Gonyaulax monilata: Population Growth and Development of Toxicity in Cultures

SYNOPSIS. The development of three 8-liter and four 12-liter cultures of the photosynthetic dinoflagellate Gonyaulax monilata was followed for 4 months. Weekly estimates were made of population levels of this chain-forming flagellate, along with incidence of cells in chains and toxicity to fish. Guppies ( Lebistes reticulatus ) were used to assay toxicity. Populations reached a peak when cultures were 3–5 weeks old, declined during weeks 6–10, and tended to stabilize thereafter thru the 17th (final week). The percentage of cells in chains was related to the slope of the population curve; rapidly increasing populations had the highest proportion of long chains, suggesting that incidence of chains is an index of the growth phase in G. monilata. Peak toxicity was not reached until culture populations had been steadily declining for a month, indicating that most toxin is released by autolysis. The reproducibility of culture population and toxicity levels recommend the methods used for future studies.     

CELL CYCLE REGULATORS IN THE FLORIDA RED TIDE DINOFLAGELLATE, GYMNODINIUM BREVE

The diel cycle is a key regulator of the cell cycle in many dinoflagellates, and may play a rate limiting role in bloom formation. Diel phasing of the cell cycle in the Florida red tide dinoflagellate, Gymnodinium breve Davis was previously described in our laboratory. In cultures grown on a 16:8 light:dark cycle, S-phase began 6–8 h into the light phase, and mitosis followed 12–14 h later. The dark/light "dawn" transition was found to provide the diel cue that serves to entrain the G. breve cell cycle. However the cell cycle mechanisms and regulators acted upon by this cue are poorly understood in dinoflagellates. The cell cycle regulatory complex, CDK1-cyclinB, is therefore currently being investigated. Cyclin dependent kinase (CDK) was first identified in G. breve using two approaches: (1) identification of a 34 kDa protein immunoreactive to an antibody raised against a conserved amino acid sequence unique to the CDK protein family (PSTAIR) and (2) inhibition of the cell cycle by olomoucine, a selective CDK inhibitor. Several approaches are currently being employed in order to describe its partner, cyclin B: (1) PCR on genomic DNA with primers deduced from known cyclin box sequences, (2) G. breve expression library screening with an antibody raised against the fission yeast cyclin B (3) western blot analysis on whole protein extracts and cyclin B immunoprecipitated proteins. Current work focuses on the differential expression of the cyclin B homologue in G. breve during its cell cycle and its relation to diel cycle control.     

A RE-EVALUATION OF CORALLINE RED ALGAL TAXONOMY USING ULTRA-STRUCTURAL INFORMATION

The marine dinoflagellate, Gymnodinium breve (Davis), produces several neurotoxins that cause neurotoxic shellfish poisoning (nsp), massive fish kills and respiratory irritation in marine mammals and humans. The common method for discerning toxic levels of G. breve for public health advisories is enumeration of live cells in a given water mass. In this study, laboratory cultures, as well as natural blooms, were added to a stirred ultra-filtration cell concentrator to separate viable cells containing intra-cell toxins from ambient water containing extra-cell toxins. Methods were validated using various mixtures of lysed and whole G. breve laboratory culture. Extractions and recovery of brevetoxins were done using a C-18 bonded-phase glass fiber extraction disc eluted with methanol. Total PbTx toxin concentrations were quantified by HPLC/UV using a C-18 column and an 85:15 methanol:water (1 ml min⁻¹) isocratic elution at 215 nm. This method of separation and extraction was subsequently applied to water samples collected during natural blooms along two different areas of the Florida Gulf coast. The results indicated that early stages of G. breve blooms contained primarily intra-cell toxins with extra-cell toxins increasing as the bloom progressed, even though very few viable G. breve cells were present. This suggests that enumeration of cells alone may be insufficient and additional toxin quantitation is necessary.     

DIEL PHASING OF THE CELL-CYCLE IN THE FLORIDA RED TIDE DINOFLAGELLATE, GYMNODINIUM BREVE

The diel cycle is a key regulator of the cell-cycle in many dinoflagellates, but the mechanisms by which the diel cycle entrains the cell-cycle remain poorly understood. In this study, we describe diel phasing of the cell-cycle in the Florida red tide dinoflagellate Gymnodinium breve Davis, determine the diel cue which serves to entrain the cell-cycle, and provide evidence for the presence of cyclin-dependent kinase (CDK), a cell-cycle regulator which may be responsive to this cue. Four laboratory isolates from the West Coast of Florida were compared. When grown on a 16:8 h LD cycle, all isolates displayed phased cell division, with the S-phase beginning 6–8 h into the light phase, and mitosis following 12–14 h later, as determined by flow cytometry. A naturally occurring bloom of G. breve, studied over one diel cycle, displayed diel cell-cycle phasing similar to that in the laboratory cultures, with the S-phase beginning during daylight and the peak of mitosis occurring approximately 4 h after sunset. In the laboratory cultures, the dark/light "dawn" transition was found to provide the diel cue which serves to entrain the G. breve cell-cycle, whereas the light/ dark "dusk" transition did not appear to be involved. Evidence for the presence of CDK in G. breve was obtained using two approaches: (1) identification of a 34-kDa protein, immunoreactive to an antibody against a conserved amino acid sequence (α-PSTAIR) unique to the CDK protein family and (2) inhibition of the cell-cycle by olomoucine, a selective CDK inhibitor. Together, these results provide the basis from which one can begin addressing mechanisms by which the diel cycle regulates the cell-cycle in G. breve.     

A critical analysis of the biological impacts of plasticizers on wildlife

This review provides a critical analysis of the biological effects of the most widely used plasticizers, including dibutyl phthalate, diethylhexyl phthalate, dimethyl phthalate, butyl benzyl phthalate and bisphenol A (BPA), on wildlife, with a focus on annelids (both aquatic and terrestrial), molluscs, crustaceans, insects, fish and amphibians. Moreover, the paper provides novel data on the biological effects of some of these plasticizers in invertebrates, fish and amphibians. Phthalates and BPA have been shown to affect reproduction in all studied animal groups, to impair development in crustaceans and amphibians and to induce genetic aberrations. Molluscs, crustaceans and amphibians appear to be especially sensitive to these compounds, and biological effects are observed at environmentally relevant exposures in the low ng l⁻¹ to µg l⁻¹ range. In contrast, most effects in fish (except for disturbance in spermatogenesis) occur at higher concentrations. Most plasticizers appear to act by interfering with the functioning of various hormone systems, but some phthalates have wider pathways of disruption. Effect concentrations of plasticizers in laboratory experiments coincide with measured environmental concentrations, and thus there is a very real potential for effects of these chemicals on some wildlife populations. The most striking gaps in our current knowledge on the impacts of plasticizers on wildlife are the lack of data for long-term exposures to environmentally relevant concentrations and their ecotoxicity when part of complex mixtures. Furthermore, the hazard of plasticizers has been investigated in annelids, molluscs and arthropods only, and given the sensitivity of some invertebrates, effects assessments are warranted in other invertebrate phyla.     

Cleavage patterns, cell-lineages and cell specification are clues to phyletic lineages in Spiralia

Embryos of molluscs, annelids, nemerteans and platyhelminthes show remarkable intra- and interphyletic resemblances and differences in mesentoblast, dorso-ventral axis and trochoblast specification. These variations have been used to investigate their evolutionary relationship. In molluscs and annelids a heterochronic shift parallels evolutionary relations based on adult characters. Nemerteans and platyhelminthes lack trochal cells and differ in the specification of the mesodermal precursor cell. Nemerteans also differ fundamentally with respect to axis specification related to the first cleavage. Therefore, close phylogenetic relations exist between molluscs and annelids, whereas nemerteans and platyhelminthes are only remotely related with each other and with molluscs and annelids.     

Phylogenetic relationships of annelids,molluscs, and arthropods evidenced from molecules and morphology

Annelids and arthropods have long been considered each other's closest relatives, as evidenced by similarities in their segmented body plans. An alternative view, more recently advocated by investigators who have examined partial 18S ribosomal RNA data, proposes that annelids, molluscs, and certain other minor phyla with trochophore larva stages share a more recent common ancestor with one another than any do with arthropods. The two hypotheses are mutually exclusive in explaining spiralian relationships. Cladistic analysis of morphological data does not reveal phylogentic relationships among major spiralian taxa but does suggest monophyly for both the annelids and molluscs. Distance and maximum-likelihood analyses of 18S rRNA gene sequences from major spiralian taxa suggest a sister relationship between annelids and molluscs and provide a clear resolution within the major groups of the spiralians. The parsimonious tree based on molecular data, however, indicates a sister relationship of the Annelida and Bivalvia, and an earlier divergence of the Gastropoda than the Annelida–Bivalvia clade. To test further hypotheses on the phylogenetic relationships among annelids, molluscs, and arthropods, and the ingroup relationships within the major spiralian taxa, we combine the molecular and morphological data sets and subject the combined data matrix to parsimony analysis. The resulting tree suggests that the molluscs and annelids form a monophyletic lineage and unites the molluscan taxa to a monophyletic group. Therefore, the result supports the Eutrochozoa hypothesis and the monophyly of molluscs, and indicates early acquisition of segmented body plans in arthropods. Received: 25 September 1995 / Accepted: 15 March 1996     

USE OF THE CRICKET ACHETA DOMESTICA L. AS A BIOASSAY ORGANISM FOR THE TOXIC EXTRACT FROM GONYAULAX MONILATA (DINOPHYCEAE)1

A toxic extract from unialgal cultures, of Gonyaulax monilata Howell was tested for insecticidal activity on the cricket Acheta domestica L The regression line of the activity was compared with that of ouabain in this insect as well as with that of the toxic extract in mice. Although mice are approximately 2.5°as sensitive as the cricket an a body weight basis, because of the insect's smaller mass‘(1/50 that of the mouse), one can treat 26° as many crickets with the same amount of toxic material. This fact, coupled with the simple rearing requirements of the insect, makes it an ideal bioassay organism as well as a useful subject far further investigations of the physiological properties of the toxic extract and other bioactive compounds available in limited quantities.     

Distribution and localization of the digestive laminarinases in animals

The presence of laminarinases in the digestive system of a number of invertebrate and vertebrate species has been investigated. The enzymes were detected in coelenterates, annelids, sipunculids, bryozoans, molluscs, arthropods (crustaceans and insects), echinoderms, tunicates and fishes. Since it could be demonstrated that, in fishes, at least, laminarinases are secreted by the intestinal mucosa rather than by associated gut bacteria, an attempt was made to correlate the distribution of these digestive enzymes with the feeding habits of the animals. The correlation is particularly evident among fishes where in the genus Leuciscus carnivores lack laminarinases in contrast to omnivores. From an evolutionary point of view, we can postulate that the possession of laminarinases is ancient in Metazoa and has been either kept or lost by more evolved taxa depending on their feeding specialization, as has already been demonstrated for chitinases and cellulases.     

THE COMPOSITION AND TOXICITY OF DINOFLAGELLATE BLOOMS IN THE SALTON SEA, CALIFORNIA

Phytoplankton blooms have been implicated in mortality events of diverse groups of organisms including fish, birds and humans. About 300 species have been reported to form "red tides," or surface discolorations due to high densities, but only 60–80 of these species produce harmful blooms. In marine systems, dinoflagellates account for 75% of all harmful algal bloom species. The Salton Sea is a large saline lake located in southeastern California, USA. The lake is eutrophic largely because it is in a closed basin and receives most of its input from agricultural and municipal wastewaters. Dinoflagellates comprise a significant portion of the phytoplankton biomass, particularly in winter, often resulting in "red" or "brown" tides. To date, 16 species of dinoflagellates have been identified from the Salton Sea, and many other unidentified forms have also been documented. In 1992, 150,000 eared grebes were found dead over a period of several months at the Salton Sea. This mortality event was among the largest of any bird species. The principal cause remains unknown, but algal toxins were suspected. A survey of the composition and toxicity of algal blooms was undertaken in 1999, and we report results from blooms where dinoflagellates dominated. Dominant species included Gonyaulax grindleyi , Gymnodinium spp., Gyrodinium uncatenum , Heterocapsa niei , and an unidentified scrippsielloid. Although most samples showed activity in a brine shrimp lethality assay, all were negative in a mouse bioassay. This evidence suggests that toxins from dinoflagellate blooms in the Salton Sea are not responsible for eared grebe mortality events.     

Food and feeding habits of Ilisha africana (Bloch) (Pisces: Clupeidae) off the Lagos coast, Nigeria

A total of 2215 specimens of Ilisha africana (Bloch) were examined for the food habits of the species off the Lagos coast, Nigeria. Their food consisted mainly of crustaceans, fish and molluscs; less important in the stomach contents were insects, annelids, chaetognaths, coelenterates, urochordates and phytoplankton. The incidence of nematodes in the stomachs was low. Monthly variation of food as well as variation with fish size and water depth were examined, as was the relationship of food composition to components of the plankton fauna. Feeding habits of I. africana were compared with those Sardinella maderensis (Lowe), Galeoides decadatylus (Bloch), Brachydeuterus auritus (Val.) and Vomer setapinnis (Mitchill); there was an overall diversity in the diets of these fish species.     

Animals associated with sponges at North Hayling, Hampshire

Sponges were collected over a period of two and a half years from the shore at North Hayling, Hampshire and their associated faunas studied.
Species of nematode, annelids, crustaceans, pyenogonids, echinoderms and fish associate with sponges Halichondria panicea, Hymeniacidon perleve and Mycale macilenta; other sponge species attract noticably very few. No animals were sponge specific.     

Dinoflagellate sterols I: Sterol c omposition of the dinoflagellates of Gonyaulax species

The sterol composition of five species of dinoflagellates of the family Gonyaulacaeae (Div. Pyrrhophyta) were examined. All the five species (Gonyaulax acatenella, G. tamarensis, G. catennela, G. washing-tonesis, and G. polyedra) were found to contain 4 alpha-23,24(R)-trimethyl-5 alpha-cholest-22-en-3 -ol (dinosterol) and cholesterol as major sterols.     

Phylogenetic analyses indicate that the 19'Hexanoyloxy-fucoxanthin-containing dinoflagellates have tertiary plastids of haptophyte origin

The three anomalously pigmented dinoflagellates Gymnodinium galatheanum, Gyrodinium aureolum, and Gymnodinium breve have plastids possessing 19'-hexanoyloxy-fucoxanthin as the major carotenoid rather than peridinin, which is characteristic of the majority of the dinoflagellates. Analyses of SSU rDNA from the plastid and the nuclear genome of these dinoflagellate species indicate that they have acquired their plastids via endosymbiosis of a haptophyte. The dinoflagellate plastid sequences appear to have undergone rapid sequence evolution, and there is considerable divergence between the three species. However, distance, parsimony, and maximum-likelihood phylogenetic analyses of plastid SSU rRNA gene sequences place the three species within the haptophyte clade. Pavlova gyrans is the most basal branching haptophyte and is the outgroup to a clade comprising the dinoflagellate sequences and those of other haptophytes. The haptophytes themselves are thought to have plastids of a secondary origin; hence, these dinoflagellates appear to have tertiary plastids. Both molecular and morphological data divide the plastids into two groups, where G. aureolum and G. breve have similar plastid morphology and G. galatheanum has plastids with distinctive features.     

Some studies on the biosynthesis of ubiquinone, isoprenoid alcohols, squalene and sterols by marine invertebrates

The ability of fourteen marine invertebrates to utilize [(14)C]mevalonate for the biosynthesis of isoprenoid compounds was investigated. Several of the animals, in particular crustaceans, bivalve molluscs, a coelenterate and a sponge, were unable to synthesize squalene and sterols, whereas gastropod molluscs, echinoderms, an annelid and a sponge could. Regardless of sterol-synthesizing ability the animals (with the exception of a sponge) always made dolichol and ubiquinone, and thus a specific block in squalene and sterol synthesis was indicated in some animals. Radioactivity accumulated in relatively large amounts in farnesol and geranylgeraniol in those animals incapable of making sterols.     

Dinoflagellate bioluminescence: a comparative study of invitro components.

In vitro bioluminescence components of the dinoflagellates Gonyaulax polyedra, G. tamarensis, Dissodinium lunual, and Pyrocystis noctiluca were studied. The luciferases and luciferins of the four species cross-react in all combinations. All of these species possess high-molecular weight luciferases (200,000-400,000 daltons) with similar pH activity profiles. The active single chains of luciferases from the Gonyaulax species have a MW of 130,000 while those from P. noctiluca and D. lunula have a MW of 60,000. Extractable luciferase activity varies with time of day in the two Gonyaulax species, but not in the other two. A luciferin binding protein (LBP) can easily be extracted from the two Gonyaulax species (MW approximately 120,000 daltons), but none could be detected in extracts of either D. lunula or P. noctiluca. Scintillons are extractable from all four species, but they vary in density and the degree to which activity can be increased by added luciferin. Although the biochemistry of bioluminescence in these dinoflagellates is generally similar, the observations that D. lunula and P. noctiluca apparently lack LBP and have luciferases with low MW single chains require further clarification.     

Nuclear Structure and Mitosis in the Dinoflagellate Gonyaulax monilata*

SYNOPSIS. Nuclear structure and mitosis in Gonyaulax monilata were studied. Between the arms of the U-shaped interphase nucleus is a band of material which, it is suggested, is homologous with the “central body” of G. tamarensis. Histochemical studies of whole cells, extruded nuclei and Epon-embedded sections indicate that the band is composed at least in part of protein, phospholipid and possibly RNA. The chromosomes of interphase nuclei are attached to the ends of the band. The band is also attached to a proteinaceous, extra-chromosomal matrix which pervades the nucleus. Studies on material treated with trypsin indicate that the matrix may be a factor in maintaining chromosomal integrity and orientation and nuclear form. During mitosis the band acts as a point of attachment for the chromosomes, and appears to control anaphase and telophase movements. It is suggested that the band is a component of the mitotic apparatus in G. monilata.     

The influence of Gomphosphaeria aponina on the growth of Gymnodinium breve and the effect of aponin on the ichthyotoxicity of Gymnodinium breve.

Cells of the marine blue-green alga Gomphosphaeria aponina survive in mixed culture with the marine dinoflagellate, Gymnodinium breve (the Florida red tide organism), but G. breve cells lysed within 4-7 days. It has been established that the cytolytic effect of G. aponina, not nutrient competition, is responsible for the decrease in number of cells. The material elaborated by G. aponina has been termed aponin and has been extracted from the cells. The effect of aponin on the ichthyotoxicity of G. breve cultures was measured using Poecilia sphenops, adapted to sea water, as the assay organism. Aponin is not ichthyotoxic toward P. sphenops, though this material, when incubated with G. breve cultures does destroy the cells and increases the ichthyotoxicity of the cultures. At certain concentrations of aponin, the ichthyotoxicity of G. breve cultures appeared to be mitigate d.     

Nitrate metabolism of the red tide dinoflagellate Gonyaulax polyedra Stein

Certain idiosyncracies in the metabolism of nitrogen and diurnal vertical migration may account for the development and persistence of Gonyaulax polyedra Stein red tides along the Southern California Coast.In culture, G. polyedra has the ability for both uptake and assimilation of nitrate in the dark and this together with its enhancement by previous N-starvation could enable these dinoflagellates to meet 50–100 % of their daily nitrogen requirements for growth from dark assimilation alone. Less pronounced light-dark variations in nitrate assimilation and a greater stability of the nitrate assimilatory enzymes, together with the ability to migrate into nitrate-rich subsurface waters at night, probably give G. polyedra and other red tide dinoflagellates a competitive advantage over coastal diatoms during the ‘upwelling season’, when most red tides occur.     

Three nuclear genes for phylogenetic, SNP and population genetic studies of molluscs and other invertebrates

The study reports new primers capable of amplifying fragments from three nuclear protein-coding genes in a variety of deep-sea molluscs and annelids - adenine nucleotide translocase (Ant), calmodulin (Cal) and cyclophilin A (CycA). The Ant primers appear to be restricted to bivalve molluscs, whereas the Cal and CycA primers also amplified appropriate gene fragments from Lepetodrilus gastropod molluscs and Osedax polychaete worms. The amplified fragment of Cal contains an intron in the molluscs, but no intron was detected in the Ant and CycA fragments from any of the tested animals. DNA sequences generated by the three primer sets exhibited one to 15 single nucleotide polymorphism sites in deep-sea vesicomyid clams and Osedax boneworms. The observed levels of polymorphism indicate that the genes are likely to be useful in both population genetic and phylogenetic analyses of different invertebrate taxa.