Karlodinium Micrum Research Articles

Bioinformatics evidence for the transfer of mycosporine-like amino acid core (4-deoxygadusol) synthesizing gene from cyanobacteria to dinoflagellates and an attempt to mutate the same gene (YP_324358) in Anabaena variabilis PCC 7937

We have identified a homologue of 4-deoxygadusol (core of mycosporine-like amino acids) synthesizing gene (ZP_05036788) from Synechococcus sp. PCC 7335 that was found to have additional functionally unknown N-terminal domain similar to homologues from dinoflagellates based on the ClustalW analysis. Phylogenetic analysis revealed that Synechococcus sp. (ZP_05036788) makes a clade together with dinoflagellates and was closest to the Oxyrrhis marina. This study shows for the first time that N-terminal additional sequences that possess upstream plastid targeting sequence in Heterocapsa triquetra and Karlodinium micrum were already evolved in cyanobacteria, and plastid targeting sequence were evolved later in dinoflagellates after divergence from chloroplast lacking Oxyrrhis marina. Thus, MAAs synthesizing genes were transferred from cyanobacteria to dinoflagellates and possibly Synechococcus sp. PCC 7335 acted as a donor during lateral gene transfer event. In addition, we also tried to mutate 4-deoxygadusol synthesizing gene (YP_324358) of Anabaena variabilis PCC 7937 by homologous recombination, however, all approaches to get complete segregation of the mutants from the wild-type were unsuccessful, showing the essentiality of YP_324358 for A. variabilis PCC 7937.

An Assessment of Vertical Inheritance versus Endosymbiont Transfer of Nucleus-encoded Genes for Mitochondrial Proteins Following Tertiary Endosymbiosis in Karlodinium micrum

Most photosynthetic dinoflagellates harbour a red alga-derived secondary plastid. In the dinoflagellate Karlodinium micrum, this plastid was replaced by a subsequent endosymbiosis, resulting in a tertiary plastid derived from a haptophyte. Evolution of endosymbionts entails substantial relocation of endosymbiont genes to the host nucleus: a process called endosymbiotic gene transfer (EGT). In K. micrum, numerous plastid genes from the haptophyte nucleus are found in the host nucleus, providing evidence for EGT in this system. In other cases of endosymbiosis, notably ancient primary endosymbiotic events, EGT has been inferred to contribute to remodeling of other cell functions by expression of proteins in compartments other than the endosymbiont from which they derived. K. micrum provides a more recently derived endosymbiotic system to test for evidence of EGT and gain of function in non-plastid compartments. In this study, we test for gain of haptophyte-derived proteins for mitochondrial function in K. micrum. Using molecular phylogenies we have analysed whether nucleus-encoded mitochondrial proteins were inherited by EGT from the haptophyte endosymbiont, or vertically inherited from the dinoflagellate host lineage. From this dataset we found no evidence of haptophyte-derived mitochondrial genes, and the only cases of non-vertical inheritance were genes derived from lateral gene transfer events.

Analysis of Dinoflagellate Mitochondrial Protein Sorting Signals Indicates a Highly Stable Protein Targeting System across Eukaryotic Diversity

Protein targeting into mitochondria from the cytoplasm is fundamental to the cell biology of all eukaryotes. Our understanding of this process is heavily biased towards “model” organisms, such as animals and fungi, and it is less clear how conserved this process is throughout diverse eukaryotes. In this study, we have surveyed mitochondrial protein sorting signals from a representative of the dinoflagellate algae. Dinoflagellates are a phylum belonging to the group Alveolata, which also includes apicomplexan parasites and ciliates. We generated 46 mitochondrial gene sequences from the dinoflagellate Karlodinium micrum and analysed these for mitochondrial sorting signals. Most of the sequences contain predicted N-terminal peptide extensions that conform to mitochondrial targeting peptides from animals and fungi in terms of length, amino acid composition, and propensity to form amphipathic α-helices. The remainder lack predicted mitochondrial targeting peptides and represent carrier proteins of the inner mitochondrial membrane that have internal targeting signals in model eukaryotes. We tested for functional conservation of the dinoflagellate mitochondrial sorting signals by expressing K. micrum mitochondrial proteins in the fungus Saccharomyces cerevisiae. Both the N-terminal and internal targeting signals were sufficiently conserved to operate in this distantly related system. This study indicates that the character of mitochondrial sorting signals was well established prior to the radiation of major eukaryotic lineages and has shown remarkable conservation during long periods of evolution.

Predicting the population dynamics in Amoebophrya parasitoids and their dinoflagellate hosts using a mathematical model

Free-living, photosynthetic marine dino- flagellates are frequently infected by microparasites of the genus Amoebophrya. Attacks by Amoebo- phrya can contribute to the termination of dino- flagellate blooms and have been suggested to in- fluence the geographical distribution of certain host species. Because infection terminates with the killing of the host (i.e. Amoebophrya behaves like a parasitoid), the interaction can be considered, from a modeling point of view, similar to the process of predation, with the difference that it takes a longer time for the parasitoid to kill the host as compared to typical predator-prey interactions. In the present work, we explored the population dynamics in Amoebophrya and their dinoflagellate hosts using the Rosenzweig-MacArthur modification of the tra- ditional Lotka-Volterra predation model. The model was parameterized for 3 systems, Akashiwo san- guinea, Gymnodinium instriatum, and Karlodinium micrum, and their respective Amoebophrya para- sitoids, using published experimental data. Parame- ter validation was possible for parasitoid search rate and mortality. The potential for host control by Amoebophrya and the probability for host extinction were studied with respect to carrying capacity, a parameter that is influenced by e.g. eutrophication. The model may be useful to predict conditions under which Amoebophrya can control populations of its dinoflagellate hosts. . Parasitism . Marine dinoflagellates . Mathematical model .

Toxicological studies of Karlodinium micrum (Dinophyceae) isolated from East China Sea

Karlodinium micrum (Strain NMBjah047) was isolated from the water samples of East China Sea (ECS). The hemolytic, ichthyotoxic, and cytotoxic activities of the algae was characterized. Embryotoxicity of both intra and extracellular extracts were also tested on a local sea urchin species. The algal intracellular hemolytic toxicity averaged about 87.5% at different algal growth phases. However, extracellular hemolytic activity depended on the population growth phase. The toxicity increased with the increase in the population size, reaching the highest hemolytic activity during the stationary phase, and maintained a relatively high activity even when the population declined. Time and density dependent ichthyotoxicity to Lateolabrax maculates juveniles was also detected. The LD50 in 24 h was 1.1 × 105 cells/mL. Inhibition of the fertilized egg hatching was also observed and estimated the IC50 in 40 h with 3.5 × 104 cells/mL. Extracellular extracts of K. micrum dense culture also showed significant cytotoxic activity on HUVEC (IC50 = 70.8 μg/mL). A dose dependent acute toxicity to embryos of sea urchin was also determined. The algal intracellular and extracellular extracts delayed or even restricted the embryological development of the sea urchin, illustrating the potential toxicity of K. micrum not only to vertebrates, but also to marine invertebrates. The hemolytic compounds in the ECS strain were extracted and analyzed. At least two fractions had significant hemolytic activities. A lipid-like compound, named Digalactosyldiacylglycerol (DGDG), was suggested to be responsible for the hemolytic activity in one of these fractions. From the results of the present studies, this strain of K. micrum isolated from the East China Sea might be considered a toxic strain with hemolytic activity, ichthyotoxicity, cytotoxicity and embryotoxicity.

Development of a rapid and effective method for preparing delicate dinoflagellates for scanning electron microscopy

We developed a rapid and effective procedure for scanning electron microscopy of three delicate dinoflagellates, Karlodinium micrum, Akashiwo sanguinea, and Heterocapsa niei. Good results were obtained when the specimens were fixed with a modified Parducz’s fixative (2% osmium tetroxide:saturated mercuric chloride = 5:1 v/v) for 10 min, washed in 0.05 M sodium cacodylate trihydrate buffer for 2 min, dehydrated in tert-butanol for 10 min and dried with hexamethyldisilazane in air for 3 min in a fume hood because reagents are very toxic. This method could be completed in 25 min. Compared with other preparative techniques, the present protocol has significant advantages for SEM observation by limiting distortion of delicate specimens and reducing the preparation time.

Anti-grazing properties of the toxic dinoflagellate Karlodinium veneficum during predator–prey interactions with the copepod Acartia tonsa

Karlodinium veneficum (syn. Karlodinium micrum, Bergholtz et al. 2006; J Phycol 42: 170-193) is a small athecate dinoflagellate commonly present in low levels in temperate, coastal waters. Occasionally, K. veneficum forms ichthyotoxic blooms due to the presence of cytotoxic, hemolytic compounds, putatively named karlotoxins. To evaluate the anti-grazing properties of these karlotoxins, we conducted food removal experiments using the cosmopolitan copepod grazer Acartia tonsa. Wild-caught, adult female A. tonsa were exposed to 6 monoalgal or mixed algal diets made using bloom concentrations of toxic (CCMP 2064) and non-toxic (CSIC1) strains of K. veneficum. Ingestion and clearance rates were calculated using the equations of Frost (1972). Exposure to the toxic strain of K. veneficum did not contribute to an increased mortality of the copepods and no sig- nificant differences in copepod mortality were found among the experimental diets. However, A. tonsa had significantly greater clearance and ingestion rates when exposed to a monoalgal diet of the non-toxic strain CSIC1 than when exposed to the monoalgal diet of toxic strain CCMP 2064 and mixed diets dominated by this toxic strain. These results support the hypothesis that karlotoxins in certain strains of K. veneficum deter grazing by potential predators and contribute to the formation and continuation of blooms.

Broad genomic and transcriptional analysis reveals a highly derived genome in dinoflagellate mitochondria

BackgroundDinoflagellates comprise an ecologically significant and diverse eukaryotic phylum that is sister to the phylum containing apicomplexan endoparasites. The mitochondrial genome of apicomplexans is uniquely reduced in gene content and size, encoding only three proteins and two ribosomal RNAs (rRNAs) within a highly compacted 6 kb DNA. Dinoflagellate mitochondrial genomes have been comparatively poorly studied: limited available data suggest some similarities with apicomplexan mitochondrial genomes but an even more radical type of genomic organization. Here, we investigate structure, content and expression of dinoflagellate mitochondrial genomes.ResultsFrom two dinoflagellates, Crypthecodinium cohnii and Karlodinium micrum, we generated over 42 kb of mitochondrial genomic data that indicate a reduced gene content paralleling that of mitochondrial genomes in apicomplexans, i.e., only three protein-encoding genes and at least eight conserved components of the highly fragmented large and small subunit rRNAs. Unlike in apicomplexans, dinoflagellate mitochondrial genes occur in multiple copies, often as gene fragments, and in numerous genomic contexts. Analysis of cDNAs suggests several novel aspects of dinoflagellate mitochondrial gene expression. Polycistronic transcripts were found, standard start codons are absent, and oligoadenylation occurs upstream of stop codons, resulting in the absence of termination codons. Transcripts of at least one gene, cox3, are apparently trans-spliced to generate full-length mRNAs. RNA substitutional editing, a process previously identified for mRNAs in dinoflagellate mitochondria, is also implicated in rRNA expression.ConclusionThe dinoflagellate mitochondrial genome shares the same gene complement and fragmentation of rRNA genes with its apicomplexan counterpart. However, it also exhibits several unique characteristics. Most notable are the expansion of gene copy numbers and their arrangements within the genome, RNA editing, loss of stop codons, and use of trans-splicing.

Species specificity and potential roles of Karlodinium micrum toxin

Karlodinium micrum is a toxic mixotrophic dinoflagellate that has been responsible for fish kills in coastal environments worldwide. The role that karlotoxins play in the life history of K. micrum is unknown, but may contribute to its bloom-forming ability. We tested the hypothesis that karlotoxins could inhibit the growth of other protists depending on the sterol composition of target cell membranes. We also examined the effect of toxin addition on feeding rates of K. micrum on a flagellated prey, Storeatula major. Dose-dependent effects of isolated karlotoxin (KmTX2) were tested in growth bioassays (24–48h) of K. micrum, three raphidophytes (Heterosigma akashiwo, Fibrocapsa japonica and Chattonella subsalsa), two cryptophytes (S. major andPyrenomonas salina), and the dinoflagellates Amphidinium carterae, Pfiesteria piscicida andP. shumwayae. Growth of K. micrum, P. salina, A. carterae and P. piscicida were not affected by karlotoxin additions up to 1 000ng ml−1. Other organisms showed growth inhibition at concentrations between 500ng ml−1 and 1 000ng ml−1. Predation by K. micrum on S. major was significantly higher in the presence of 25ng ml−1 KmTX2. The results are consistent with a role for karlotoxin in allelopathic inhibition of competitors and/or prey immobilisation depending on sterol composition.

Sneaking under the toxin surveillance radar: parasitism and sterol content

Parasitic dinoflagellates of the genus Amoebophrya infect and kill bloom-forming dinoflagellates, including the toxic species Karlodinium micrum. Strains of K. micrum produce cytotoxic compounds (KmTX) that render cell membranes permeable to a range of small ions and molecules, resulting in cell death through osmotic lysis. Membrane sterol composition appears to play a role in the sensitivity of different algal species to the membrane-disrupting effects of KmTX. This sterol specificity also appears to be responsible for the apparent immunity of K. micrum to its own toxins. K. micrum has a unique sterol profile, shared only by the congeneric dinoflagellates Karenia brevis and K. mikimotoi, dominated by (24S)-4α-methyl-5α-ergosta-8(14), 22-dien-3β-ol (72% by weight). This sterol has recently been named gymnodinosterol. Analysis of the sterol content in Amoebophrya sp. infecting K. micrum showed gymnodinosterol also to be dominant (62%). This was not simply a reflection of retaining host lipid content because K. micrum contains octadecapentaenoic acid (18:5n3), largely in galactolipids of the chloroplast, whereas Amoebophrya sp. contained little to no 18:5n3. By having a sterol content similar to its host, Amoebophrya sp. is able to avoid cell lysis caused by the cytotoxic compounds produced by the host.

Impact of tropical storms and drought on the dinoflagellates Karlodinium micrum and Prorocentrum minimum in estuarine rivers of North Carolina, USA

The dinoflagellates Karlodinium micrum and Prorocentrum minimum are harmful algal bloom taxa from different parts of the world. They are routinely documented in estuarine rivers in North Carolina, but there have been no known toxic outbreaks of these taxa in these rivers. P. minimum usually blooms during January–March and K. micrum is most likely to bloom during June–August. North Carolina experienced three hurricanes during autumn 1999, an ongoing drought from October 2001 to October 2002, one hurricane during autumn 2003, and remnants from seven tropical systems during August–September 2004. These weather events impacted the abundance patterns of both dinoflagellates. In the months following the 1999 storms, cell concentrations of both species were abnormally low or absent during their respective bloom seasons. During the first few months of the drought, cell concentrations of both dinoflagellates were unusually high but decreased as the drought continued. The abundance of both species seemed to be unaffected by the 2003 storm. K. micrum concentrations were unusually low during the late summer growing season of 2004. Extreme weather events may impact the presence of these potentially harmful dinoflagellates.

EFL GTPase in Cryptomonads and the Distribution of EFL and EF-1α in Chromalveolates

EFL (EF-like protein) is a member of the GTPase superfamily that includes several translation factors. Because it has only been found in a few eukaryotic lineages and its presence correlates with the absence of the related core translation factor EF-1alpha, its distribution is hypothesized to be the result of lateral gene transfer and replacement of EF-1alpha. In one supergroup of eukaryotes, the chromalveolates, two major lineages were found to contain EFL (dinoflagellates and haptophytes), while the others encode EF-1alpha (apicomplexans, ciliates, heterokonts and cryptomonads). For each of these groups, this distribution was deduced from whole genome sequence or expressed sequence tag (EST) data from several species, with the exception of cryptomonads from which only a single EF-1alpha PCR product from one species was known. By sequencing ESTs from two cryptomonads, Guillardia theta and Rhodomonas salina, and searching for all GTPase translation factors, we revealed that EFL is present in both species, but, contrary to expectations, we found EF-1alpha in neither. On balance, we suggest the previously reported EF-1alpha from Rhodomonas salina is likely an artefact of contamination. We also identified EFL in EST data from two members of the dinoflagellate lineage, Karlodinium micrum and Oxyrrhis marina, and from an ongoing genomic sequence project from a third, Perkinsus marinus. Karlodinium micrum is a symbiotic pairing of two lineages that would have both had EFL (a dinoflagellate and a haptophyte), but only the dinoflagellate gene remains. Oxyrrhis marina and Perkinsus marinus are early diverging sister-groups to dinoflagellates, and together show that EFL originated early in this lineage. Phylogenetic analysis confirmed that these genes are all EFL homologues, and showed that cryptomonad genes are not detectably related to EFL from other chromalveolates, which collectively form several distinct groups. The known distribution of EFL now includes a third group of chromalveolates, cryptomonads. Of the six major subgroups of chromalveolates, EFL is found in half and EF-1alpha in the other half, and none as yet unambiguously possess both genes. Phylogenetic analysis indicates EFL likely arose early within each subgroup where it is found, but suggests it may have originated multiple times within chromalveolates as a whole.

The balance of autotrophy and heterotrophy during mixotrophic growth of Karlodinium micrum (Dinophyceae)

We studied autotrophic and heterotrophic C metabolism during mixotrophic growth of Karlodinium micrum (Leadbeter et Dodge) Larsen (Dinophyceae) on prey Storeatula major (Cryptophyceae). Our goal was to determine the balance of autotrophy and heterotrophy that supports mixotrophic growth in K. micrum. Assimilation of inorganic 14 C and 14 C-labeled prey was used to separate the quantity and quality (i.e., lipid, polysaccharide and protein) of C obtained by autotrophy and heterotrophy, respectively. Growth rates (μ) of mixotrophic K. micrum were 0.52-0.75 div.·day -1 , equal to or greater than the maximum autotrophic growth rate (0.55 div.·day -1 ) of K. micrum. Autotrophy represented 27-69% of gross C uptake during mixotrophic growth. Cellular photosynthetic performance (PP cell , pg C cell -1 ·day -1 ) was 24-52% lower during mixotrophic growth than during autotrophic growth of K. micrum. Mixotrophic K. micrum assimilated 16% less photosynthate as protein compared to autotrophic K. micrum, while protein was the major net assimilation product (52%) from ingested prey C. Growth efficiency (%GE) of mixotrophic cultures, based on both autotrophic and heterotrophic C sources, averaged 36 ± 2.9%, slightly lower than the 40-50% GE typical of purely autotrophic K. micrum, but higher C gains associated with heterotrophic feeding more than compensated for the decrease in %GE in mixotrophic K. micrum. We conclude that mixotrophic growth of K. micrum is dominated by heterotrophic metabolism, although photosynthesis continues at a lowered rate. This is consistent with a shift toward secondary production in plankton assemblages dominated by mixotrophically growing K. micrum.

Histopathological Effects in Fish Exposed to the Toxins from Karlodinium micrum

AbstractKarlodinium micrum (family Dinophyceae) produces toxic compounds (KmTx's) that are associated with fish kills. For zebrafish Danio rerio larvae (24 h old) exposed to either KmTx 1 or KmTx 2, mortality (100% in 24 h) was observed at toxin concentrations of 1 μg/mL or more, whereas no mortality occurred after 24 h at concentrations of 0.5 μg/mL or less. Zebrafish and sheepshead minnow Cyprinodon variegatus juveniles (60–90 d old) exposed to KmTx 2 were more sensitive to the toxin's effects than larvae were; mortalities in the juveniles began at 0.1–0.5 μg/mL. In whole, sectioned juvenile zebrafish, gills were the primary site showing injury by light microscopy. Histology of gills in both species treated with 0.5 μg KmTx 2/mL (100% mortality in 1 h) showed epithelial necrosis and shortening or loss of secondary lamellae. Histology of zebrafish gills treated with 0.05 and 0.1 μg/mL (0–44% mortality in 4 h) showed clubbing and bridging between secondary lamellae within 4 h of exposure. Sheepshead minnow exposed to 0.1 μg/mL showed similar gill pathology but no mortality after 6 h. Sheepshead minnow exposed to KmTx 2 at 0.5 μg/mL or more all died in less than 1 h. Transmission electron microscopy of gills of moribund zebrafish exposed to 0.1 μg/mL revealed extensive cellular hypertrophy and lysis of epithelial and chloride cells. Because concentrations of KmTx 1 and KmTx 2 range from 0.1 to 0.8 μg/mL in filtered water samples from K. micrum–associated fish kills, these results suggest that the concentrations of KmTx 1 and KmTx 2 found during fish kills are acutely toxic to fish and that gills are a primary target.

Combined Heat Shock Protein 90 and Ribosomal RNA Sequence Phylogeny Supports Multiple Replacements of Dinoflagellate Plastids

Dinoflagellates harbour diverse plastids obtained from several algal groups, including haptophytes, diatoms, cryptophytes, and prasinophytes. Their major plastid type with the accessory pigment peridinin is found in the vast majority of photosynthetic species. Some species of dinoflagellates have other aberrantly pigmented plastids. We sequenced the nuclear small subunit (SSU) ribosomal RNA (rRNA) gene of the "green" dinoflagellate Gymnodinium chlorophorum and show that it is sister to Lepidodinium viride, indicating that their common ancestor obtained the prasinophyte (or other green alga) plastid in one event. As the placement of dinoflagellate species that acquired green algal or haptophyte plastids is unclear from small and large subunit (LSU) rRNA trees, we tested the usefulness of the heat shock protein (Hsp) 90 gene for dinoflagellate phylogeny by sequencing it from four species with aberrant plastids (G. chlorophorum, Karlodinium micrum, Karenia brevis, and Karenia mikimotoi) plus Alexandrium tamarense, and constructing phylogenetic trees for Hsp90 and rRNAs, separately and together. Analyses of the Hsp90 and concatenated data suggest an ancestral origin of the peridinin-containing plastid, and two independent replacements of the peridinin plastid soon after the early radiation of the dinoflagellates. Thus, the Hsp90 gene seems to be a promising phylogenetic marker for dinoflagellate phylogeny.

A Tertiary Plastid Uses Genes from Two Endosymbionts

The origin and subsequent spread of plastids by endosymbiosis had a major environmental impact and altered the course of a great proportion of eukaryotic biodiversity. The ancestor of dinoflagellates contained a secondary plastid that was acquired in an ancient endosymbiotic event, where a eukaryotic cell engulfed a red alga. This is known as secondary endosymbiosis and has happened several times in eukaryotic evolution. Certain dinoflagellates, however, are unique in having replaced this secondary plastid in an additional (tertiary) round of endosymbiosis. Most plastid proteins are encoded in the nucleus of the host and are targeted to the organelle. When secondary or tertiary endosymbiosis takes place, it is thought that these genes move from nucleus to nucleus, so the plastid retains the same proteome. We have conducted large-scale expressed sequence tag (EST) surveys from Karlodinium micrum, a dinoflagellate with a tertiary haptophyte-derived plastid, and two haptophytes, Isochrysis galbana and Pavlova lutheri. We have identified all plastid-targeted proteins, analysed the phylogenetic origin of each protein, and compared their plastid-targeting transit peptides. Many plastid-targeted genes in the Karlodinium nucleus are indeed of haptophyte origin, but some genes were also retained from the original plastid (showing the two plastids likely co-existed in the same cell), in other cases multiple isoforms of different origins exist. We analysed plastid-targeting sequences and found the transit peptides in K.micrum are different from those found in either dinoflagellates or haptophytes, pointing to a plastid with an evolutionarily chimeric proteome, and a massive remodelling of protein trafficking during plastid replacement.

Photoacclimation in phytoplankton: implications for biomass estimates, pigment functionality and chemotaxonomy

Chl a and C-normalized pigment ratios were studied in two dinophytes (Prorocentrum minimum and Karlodinium micrum), three haptophytes (Chrysochromulina leadbeateri, Prymnesium parvum cf. patelliferum, Phaeocystis globosa), two prasinophytes (Pseudoscourfieldia marina, Bathycoccus prasinos) and the raphidophyte Heterosigma akashiwo, in low (LL, 35 μmol photons m−2 s−1) and high light (HL, 500 μmol photons m−2 s−1). Pigment ratios in LL and HL were compared against a general rule of photoacclimation: LL versus HL ratios ≥1 are typical for light-harvesting pigments (LHP) and 1 with some exceptions such as Chl c3 in P. globosa and MV Chl c3 in C. leadbeateri. LL/HL to Chl a ratios of photosynthetic carotenoids were close to 1, except Hex-fuco in P. globosa (four-fold higher Chl a ratio in HL vs LL). Although pigment ratios in P. globosa clearly responded to the light conditions the diadinoxanthin-diatoxanthin cycle remained almost unaltered at HL. Total averaged pigment and LHP to C ratios were significantly higher in LL versus HL, reflecting the photoacclimation status of the studied species. By contrast, the same Chl a-normalized ratios were weakly affected by the light intensity due to co-variation with Chl a. Based on our data, we suggest that the interpretation of PPC and LHP are highly dependent on biomass normalization (Chl a vs. C).

Relative Uptake of Urea and Ammonium by Dinoflagellates or Cyanobacteria in Shrimp Mesocosms

The relative role of the organic nitrogen source, urea, versus ammonium as a nitrogen source for two species of dinoflagellates was compared with one species of cyanobacteria. Experiments were conducted opportunistically in nutrient-rich marine water during blooms of 34either cyanobacteria or dinoflagellates in outdoor mesocosms. These replicate mesocosms, which were stocked with shrimp fed high-protein formulated feeds, contained high biomasses of phytoplankton (mean chlorophyll a concentrations, 439.2–811.2 μg l−1). 15N-urea and ammonium uptake rates for dinoflagellate-dominated blooms (Gymnodinium pulchellum-complex (Larsen), Karlodinium micrum (Larsen) (Dinophyceae)) were compared with blooms of the cyanobacterium, Romeria sp. (Cyanophyceae) in mesocosms with mean urea and ammonium concentrations ranging from 2.32 to 3.24 μM, and 7.39 to 64.85 μM, respectively. Urea uptake rates were significantly (p < 0.005) lower than ammonium uptake rates irrespective of which algal species dominated the bloom. Additionally urea uptake rates were not significantly higher in G. pulchellum-complex or K. micrum-dominated blooms than in Romeria sp. blooms. These results suggest that G pulchellum complex and K. micrum may not be gaining a competitive advantage in waters high in dissolved organic matter simply by preferentially utilizing urea. The periodic dominance of these species in highly organic environments, such as shrimp ponds, is likely to have a more complex explanation.

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.

COMPARATIVE MORPHOLOGY AND MOLECULAR PHYLOGENETIC ANALYSIS OF THREE NEW SPECIES OF THE GENUS KARENIA (DINOPHYCEAE) FROM NEW ZEALAND1

Three new dinoflagellate species, Karenia papilionacea sp. nov., Karenia selliformis sp. nov., and Karenia bidigitata sp. nov., were compared with the toxic species Karenia mikimotoi (Miyake &amp; Kominami ex Oda) G. Hansen &amp; Moestrup, Karenia brevis (Davis) G. Hansen &amp; Moestrup, and Karenia brevisulcata (Chang) G. Hansen &amp; Moestrup using the same fixative. Distinguishing morphological characters for the genus Karenia included a smooth theca and a linear apical groove. The new species can be distinguished on the basis of morphological characters of vegetative cells that include the location and shape of the nucleus; the relative excavation of the hypotheca; the characteristics of apical and sulcal groove extensions on the epitheca; the cellular shape, size, and symmetry; the degree of dorsoventral compression; and the presence of an apical protrusion or carina. Species with pronounced dorsoventral compression swim in a distinctive fluttering motion. An intercingular tubular structure traversing the proximal and distal ends of the cingulum is common to the species of Karenia, Karlodinium micrum (Leadbeater &amp; Dodge) J. Larsen, Gymnodinium pulchellum J. Larsen, and Gyrodinium corsicum Paulmier. Molecular phylogenetic analyses of rDNA sequence alignments show that the new species are phylogenetically distinct but closely related to K. mikimotoi and K. brevis.