Specific Detection of Pacific Oyster (Crassostrea gigas) Larvae in Plankton Samples Using Nested Polymerase Chain Reaction

Management of sustainable Pacific oyster fisheries would be assisted by an early, rapid, and accurate means of detecting their planktonic larvae. Reported here is an approach, based on polymerase chain reaction (PCR), for the detection of Pacific oyster larvae in plankton samples. Species-specific primers were designed by comparing partial mitochondrial cytochrome oxidase subunit I (COI) sequences from Crassostrea gigas, with other members of the family Ostreidae including those of Crassostrea angulata. Assay specificity was empirically validated through screening DNA samples obtained from several species of oysters. The assay was specific as only C. gigas samples returned PCR-positive results. A nested PCR approach could consistently detect 5 or more D-hinge-stage larvae spiked into a background of about 146 mg of plankton. The assay does not require prior sorting of larvae. We conclude that the assay could be used to screen environmental and ballast water samples, although further specificity testing against local bivalve species is recommended in new locations.     

Cyst and radionuclide evidence demonstrate historic Gymnodinium catenatum dinoflagellate populations in Manukau and Hokianga Harbours, New Zealand

Between May 2000 and February 2001, a major bloom of the toxic dinoflagellate Gymnodinium catenatum (a causative organism of Paralytic Shellfish Poisoning, PSP) affected over 1500 km of coastline of New Zealand’s North Island. As this was the first record of this species in New Zealand, we aimed to resolve whether this represented a recent introduction/spreading event or perhaps an indigenous cryptic species stimulated by environmental/climatic change. To answer this question, we analysed for G. catenatum resting cysts in ²¹⁰Pb dated sediment cores (18–34 cm long; sedimentation rates 0.34–0.69 cm per year) collected by SCUBA divers from Manukau Harbour, where the species was first detected, and from Hokianga Harbour, where the highest shellfish toxicity was recorded, while using Wellington Harbour as a well-monitored control site. The results of this study conclusively demonstrate that abundant G. catenatum has been in northern New Zealand at least since the early 1980s, increasing up to 1200 cysts/g around the year 2000, but with low cyst concentrations possibly present since at least 1937. In contrast, Wellington Harbour cores contained only very sparse G. catenatum cysts (8 cysts/g), present only to a depth of 7 cm (surface mixed layer depth), reflecting an apparent recent range expansion of this dinoflagellate in New Zealand, possibly stimulated by unusual climatic conditions associated with the 2000 La Nina event. The significant increases since the early 1980s also of Protoperidinium cysts at Hokianga Harbour and of Gonyaulax, Protoperidinium and Protoceratium cysts at Manukau Harbour suggest a broad scale environmental change has occurred in Northland, New Zealand.     

Efficacy of three commercially available ballast water biocides against vegetative microalgae, dinoflagellate cysts and bacteria

One proposed solution to the problem of ballast-mediated aquatic invasions involves chemically treating ballast water to kill key target organisms. Here, we examine the efficacy of three commercially available ballast water biocides using vegetative microalgae, dinoflagellate resting cysts and bacteria as test organisms. Chemicals tested were the ballast water biocides SeaKleen^® and Peraclean^® Ocean, and the chlorine dioxide biocide Vibrex^®. Results demonstrate that the applicability of each of the three chemical biocides as a routine ballast water treatment is limited by factors such as cost, biological effectiveness and possible residual toxicity of the discharged ballast water (assessed on the basis of impact on motility of vegetative marine microalgae). Of the three biocides tested, Peraclean^® Ocean appears to hold the most potential; however its effectiveness in shipboard trials is yet to be proven. Peraclean^® Ocean was biodegradable within 2–6 weeks (initial concentration of 200 ppm), could effectively inactivate resting cysts of the marine dinoflagellates Gymnodinium catenatum, Alexandrium catenella and Protoceratium reticulatum at 400 ppm, could control bacterial growth of Escherichia coli, Staphylococcus aureus, Listeria innocua and Vibrio alginolyticus at 125–250 ppm, and could eliminate vegetative dinoflagellate cells at a concentration of 100 ppm. SeaKleen^® eliminated vegetative microalgae at 2 ppm and could control resting cysts of the dinoflagellates G. catenatum and P. reticulatum at a concentration of 6 and 10 ppm, respectively, when exposed for a period of 2 weeks. SeaKleen^® did not inactivate resting cysts of A. catenella at a concentration of 10 ppm and was found to degrade at a rate that could result in the discharge of residual toxic water into the marine environment. Together with the poor bactericidal properties of SeaKleen^® (100–200 ppm required), this may limit the use of this biocide as a routine treatment option. Vibrex^® is not a suitable ballast water treatment option due to the need for hydrochloric acid as an activator, however it was found to be the most effective against bacteria (complete inhibition at 15 ppm) indicating that onboard chlorine dioxide generators may provide an effective bacterial treatment option. The performance of these biocides was adversely influenced by a variety of factors including low water temperatures (6 °C compared to 17 °C), light versus dark conditions, and the presence of humus-rich seawater and ballast water sediments.     

A review of the molecular evidence for ballast water introduction of the toxic dinoflagellates Gymnodinium catenatum and the Alexandrium “tamarensis complex” to Australasia

The potential of ballast water to act as a major introduction vector for toxic dinoflagellates and other phytoplankton is beyond doubt; however, evidence that links the suspected introduced species with a source population is less convincing, especially without supporting historical and biochemical data, or consideration of palaeobiogeographical scenarios that may explain current species distributions. This paper presents new molecular data based on LSU-rDNA and rDNA-ITS sequences that demonstrate an unequivocal and recent link between Temperate Asian and Australasian populations of the toxic dinoflagellates Gymnodinium catenatum and toxic strains of the Alexandrium “tamarensis complex”. We integrate our data with supporting evidence from historical distribution records, sediment dating studies, toxin profiles, mating studies and previous molecular studies. We contrast the observed patterns of genetic and biochemical variation with those expected from various palaeobiogeographical scenarios explaining the evolution and natural dispersal of both species. While definitive proof is impossible, the total evidence indicates that these toxic dinoflagellates were introduced to Australasia during the past 100 years, most probably via ballast water from bulk-cargo shipping from Japan and/or south-east Asia.     

Transport of diatom and dinoflagellate resting spores in ships' ballast water: implications for plankton biogeography and aquaculture

Diatom and dinoflagellate species that are not endemic to aregion can be inadvertently introduced when their resistantresting stages are discharged with the ballast-tank waters andsediments of bulk cargo vessels. A survey of 343 cargo vesselsentering 18 Australian ports showed that 65% of ships were carryingsignificant amounts of sediment on the bottom of their ballasttanks. All of these samples contained diatoms, including speciesthat are not endemic to Australian waters. Diatom resting spores,especially of Chaetoceros, were also detected. Dinoflagellateresting spores (cysts) were present in 50% of the sediment samples.Of the 53 cyst species identified, 20 (including Diplopelta,Diplopsalopsis, Gonyaulax, Polykrikos, Protoperidinium, Scrippsiellaand Zygabikodinium spp.) were successfully germinated to produceviable cultures. Such diversity of diatom and dinoflagellatespecies in ships' ballast water suggests that the apparent cosmopolitanismof many coastal phytoplankton species may be due partly to theglobal transport of seawater ballast. Of considerable concernwas the detection in 16 ships of cysts of the toxic dinoflagellatesAlexandrium catenella, Alexandrium tamarense and Gymnodiniumcatenatum. One single ballast tank was estimated to contain>300 million viable A.tamarense cysts, some of which weresuccessfully germinated in the laboratory to produce toxic cultures.These toxic dinoflagellate species, which can contaminate shellfishwith paralytic shellfish poisons, pose a serious threat to humanhealth and the aquaculture industry. Ballast-water quarantinemeasures recently introduced in Australia are discussed. Mid-oceanexchange of ballast water is only partially effective in removingdinoflagellate cysts which have settled to the bottom of ballasttanks. The present work indicates that the most effective measureto prevent the spreading of toxic dinoflagellate cysts via ships'ballast water would be to avoid taking on ballast water duringdinoflagellate blooms in the water column of the world's ports.     

Revised dinoflagellate phylogeny inferred from molecular analysis of large-subunit ribosomal RNA gene sequences

The nucleotide sequence analysis of the PCR products corresponding to the variable large-subunit rRNA domains D1, D2, D9, and D10 from ten representative dinoflagellate species is reported. Species were selected among the main laboratory-grown dinoflagellate groups: Prorocentrales, Gymnodiniales, and Peridiniales which comprise a variety of morphological and ecological characteristics. The sequence alignments comprising up to 1,000 nucleotides from all ten species were employed to analyze the phylogenetic relationships among these dinoflagellates. Maximum parsimony and neighbor joining trees were inferred from the data generated and subsequently tested by bootstrapping. Both the D1/D2 and the D9/D10 regions led to coherent trees in which the main class of dinoflagellates, Dinophyceae, is divided in three groups: prorocentroid, gymnodinioid, and peridinioid. An interesting outcome from the molecular phylogeny obtained was the uncertain emergence of Prorocentrum lima. The molecular results reported agreed with morphological classifications within Peridiniales but not with those of Prorocentrales and Gymnodiniales. Additionally, the sequence comparison analysis provided strong evidence to suggest that Alexandrium minutum and Alexandrium lusitanicum were synonymous species given the identical sequence they shared. Moreover, clone Gg1V, which was determined Gymnodinium catenatum based on morphological criteria, would correspond to a new species of the genus Gymnodinium as its sequence clearly differed from that obtained in G. catenatum. The sequence of the amplified fragments was demonstrated to be a valuable tool for phylogenetic and taxonomical analysis among these highly diversified species. Correspondence to: J. M. Bautista     

Application of real-time PCR assay for detection and quantification of Alexandrium tamarense and Alexandrium catenella cysts from marine sediments

The dinoflagellates Alexandrium tamarense (Lebor) Balech and Alexandrium catenella (Whedon and Kofoid) Balech (Dinophyceae) are believed to be the main species responsible for paralytic shellfish poisoning (PSP) all over the world. It is necessary to identify A. tamarense and A. catenella cysts and to monitor their distribution in sediment in order to minimize the damages caused by PSP to the economy and food quality because cysts are the seed population for blooms caused by motile vegetative cells. In this study, we developed an efficient DNA extraction method from the natural cysts present in marine sediments after they were size fractionated with a plankton net (mesh size of 20–150 μm). The 10–3000 cysts were added to the sediments collected from the Ariake Sea, and for which the primuline-staining method did not reveal any cysts. DNA was then extracted from each sample, and linear standard curves for A. tamarense and A. catenella cysts were obtained from the correlation between the Ct values by real-time PCR and the log of the initial densities of cysts. We monitored the A. tamarense and A. catenella cyst densities in the environmental samples. This assay was demonstrated to be a powerful tool for the identification, detection, and quantification of the cysts of the toxic dinoflagellates.     

PIGMENTS,FATTY ACIDS,AND STEROLS OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM1

Cultures and field samples of the toxic dinoflagellate Gymnodinium catenatum Graham from Tasmania, Australia, were analyzed for pigment, fatty acid, and sterol composition. Gymnodinium catenatum contained the characteristic pigments of photosynthetic dinoflagellates, including chlorophyll a, chlorophyll c₂, and the carotenoids peridinin, dinoxanthin, diadinoxanthin, diatoxanthin, and β,β-carotene. In midlogarithmic and early stationary phase cultures, the chlorophyll a content ranged 50–72 pg · cell^?1, total lipids 956–2084 pg · cell^?1, total fatty acids 426–804 pg · cell^?1, and total sterols 8–20 pg · cell^?1. The major fatty acids (in order of decreasing abundance) were 16:0, 22:6(n-3), and 20:5(n-3) (collectively 65–70% of the total fatty acids), followed by 16:1(n-7), 18:2(n-6), and 14:0. This distribution is characteristic of most dinoflagellates, except for the low abundance (<3%) of the fatty acid 18:5(n-3), considered by some authors to be a marker for dinoflagellates. The three major sterols were 4α-methyl-5α-cholest-7-en-3β-ol, 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (the dinoflagellate sterol, dinosterol), and 4α,23,24-trimethyl-5α-cholest-7-en-3β-ol. These three sterols comprised about 75% of the total sterols in both logarithmic and early stationary phase cultures, and they were also found in high proportions (22–25%) in natural dinoflagellate bloom samples. 4-Desmethyl sterols, which are common in most microalgae, were only present in trace amounts in G. catenatum. The chemotaxonomic affinities of G. catenatum and the potential for using specific signature lipids for monitoring toxic dinoflagellate blooms are discussed.     

THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM (DINOPHYCEAE) REQUIRES MARINE BACTERIA FOR GROWTH1

Interactions with the bacterial community are increasingly considered to have a significant influence on marine phytoplankton populations. Here we used a simplified dinoflagellate‐bacterium experimental culture model to conclusively demonstrate that the toxic dinoflagellate Gymnodinium catenatum H. W. Graham requires growth‐stimulatory marine bacteria for postgermination survival and growth, from the point of resting cyst germination through to vegetative growth at bloom concentrations (10³ cells · mL^?1). Cysts of G. catenatum were germinated and grown in unibacterial coculture with antibiotic‐resistant or antibiotic‐sensitive Marinobacter sp. DG879 or Brachybacterium sp., and with mixtures of these two bacteria. Addition of antibiotics to cultures grown with antibiotic‐sensitive strains of bacteria resulted in death of the dinoflagellate culture, whereas cultures grown with antibiotic‐resistant bacteria survived antibiotic addition and continued to grow beyond the 21 d experiment. Removal of either bacterial type from mixed‐bacterial dinoflagellate cultures (using an antibiotic) resulted in cessation of dinoflagellate growth until bacterial concentration recovered to preaddition concentrations, suggesting that the bacterial growth factors are used for dinoflagellate growth or are labile. Examination of published reports of axenic dinoflagellate culture indicate that a requirement for bacteria is not universal among dinoflagellates, but rather that species may vary in their relative reliance on, and relationship with, the bacterial community. The experimental model approach described here solves a number of inherent and logical problems plaguing studies of algal‐bacterium interactions and provides a flexible and tractable tool that can be extended to examine bacterial interactions with other phytoplankton species.     

IDENTIFICATION OF MARINE DINOFLAGELLATES USING FLUORESCENT LECTINS1

Toxic and nontoxic species of marine dinoflagellates were characterized using fluorescent lectins. Lectin binding was detected by epifluorescence as well as spectrofluorometry. The binding assay of fluorescent lectins readily differentiated between morphologically similar species (i.e the toxic dinoflagellate Gymnodinium catenatum and the nontoxic Gymnodinium sp.). Lectins appear to be a useful tool to distinguish among different clones of the same species and, thus, possibly as a tool in dinoflagellate identification. Moreover, the lectins used show that thecate species have more binding sites and diversity in glycan moieties than athecate species.     

Cyst formation,sedimentation, and preservation: Factors affecting dinoflagellate assemblages in recent sediments from trondheimsfjord,Norway

Plankton records and 25 samples of Recent sediment from Trondheimsfjord and the adjoining shelf were studied to investigate production, sedimentation, and preservation of cysts, as factors which influence the eventual composition of dinoflagellate cyst assembleges. All sediment samples were examined for dinoflagellate cysts using routine semiquantitative palynological procedures. In addition, fjord sediments were subjected to a limited sediment analysis, and, for three samples, results from preparations both with and without acid treatments were compared. For the first time, cyst assembleges from Recent sediments were directly compared with extensive plankton records from overlying waters. Results indicate that approximately 20% of the 55 locally recorded dinoflagellate species contribute cysts to bottom sediments. Once formed, cysts behave as fine silt particles in the sedimentary regime, increasing in abundance as the percentage abundance of finer sediment increases, usually with increased water depth. Cyst-forming species are almost entirely restricted to a few genera, particularly Gonyaulax and Peridinium, within the order Peridiniales. For some groups, reasonably good correspondence was found between percentage abundances of dinoflagellates in plankton and their cysts in sediment, though plankton records covering at least five years were required to establish this. Gonyaulax grindleyi Reinecke (Von Stosch 1969) appeared to be consistently overrepresented by cysts in sediment relative to available plankton evidence; possible explanations are suggested. At least 30% of the cyst species present, including most Peridinium species, were eliminated, or rendered unreliable for semiquantitative palynology, by application of routine palynological preparation treatments. Such cysts may provide useful, non-quantitative, palynological information from Recent and possibly Quaternary sediments, but their persistence would seem unlikely. Thus, factors of preservation probably further restrict the dinoflagellate fossil record. Cyst assemblages from Trondheimsfjord are comparable with those previously recorded from the northeastern coast of U.S.A., and from Scotland and northeastern England. Fjord assemblages are dominated by small, simple, spinose cysts which would be regarded as acritarchs if culture experiments had not proved that they are dinoflagellate cysts. Much potential biogeographic and palaeoenvironmental information was included within the less abundant species.Attention is drawn to the role which future culture experiments may be expected to play in helping to resolve taxonomic difficulties currently affecting dinoflagellate studies. Palynological significance of results from the present study is discussed especially with reference to recent work by Von Stosch which strongly suggests that cysts may be hypnozygotes formed routinely in sexual cycles of dinoflagellates.     

Identification of the dinoflagellate community during Cochlodinium polykrikoides (Dinophyceae) blooms using amplified rDNA melting curve analysis and real-time PCR probes

The dinoflagellate community present during blooms of the fish killing dinoflagellate Cochlodinium polykrikoides was characterized by DNA melting curve analysis and direct sequencing of the SSU rDNA amplified from environmental sample extracts. PCR amplification of genomic DNA from Gaedo water samples using dinoflagellate-specific SSU rDNA primers yielded 280 clones, which were screened by closed tube PCR-melting curve analysis targeting a region of the SSU rDNA, enabling high throughput analysis. Twenty-eight clones producing distinct melting curve patterns were sequenced, and their phylogenetic information revealed that C. polykrikoides co-occurred with morphologically similar species including Gymnodinium impudicum and Gymnodinium catenatum. Temporal variations of C. polykrikoides and G. impudicum abundances in South Sea were also examined by species-specific real-time TaqMan-based PCR probes developed in this study. C. polykrikoides- and G. impudicum-specific real-time PCR probes were designed targeting the internal transcribed spacer 2 ribosomal DNA region. The probe specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR products from environmental samples. The real-time PCR assays showed that C. polykrikoides cell densities peaked in August at 16,928 cells mL^?1, while G. impudicum was present at low abundances (below 25 cells mL^?1). Our amplified rDNA melting curve protocol provides a facile method for the characterization of the dinoflagellate community, and the real-time PCR assay could be an alternative method for rapid and sensitive enumeration of harmful dinoflagellates in the marine environment.     

Global toxicology,ecophysiology and population relationships of the chainforming PST dinoflagellate Gymnodinium catenatum

Increasing scientific awareness since the 1980s of the chain-forming dinoflagellate Gymnodinium catenatum has led to this species being reported with increased frequency in a globally increasing number of countries (23 at present). G. catenatum exhibits little molecular genetic variation in rDNA over its global range, in contrast to RAPD fingerprinting which points to high genetic variation within regional populations even between estuaries 50 km apart. All Australian and New Zealand strains possess a thymine nucleotide (T-gene) near the start of the 5.8S rRNA whereas all other global populations examined to date possess cytosine-nucleotide (C-gene), except for southern Japan which harbours both C-gene and T-gene strains. Together with cyst and plankton evidence this strongly suggests that both Australian and New Zealand populations have derived from southern Japan. Global dinoflagellate populations and cultures exhibit an extraordinary variation in PST profiles (STX and 21 analogues), but consistent regional patterns are evident with regard to the production of C1,2; C3,4; B1,2; and neoSTX analogues. PST profiles of cyst-derived cultures are deemed unrepresentative. Distinct ecophysiological differences exist between tropical (21–32 °C) and warm-temperate ecotypes (12–18 °C), but these appear unrelated to ITS genotypes and PST toxin phenotypes. On current evidence, cyst germination appears to play a minimal role in the bloom dynamics of this species, while seasonal and inter-annual bloom variations result from the physical constraints (temperature and light) on the growth of the dinoflagellates in the water column. G. catenatum exhibits a capacity to utilize many forms of nitrogen. Its chain formation and strong motility allow it to undergo retrieval migrations to exploit light and nutrient resource gradients in both stratified and mixed environments. Subtle strain-level variations in micronutrient (Se, humics) requirements and interaction with associated bacterial flora may provide a partial explanation for the contrasting inshore (Tasmanian), and offshore (Spain, Mexico) bloom patterns by the same species in different geographic regions.     

GREEN AUTOFLUORESCENCE OF DINOFLAGELLATE CYSTS CAN BE USED INSTEAD OF PRIMULINE STAINING FOR CYST VISUALIZATION AND ENUMERATION IN SEDIMENTS1

Primuline staining is widely used to visualize and enumerate dinoflagellate cysts in marine sediments. In staining cysts of Gymnodinium catenatum H. W. Graham, Scrippsiella trochoidea (F. Stein) A. R. Loebl., and cysts from estuarine sediments, we found their green fluorescence after primuline treatment to be seemingly no different from the green autofluorescence (GAF) inherent in vegetative cells and cysts of dinoflagellates fixed in formaldehyde. Although primuline subsequently proved to enhance green fluorescence of both species quantitatively, we nonetheless recommend taking advantage of dinoflagellates' GAF to detect and count their cysts in sediments. Doing so will reduce the time, chemical consumption, and possible loss of cells involved in the primuline‐staining procedure.     

Dinoflagellate community analysis of a fish kill using denaturing gradient gel electrophoresis

A molecular method using the polymerase chain reaction (PCR) amplification of small subunit gene sequences (18S rDNA) and denaturing gradient gel electrophoresis (DGGE) was used to determine both the population complexity and species identification of organisms in harmful algal blooms. Eighteen laboratory cultures of dinoflagellates, including Akashiwo, Gymnodinium, Heterocapsa, Karenia, Karlodinium, Pfiesteria, and Pfiesteria-like species were analyzed using dinoflagellate-specific oligonucleotide primers and DGGE. The method is sensitive and able to determine the number of species in a sample, as well as the taxonomic identity of each species, and is particularly useful in detecting differences between species of the same genus, as well as differences between morphologically similar species. Using this method, each of eight Pfiesteria-like species was verified as being clonal isolates of Pfiesteria piscicida. The sensitivity of dinoflagellate DGGE is approximately 1000 cells/ml, which is 100-fold less sensitive than real-time PCR. However, the advantage of DGGE lies in its ability to analyze dinoflagellate community structure without needing to know what is there, while real-time PCR provides much higher sensitivity and detection levels, if probes exist for the species of interest, attributes that complement DGGE analysis. In a blinded test, dinoflagellate DGGE was used to analyze two environmental fish kill samples whose species composition had been previously determined by other analyses. DGGE correctly identified the dominant species in these samples as Karlodinium micrum and Heterocapsa rotundata, proving the efficacy of this method on environmental samples. Toxin analysis of a clonal isolate obtained from the fish kill samples confirmed the presence of KmTx2, corroborating the earlier genetic identification of toxic K. micrum in the fish kill water sample.     

Morphological and molecular genetic characterization of Cryptoperidiniopsis brodyi (Dinophyceae) from Australia-wide isolates

Cryptoperidiniopsis brodyi is a common heterotrophic dinoflagellate known to often co-occur with Pfiesteria species in eastern U.S. estuaries. In this study, C. brodyi from Australia and Pfiesteria piscicida from ballast water from Indonesia were characterized by morphological and genetic analyses. Two P. piscicida strains originating from ballast water samples showed little genetic differences compared to P. piscicida from other countries and their morphology was identical. This finding indicates a potential inflow of P. piscicida into Australian estuaries via ballast water. Nine cultures of C. brodyi were established from Tasmania, South Australia and Western Australia. All C. brodyi cultures exhibited identical thecal plate patterns and could not be discriminated from other non-Australian strains. In contrast, two distinct genotypes could be identified by rDNA sequence analyses which were distinct from the U.S. genotype of C. brodyi. A previous survey using PCR-based methods reported a wide distribution of Pfiesteria shumwayae in Australia. However, the present study demonstrated that SSU rDNA-based P. shumwayae-specific primers produce false-positive PCR reactions with Australian C. brodyi. These results suggest that genetic variants of C. brodyi are widely distributed in Australia and Australian genotypes of C. brodyi had previously been misidentified as P. shumwayae. This finding also indicates that previous Australian distribution studies of P. shumwayae using SSU rDNA-based primers are potentially erroneous and need to be revisited.     

GYMNODINIUM COROLLARIUM SP. NOV. (DINOPHYCEAE)—A NEW COLD‐WATER DINOFLAGELLATE RESPONSIBLE FOR CYST SEDIMENTATION EVENTS IN THE BALTIC SEA1

A naked dinoflagellate with a unique arrangement of chloroplasts in the center of the cell was isolated from the northern Baltic proper during a spring dinoflagellate bloom (March 2005). Morphological, ultrastructural, and molecular analyses revealed this dinoflagellate to be undescribed and belonging to the genus Gymnodinium F. Stein. Gymnodinium corollarium A. M. Sundström, Kremp et Daugbjerg sp. nov. possesses features typical of Gymnodinium sensu stricto, such as nuclear chambers and an apical groove running in a counterclockwise direction around the apex. Phylogenetic analyses based on partial nuclear‐encoded LSU rDNA sequences place the species in close proximity to G. aureolum, but significant genetic distance, together with distinct morphological features, such as the position of chloroplasts, clearly justifies separation from this species. Temperature and salinity experiments revealed a preference of G. corollarium for low salinities and temperatures, confirming it to be a cold‐water species well adapted to the brackish water conditions in the Baltic Sea. At nitrogen‐deplete conditions, G. corollarium cultures produced small, slightly oval cysts resembling a previously unidentified cyst type commonly found in sediment trap samples collected from the northern and central open Baltic Sea. Based on LSU rDNA comparison, these cysts were assigned to G. corollarium. The cysts have been observed in many parts of the Baltic Sea, indicating the ecologic versatility of the species and its importance for the Baltic ecosystem.     

OLIGONUCLEOTIDE PRIMERS FOR THE DETECTION OF BIOLUMINESCENT DINOFLAGELLATES REVEAL NOVEL LUCIFERASE SEQUENCES AND INFORMATION ON THE MOLECULAR EVOLUTION OF THIS GENE1

Bioluminescence is reported in members of 18 dinoflagellate genera. Species of dinoflagellates are known to have different bioluminescent signatures, making it difficult to assess the presence of particular species in the water column using optical tools, particularly when bioluminescent populations are in nonbloom conditions. A “universal” oligonucleotide primer set, along with species and genus‐specific primers specific to the luciferase gene were developed for the detection of bioluminescent dinoflagellates. These primers amplified luciferase sequences from bioluminescent dinoflagellate cultures and from environmental samples containing bioluminescent dinoflagellate populations. Novel luciferase sequences were obtained for strains of Alexandrium cf. catenella (Whedon et Kof.) Balech and Alexandrium fundyense Balech, and also from a strain of Gonyaulax spinifera (Clap. et Whitting) Diesing, which produces bioluminescence undetectable to the naked eye. The phylogeny of partial luciferase sequences revealed five significant clades of the dinoflagellate luciferase gene, suggesting divergence among some species and providing clues on their molecular evolution. We propose that the primers developed in this study will allow further detection of low‐light‐emitting bioluminescent dinoflagellate species and will have applications as robust indicators of dinoflagellate bioluminescence in natural water samples.