfiesteria piscicida Steid. et J. M. Burkh. and other Pfiesteria‐like dinoflagellates in size and shape, making it easily misidentified using LM. Luciella atlantis, in contrast, has a more distinctive morphology. It can be distinguished from L. masanensis and other Pfiesteria‐like organisms by a larger cell size, a more conical‐shaped epitheca and hypotheca, larger rhombic‐shaped intercalary plates, and an asymmetrical hypotheca. The genus Luciella is assigned to the order Peridiniales and the family Pfiesteriaceae based on plate tabulation, plate pattern, general morphology, and phylogenetic analysis.     

A neuroprotective role for angiogenin in models of Parkinson's disease

We previously observed marked down-regulation of the mRNA for angiogenin, a potent inducer of neovascularization, in a mouse model of Parkinson's disease (PD) based on over-expression of alpha-synuclein. Angiogenin has also been recently implicated in the pathogenesis of amyotrophic lateral sclerosis. In this study, we confirmed that mouse angiogenin-1 protein is dramatically reduced in this transgenic alpha-synuclein mouse model of PD, and examined the effect of angiogenin in cellular models of PD. We found that endogenous angiogenin is present in two dopamine-producing neuroblastoma cell lines, SH-SY5Y and M17, and that exogenous angiogenin is taken up by these cells and leads to phosphorylation of Akt. Applied angiogenin protects against the cell death induced by the neurotoxins 1-methyl-4-phenylpyridinium and rotenone and reduces the activation of caspase 3. Together our data supports the importance of angiogenin in protecting against dopaminergic neuronal cell death and suggests its potential as a therapy for PD.     

Effect of salinity on the distribution, growth, and toxicity of Karenia spp.

The first recorded bloom of Karenia spp., resulting in brevetoxin in oysters, in the low salinity waters of the Northern Gulf of Mexico (NGOMEX) occurred in November 1996. It raised questions about the salinity tolerance of Karenia spp., previously considered unlikely to occur at salinities <24 psu, and the likelihood that the bloom would reoccur in the NGOMEX. Salinity was investigated as a factor controlling Karenia spp. abundance in the field, using data from the NGOMEX 1996 bloom and Florida coastal waters from 1954 to 2004, and growth and toxin production in cultures of Karenia brevis (Davis) G. Hansen and Moestrup. During the NGOMEX bloom, Karenia spp. occurred much more frequently at low salinities than in Florida coastal waters over the last 50 years. The data suggest that the NGOMEX bloom started on the NW Florida Shelf, an area with a higher frequency of Karenia spp. at low salinities than the rest of Florida, and was transported by an unusual westward surface current caused by Tropical Storm Josephine. The minimum salinity at which growth occurred in culture ranged between 17.5 and 20 psu, but the optimal salinity ranged between low values of 20 or 25 and high values of 37.5–45 psu, depending on the clone. The effect of salinity on toxin production in one clone of K. brevis was complex, but at all salinities brevetoxin levels were highest during the stationary growth phase, suggesting that aging, high density blooms may pose the greatest public health threat. The results demonstrate that Karenia spp. can be a public health threat in low salinity areas, but the risk in the NGOMEX is relatively low. No bloom has occurred since the 1996 event, which was probably associated with a special set of conditions: a bloom along the Florida Panhandle and a tropical storm with a track that set up a westward current.     

FIRST REPORT OF GYMNODINIUM PULCHELLUM (DINOPHYCEAE) IN NORTH AMERICA AND ASSOCIATED FISH KILLS IN THE INDIAN RIVER, FLORIDA

Fish and invertebrate kills were reported from September to October 1996 in the Indian River, Florida, coincident with blooms of the dinoflagellate Gymnodinium pulchellum Larsen 1994. This is the first report of a bloom of this species in the Americas. Fish and invertebrate species affected were common snook ( Centropomus undecimalis ), striped mullet ( Mugil cephalus ), hardhead catfish ( Arius felis ), red drum ( Sciaenops ocellatus ), sheepshead ( Archosargus probatocephalus ), black drum ( Pogonias cromis ), blue crab ( Callinectes sapidus ), and shrimp ( Penaeus spp.). However, Gymnodinium pulchellum has previously caused fish kills in Japan and Australia. Examination of archived phytoplankton samples from a fish kill reported in the same area of the Indian River in August 1990 confirmed the presence of high concentrations of G. pulchellum. Fish kills associated with Alexandrium monilatum and potentially Pfiesteria -like species in the Indian River also are discussed. Scanning electron microscopy provided additional morphological detail on this distinct but little-known dinoflagellate.     

Relating Phytoplankton Dynamics to Environmental Forcing in the Lower St. Johns River Estuary: A Multivariate Assessment

The St. Johns River, a 300‐mile riverine and estuarine system located along the north‐eastern coastline of Florida, has undergone extensive eutrophication through point and nonpoint source nutrient inputs. Moreover, recent reports of sudden fish kills and a high incidence of fish with lesions suggest the potential for harmful algal blooms. As part of a NOAA‐funded project involving the characterization of water quality parameters in relation to water inflows and nutrient inputs and the development of species‐specific markers/probes for instrumental‐based monitoring efforts, we used primer‐based multivariate analyses to examine the relationship between abiotic variables and both total and phylogenetic‐group chlorophyll a concentrations (derived from photopigments and ChemTax matrix factorization of diagnostic carotenoids) during 2001. Seven sampling sites (identified through principle components ordination of physical/chemical parameters as ranging from oligo‐ to mesohaline) were sampled intensively over 2‐week periods on a seasonal basis. Chlorophyll a concentrations typically ranged from 5 to 35 µg/L with the greatest concentrations occurring at the oligohaline sites. Phytoplankton assemblages were dominated by diatoms, cryptophytes, and cyanobacteria, and together typically comprised up to 90% of the total chlorophyll a. Temporal variability in phytoplankton assemblages followed seasonal trends impacted by meteorological and hydrological forcing. Spatial variability in phylogenetic‐group abundance (illustrated through multidimensional scalar ordination of sample dissimilarity) was dramatic and associated with differences in abiotic variables along the estuarine gradient.     

IDENTIFICATION OF PFIESTERIA PISCICIDA (DINOPHYCEAE) AND PFIESTERIA‐LIKE ORGANISMS USING INTERNAL TRANSCRIBED SPACER‐SPECIFIC PCR ASSAYS1

The putative harmful algal bloom dinoflagellate, Pfiesteria piscicida (Steidinger et Burkholder), frequently co‐occurs with other morphologically similar species collectively known as Pfiesteria‐like organisms (PLOs). This study specifically evaluated whether unique sequences in the internal transcribed spacer (ITS) regions, ITS1 and ITS2, could be used to develop PCR assays capable of detecting PLOs in natural assemblages. ITS regions were selected because they are more variable than the flanking small subunit or large subunit rRNA genes and more likely to contain species‐specific sequences. Sequencing of the ITS regions revealed unique oligonucleotide primer binding sites for Pfiesteria piscicida, Pfiesteria shumwayae (Glasgow et Burkholder), Florida “Lucy” species, two cryptoperidiniopsoid species, “H/V14” and “PLO21,” and the estuarine mixotroph, Karlodinium micrum (Leadbetter et Dodge). These PCR assays had a minimum sensitivity of 100 cells in a 100‐mL sample (1 cell·mL^?1) and were successfully used to detect PLOs in the St. Johns River system in Florida, USA. DNA purification and aspects of PCR assay development, PCR optimization, PCR assay controls, and collection of field samples are discussed.