POTENTIAL UTILITY OF MITOCHONDRIAL CYTOCHROME B AND ITS MRNA EDITING IN RESOLVING CLOSELY RELATED DINOFLAGELLATES: A CASE STUDY OF PROROCENTRUM (DINOPHYCEAE)1

Difficulties often occur in separating closely related dinoflagellate species. In this study, the potential utility of mitochondrial cytochrome b (cob) gene sequence and mRNA editing characteristics was assessed using Prorocentrum Ehrenberg as a model. The cob sequences and the patterns of their mRNA editing were analyzed for several Prorocentrum taxa. Results revealed little difference in cob sequence and mRNA editing characteristics between geographic populations of P. minimum (Pavillard) Schiller, while a notable difference was detected between different species (P. minimum and P. micans Ehrenberg). Furthermore, these P. minimum populations consistently formed a tight cluster on phylogenetic trees inferred from cob sequences as well as mRNA editing characteristics, whereas different Prorocentrum species were well separated, with a genetic distance of 0.0042±0.0024 for the former and 0.0141±0.0012 for the latter (P<0.01; two‐tailed t‐test). When the analysis was applied to the case of P. donghaiense Lu et Goebel and CCMP1517 strain of P. dentatum Stein, no differences were detected between these two taxa with respect to cob mRNA editing pattern and only small differences equivalent to those between P. minimum populations were detected in terms of cob sequence. On the cob sequence‐ and editing‐based phylogenetic trees, P. donghaiense and P. dentatum CCMP1517 consistently clustered together at a position sister to P. minimum. The results suggest that cob, combined with its mRNA editing, can potentially be a useful delineator of Prorocentrum species, and that P. donghaiense and P. dentatum CCMP1517 are most likely the same species and both are closely related to P. minimum.     

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.     

ECTOCELLULAR GLUCOSIDASE AND PEPTIDASE ACTIVITY OF THE MIXOTROPHIC DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)1

The activities of the enzymes α‐ and β‐glucosidase, and leucine aminopeptidase were measured in cultures of the dinoflagellate Prorocentrum minimum (Pavill.) J. Schiller and in field samples collected during dinoflagellate blooms occurring in tributaries of the Chesapeake Bay, Maryland, USA. Activities were measured using fluorogenic artificial substrates and partitioned among the >5 μm size fraction, small microbes fraction (0.1–5 μm), and dissolved phase (<0.1 μm). P. minimum and most other photosynthetic dinoflagellates are >5 μm in size and thus can be separated from the small microbes fraction, which contains most bacteria. Little to no glucosidase activity was detected associated with the >5 μm size fraction in cultures or in field samples, with most of the activity (67% to 93% in cultures, 54% to 100% in field samples) in the small microbes size fraction for both α and β glucosidase. In contrast, 67% to 90% of the total leucine aminopeptidase (LAP) activity in cultures was measured in the >5 μm fraction. Within a culture, LAP activity in the size fraction containing P. minimum decreased in response to ammonium and urea additions, but not in response to nitrate. In field samples, LAP activity was positively correlated with dinoflagellate abundance and chl a, and negatively correlated with ammonium concentration. During blooms, up to 34% of LAP activity was associated with the >5 μm fraction, indicating that when abundant, dinoflagellates may make a substantial contribution to ectocellular LAP activity in the water column.     

MOLECULAR SYSTEMATICS OF THE GRACILARIACEAE (GRACILARIALES,RHODOPHYTA) WITH EMPHASIS ON SOUTHERN AFRICA1

Southern Africa has economically exploited populations of terete gracilarioids on the cool temperate west coast and numerous species of endemic and Indo‐Pacific tropical Gracilariaceae on the south and east coasts. Gross morphological characters have been the main means of identification, and incorrect applications have led to a number of misidentifications. In this study, small subunit rDNA and RUBISCO spacer sequences were used to determine phylogenetic relationships. Whereas rDNA sequences successfully differentiate major groups within the family as well as species belonging to the Gracilariopsis and the Curdiea/Melanthalia clade, RUBISCO spacer sequencing was required to distinguish between species of Gracilaria. The southern African gracilarioid complex (stringy, terete, elongate members of the Gracilariaceae) was resolved into three species: Gracilaria gracilis, Gracilariopsis longissima, and Gracilariopsis funicularis. South African Gracilaria protea was shown to be conspecific with tropical Indian Ocean G. corticata. Apart from G. gracilis and G. corticata, South African Gracilaria species were differentiated into a temperate‐tropical terete grouping and a temperate‐tropical flattened grouping.     

Inducible Mixotrophy in the Dinoflagellate Prorocentrum minimum

Prorocentrum minimum is a neritic dinoflagellate that forms seasonal blooms and red tides in estuarine ecosystems. While known to be mixotrophic, previous attempts to document feeding on algal prey have yielded low grazing rates. In this study, growth and ingestion rates of P. minimum were measured as a function of nitrogen (‐N) and phosphorous (‐P) starvation. A P. minimum isolate from Chesapeake Bay was found to ingest cryptophyte prey when in stationary phase and when starved of N or P. Prorocentrum minimum ingested two strains of Teleaulax amphioxeia at higher rates than six other cryptophyte species. In all cases ‐P treatments resulted in the highest grazing. Ingestion rates of ‐P cells on T. amphioxeia saturated at ~5 prey per predator per day, while ingestion by ‐N cells saturated at 1 prey per predator per day. In the presence of prey, ‐P treated cells reached a maximum mixotrophic growth rate (μ_max) of 0.5 d^?1, while ‐N cells had a μ_max of 0.18 d^?1. Calculations of ingested C, N, and P due to feeding on T. amphioxeia revealed that phagotrophy can be an important source of all three elements. While P. minimum is a proficient phototroph, inducible phagotrophy is an important nutritional source for this dinoflagellate.     

Effects of natural and field-simulated blooms of the dinoflagellate Prorocentrum minimum upon hemocytes of eastern oysters, Crassostrea virginica, from two different populations

Oysters, Crassostrea virginica, from two populations, one from a coastal pond experiencing repeated dinoflagellate blooms (native), and the other from another site where blooms have not been observed (non-native), were analyzed for cellular immune system profiles before and during natural and simulated (by adding cultured algae to natural plankton) blooms of the dinoflagellate Prorocentrum minimum. Significant differences in hemocytes between the two oyster populations, before and after the blooms, were found with ANOVA, principal components analysis (PCA) and ANOVA applied to PCA components. Stress associated with blooms of P. minimum included an increase in hemocyte number, especially granulocytes and small granulocytes, and an increase in phagocytosis associated with a decrease in aggregation and mortality of the hemocytes, as compared with oysters in pre-bloom analyses. Non-native oysters constitutively had a hemocyte profile more similar to that induced by P. minimum than that of native oysters, but this profile did not impart increased resistance. The effect of P. minimum on respiratory burst was different according to the origin of the oysters, with the dinoflagellate causing a 35% increase in the respiratory burst of the native oysters but having no effect on that of the non-native oysters. Increased respiratory burst in hemocytes of native oysters exposed to P. minimum in both simulated and natural blooms may represent an adaptation to annual blooms whereby surviving native oysters protect themselves against tissue damage from ingested P. minimum.     

PHYLOGENETIC ANALYSIS OF 32 STRAINS OF VAUCHERIA (XANTHOPHYCEAE) USING THE rbcL GENE AND ITS TWO FLANKING SPACER REGIONS1

The complete rbcL gene was sequenced for 21 species and 32 strains of Vaucheria and for five other Xanthophyceae (Asterosiphon dichotomus (Kützing) Rieth, Botrydium becharianum Vischer, B. cystosum Vischer, B. stoloniferum Mitra, Tribonema intermixtum Pascher). The psbA‐rbcL spacer, upstream of the rbcL gene, and the RUBISCO spacer between the rbcL and rbcS genes were also completely sequenced for the Vaucheria strains and Asterosiphon. The psbA‐rbcL spacer was the most variable region that was sequenced, and only the 3′ end of the spacer could be aligned. Phylogenetic analyses (maximum parsimony, neighbor joining, and maximum likelihood) were conducted using the DNA sequence and the amino acid sequence for the rbcL gene, and a second analysis was conducted using a portion of the psbA‐rbcL spacer +rbcL gene + RUBISCO spacer. All analyses showed that Vaucheria species formed monophyletic clades that corresponded with morphologically based subgeneric sections, including the section Racemosae. Species producing a gametophore (= fruiting branch, bearing both an antheridium and oogonium) formed a monophyletic clade in all analyses. The nongametophore species sometimes formed a monophyletic clade but other times formed a basal grade. Pair‐wise comparisons of nucleotides and amino acids showed that for some species, numerous nucleotide changes resulted in relatively few amino acid changes. Consequently, phylogenetic analysis of the amino acids produced numerous trees, which in a strict consensus tree resulted in numerous polychotomies. An original strain of V. terrestris that was deposited in two culture collections over 25 years ago had identical sequences, suggesting no rapid change was occurring in the sequenced regions. Two strains of V. prona, isolated from Europe and North America, had identical sequences. Other species, for which two or more strains were examined, had different sequences. These results suggest that cryptic species complexes exist within Vaucheria because the rbcL gene is a conservative gene that is identical in other protists.     

Species‐specific mitochondrial gene rearrangements in biting midges and vector species identification

Abstract Partial mitochondrial gene sequences of 16 Culicoides species were determined to elucidate phylogenetic relations among species and to develop a molecular identification method for important virus vector species. In addition, the analysis found mitochondrial gene rearrangement in several species. Sequences of the mitochondrial genome region, cox1–trnL2–cox2 (1940–3785 bp) of 16 Culicoides and additional sequences were determined in some species, including whole mitochondrial genome sequences of Culicoides arakawae. Nine species showed common organization in this region, with three genes cox1–trnL2–cox2 and a small or no intergenic region (0–30 bp) between them. The other seven species showed translocation of tRNA and protein‐coding genes and/or insertion of AT‐rich non‐coding sequences (65–1846 bp) between the genes. The varied gene rearrangements among species within a genus is very rare for mitochondrial genome organization. Phylogenetic analyses based on the sequences of cox1+cox2 suggest a few clades among Japanese Culicoides species. No relationships between phylogenetic closeness and mitochondrial gene rearrangements were observed. Sequence data were used to establish a polymerase chain reaction tool to distinguish three important vector species from other Culicoides species, for which classification during larval stages is not advanced and identification is difficult.     

Prorocentrum minimum blooms: potential impacts on dissolved oxygen and Chesapeake Bay oyster settlement and growth

The cosmopolitan dinoflagellate Prorocentrum minimum is a recurrent bloom forming species in the Chesapeake Bay and its tributaries, generally observed at its highest levels in late spring and summer. Laboratory studies were conducted to assess potential bloom impacts on diel oxygen concentrations in shallow littoral zones as well as settlement success and post-set growth of the eastern oyster Crassostrea virginica. Using light–dark and dark cultures and periodic diel sub-sampling, bloom levels of P. minimum produced supersaturated oxygen levels at the end of each day while darkened cultures were typified by rapid decreases in dissolved oxygen (DO) (1.1–1.3 mg L⁻¹ h⁻¹) to hypoxic and anoxic levels within 4 days. These data suggest shallow, poorly flushed systems and the biota in them will experience rapid and large diel variations in oxygen, implying recurrent P. minimum blooms need be considered as short-term oxygen stressors for Bay oyster spat and other living resources. Direct effects of P. minimum impacts on oysters were not as expected or previously reported. In one experiment, pre-bloom isolates of P. minimum were grown and then exposed to polyvinyl chloride (PVC) settlement plates to see whether dinoflagellate preconditioning of the hard substrate might affect oyster sets. No differences were noted between set on the PVC with P. minimum exposure to set recorded with filtered seawater, Instant Ocean^®, or Isochrysis. In the second oyster experiment, spat on PVC plates were exposed to field collected P. minimum blooms and a commercial mixture of several other food types including Isochrysis. Oyster growth was significantly higher in P. minimum exposures than noted in the commercial mix. These results, compared to results with other isolates from the same region, indicate substantial positive impact from some of the P. minimum blooms of the area while others separated in space, time, or nutrient status could severely curtail oyster success through toxin production induced by nutrient limitation.     

CLONING,EXPRESSION, AND CHARACTERIZATION OF A HISTONE‐LIKE PROTEIN FROM THE MARINE DINOFLAGELLATE LINGULODINIUM POLYEDRUM (DINOPHYCEAE)1

Although nucleosomes and histones are lacking in dinoflagellate nuclei, small basic histone‐like proteins have been reported, but their function(s) is unknown. In this study we cloned and sequenced a gene for a histone‐like protein from the dinoflagellate Lingulodinium polyedrum (Stein) Dodge (HLp) (formerly Gonyaulax polyedra Stein) and investigated its post‐translational modification and DNA‐binding activities. HLp appears to be acetylated in L. polyedrum, and we identified several L. polyedrum proteins that possess histone acetyltransferase activity and may be responsible for this modification. HLp binds weakly to L. polyedrum DNA but to certain specific sequences with higher affinity, consistent with its having a regulatory function.     

MOLECULAR PHYLOGENY OF THE HAPTOPHYTA BASED ON THE rbcL GENE AND SEQUENCE VARIATION IN THE SPACER REGION OF THE RUBISCO OPERON

A phylogeny of 21 haptophyte algae was inferred by maximum parsimony, neighbor-joining, and maximum likelihood analyses of sequences of the plastid-encoded gene, rbcL. Sequence variation in the spacer region of the RUBISCO operon was also investigated. In all the rbcL trees constructed, the haptophytes form two distinct clades: one includes the Pavlovales and the other includes the Prymnesiales, Coccosphaerales, and Isochrysidales (all sensu Parke and Green 1976 . This relationship coincides with the recent taxonomic treatment splitting the division into two subclasses, the Prymnesidae and Pavlovidae ( Cavalier-Smith 1989 ) or the Prymnesiophycidae and the Pavlovophycidae using botanical suffixes ( Jordan and Green 1994 ), or into two classes, the Patelliferea and the Pavlovea ( Cavalier-Smith 1993 ). In the Prymnesiophycidae, all the coccolithophorids examined are placed in a single clade, which suggests a single origin of the coccolithophorids and the ability of coccolith formation in the haptophytes. The genus Chrysochromulina is polyphyletic. Species of Chrysochromulina with a very long haptonema and a compressed cell body (typical of species including the type C. parva Lackey) form a clade, including Imantonia, that is often classified in the Isochrysidales in the neighbor-joining tree, whereas some species possessing a nontypical cell body and cell covering form a clade with Prymnesium and Platychrysis in all trees. It is suggested that loss of the haptonema in Imantonia and the reduction in Prymnesium and Platychrysis occurred secondarily and independently in two different lineages. Within the coccolithophorids, four clades are recognized: Pleurochrysis, Calyptrosphaera-Cruciplacolithus-Calcidiscus-Umbilicosphaera, Helicosphaera, and Emiliania-Gephyrocapsa. A non-coccolith-bearing haptophyte, Isochrysis, is an ingroup of the Emiliania-Gephyrocapsa clade, suggesting its secondary loss of the ability to form a coccolith. Sequence comparison of the spacer region of RUBISCO operon supports most results obtained in the analysis of rbcL sequences. Monophyly of the Prymnesiales sensu Parke and Green is still unclear because of low (<50%) bootstrap support for this group.     

CO2‐CONCENTRATING MECHANISMS OF THE POTENTIALLY TOXIC DINOFLAGELLATE PROTOCERATIUM RETICULATUM (DINOPHYCEAE,GONYAULACALES)1

The low CO₂ concentration in seawater poses severe restrictions on photosynthesis, especially on those species with form II RUBISCO. We found that the potentially toxic dinoflagellate Protoceratium reticulatum Clap. et J. Lachm. possesses a form II RUBISCO. To cast some light on the mechanisms this organism undergoes to cope with low CO₂ availability, we compared cells grown at atmospheric (370 ppm) and high (5000 ppm) CO₂ concentrations, with respect to a number of physiological parameters related to dissolved inorganic carbon (DIC) acquisition and assimilation. The photosynthetic affinity for DIC was about one order of magnitude lower in cells cultivated at high [CO₂]. End‐point pH‐drift experiments suggest that P. reticulatum was not able to efficiently use HCO₃^? under our growth conditions. Only internal carbonic anhydrase (CA) activity was detected, and its activity decreased by about 60% in cells cultured at high [CO₂]. Antibodies raised against a variety of algal CAs were used for Western blot analysis: P. reticulatum extracts only cross‐reacted with anti‐ß‐CA sera, and the amount of immunoreactive protein decreased in cells grown at high [CO₂]. No pyrenoids were observed under all growth conditions. Our data indicate that P. reticulatum has an inducible carbon‐concentrating mechanism (CCM) that operates in the absence of pyrenoids and with little intracellular CO₂ accumulation. Calculations on the impact of the CA activity to photosynthetic growth [CO₂] suggest that it is an essential component of the CCM of P. reticulatum and is necessary to sustain the photosynthetic rates observed at ambient CO₂.     

Prorocentrum minimum (Dinophyceae) in the Baltic Sea: morphology, occurrence—a review

The potentially toxic dinoflagellate Prorocentrum minimum (Pavillard) Schiller has successfully established in the Baltic Sea in the last two decades. A review of the invasion history is presented as well as new data on the spatial and inter-annual variability of this species and its relation to salinity, temperature, and nutrient concentrations. A short literature review of the morphological characters of the Baltic P. minimum is also included.From 1993 to 2002, P. minimum was a regular component of the summer and autumn plankton flora of the Baltic Sea proper and the Gulf of Finland. Its abundance varied considerably inter-annually and did not show any clear trends during the period. Abundance of P. minimum was significantly higher in the nutrient-enriched Bay of Mecklenburg (German coast) and the southern Baltic proper than in the central and northern Baltic proper and the Gulf of Finland, where its abundance was mostly sparse. In coastal waters P. minimum occasionally reached densities of several million cells per litre and dominated phytoplankton biomass (>90%).Abundance of the Baltic P. minimum was generally not related to salinity or temperature. It could be a dominant species at both high and low salinity (over 15 and 4.8 PSU), and its temperature range was broad (from 2.7 to 26.4 °C). However, dense populations usually occurred from July to October at temperatures above 10 °C.Further, there appears to be a positive correlation between the success of P. minimum in the Baltic Sea and high concentrations of total phosphorus and nitrogen.This tolerant and morphologically variable dinoflagellate seems to be a morphospecies without subtaxa, which can expand its range in the Baltic Sea, especially in nutrient-rich coastal waters.     

A PROPOSAL FOR A NEW RED ALGAL ORDER,THE THOREALES1

Representatives of the freshwater red algal family Thoreaceae were studied to resolve their taxonomic and phylogenetic status. Three specimens of Nemalionopsis and five collections of Thorea were examined for pit plug ultrastructure and analyzed for the sequences of the genes coding for the large subunit of RUBISCO (rbcL) and the small subunit of rRNA (18S rRNA). The phylogenetic trees generated from the two genes, and a combined tree all showed the Thoreaceae to be contained in a well‐supported monophyletic clade that is separate from the other two families currently classified in the Batrachospermales, the Batrachospermaceae and the Lemaneaceae. In addition, secondary structure elements of the 18S rRNA gene were observed at positions 650 and 1145 (Escherichia coli numbering system) that are not present in other members of the Rhodophyta. The pit plugs of the gametophytic and chantransia stages of the Thoreaceae contain two cap layers, the outer one of which is typically plate‐like, though occasionally inflated ones have been seen. No pit plug cap membrane has been observed. These findings indicate the Thoreaceae has been misclassified in the Batrachospermales and should be placed in its own order, the Thoreales. This order is characterized by having freshwater representatives with multiaxial gametophytes, a uniaxial chantransia stage, and pit plugs with two cap layers, the outer one of which is usually plate‐like.     

Polymorphism of the IGHA gene in sheep

Genetic variation in immunoglobulin A, the most abundant immunoglobulin in mammalian cells, has not been reported in ruminants. In this study, variation in the immunoglobulin heavy alpha chain constant gene (IGHA) of sheep was investigated by amplification of a fragment that included the hinge coding sequence, followed by single-strand conformational polymorphism (SSCP) analysis and DNA sequencing. Three novel sequences, each characterized by unique SSCP banding patterns, were identified. One or two sequences were detected in individual sheep and all the sequences identified shared high homology to the published ovine and bovine IGHA sequences, suggesting that these sequences represent allelic variants of the IGHA gene in sheep. Sequence alignment showed that these sequences differed mainly in the 3′ end of exon 1 and in the coding sequence of the hinge region. There was either a deletion or an insertion of two codons in the hinge coding region in these allelic variants. Codon usage in the hinge coding region was quite different from that in the non-hinge coding regions of the gene, suggesting different evolution of the IGHA hinge sequence. Three novel amino acid sequences of ovine IGHA were also predicted, and variation in these sequences might not only affect antigen recognition but also susceptibility to cleavage by bacterial or parasitic proteases. Nucleotide sequence data reported in this paper have been submitted to the NCBI GenBank nucleotide sequence database and have been assigned the accession nos. AY956424–AY956426.     

A CELLULOSE SYNTHASE (CESA) GENE FROM THE RED ALGA PORPHYRA YEZOENSIS (RHODOPHYTA)1

The cell walls of Porphyra species, like those of land plants, contain cellulose microfibrils that are synthesized by clusters of cellulose synthase enzymes (“terminal complexes”), which move in the plasma membrane. However, the morphologies of the Porphyra terminal complexes and the cellulose microfibrils they produce differ from those of land plants. To characterize the genetic basis for these differences, we have identified, cloned, and sequenced a cellulose synthase (CESA) gene from Porphyra yezoensis Ueda strain TU‐1. A partial cDNA sequence was identified in the P. yezoensis expressed sequence tag (EST) index using a land plant CESA sequence as a query. High‐efficiency thermal asymmetric interlaced PCR was used to amplify sequences upstream of the cDNA sequence from P. yezoensis genomic DNA. Using the resulting genomic sequences as queries, we identified additional EST sequences and a full‐length cDNA clone, which we named PyCESA1. The conceptual translation of PyCESA1 includes the four catalytic domains and the N‐ and C‐terminal transmembrane domains that characterize CESA proteins. Genomic PCR demonstrated that PyCESA1 contains no introns. Southern blot analysis indicated that P. yezoensis has at least three genomic sequences with high similarity to the cloned gene; two of these are pseudogenes based on analysis of amplified genomic sequences. The P. yezoensis CESA peptide sequence is most similar to cellulose synthase sequences from the oomycete Phytophthora infestans and from cyanobacteria. Comparing the CESA genes of P. yezoensis and land plants may facilitate identification of sequences that control terminal complex and cellulose microfibril morphology.