Nitrogen-fixing organelle in a marine alga.

Symbiotic interactions were key to the evolution of chloroplast and mitochondria organelles, which mediate carbon and energy metabolism in eukaryotes. Biological nitrogen fixation, the reduction of abundant atmospheric nitrogen gas (N2) to biologically available ammonia, is a key metabolic process performed exclusively by prokaryotes. Candidatus Atelocyanobacterium thalassa, or UCYN-A, is a metabolically streamlined N2-fixing cyanobacterium previously reported to be an endosymbiont of a marine unicellular alga. Here we show that UCYN-A has been tightly integrated into algal cell architecture and organellar division and that it imports proteins encoded by the algal genome. These are characteristics of organelles and show that UCYN-A has evolved beyond endosymbiosis and functions as an early evolutionary stage N2-fixing organelle, or "nitroplast."

Targeted Metabolite Fingerprints of Thirteen Gambierdiscus, Five Coolia and Two Fukuyoa Species.

The genus Gambierdiscus produces an array of bioactive hydrophilic and lipophilic secondary metabolites that range in mode of action and toxicity. In this study, the metabolite fingerprint was mapped for thirteen Gambierdiscus, five Coolia and two Fukuyoa species (34 isolates) by assessing the production of 56 characterised secondary metabolites. Gambierdiscus polynesiensis was the only species to produce Pacific-ciguatoxin-3B (P-CTX3B), P-CTX3C, iso-P-CTX3B/C, P-CTX4A, P-CTX4B and iso-P-CTX4A/B. G. australes produced maitotoxin-1 (MTX-1) and MTX-5, G. cheloniae produced MTX-6 and G. honu produced MTX-7. Ubiquitous production of 44-methylgambierone was observed amongst all the Gambierdiscus isolates, with nine species also producing gambierone. Additional gambierone analogues, including anhydrogambierone (tentatively described herein), were also detected in all Gambierdiscus species, two Coolia and two Fukuyoa species. Gambieroxide was detected in G. lewisii and G. pacificus and gambieric acid A was detected in ten Gambierdiscus species, with G. australes (CAWD381) being the only isolate to produce gambieric acids A-D. This study has demonstrated that the isolates tested to date produce the known CTXs or MTXs, but not both, and highlighted several species that produced 'unknown' compounds displaying characteristics of cyclic polyethers, which will be the focus of future compound discovery efforts.

Nuclear Transformation of the Marine Pennate Diatom Nitzschia sp. Strain NIES-4635 by Multi-Pulse Electroporation.

Nitzschia is one of the largest genera of diatoms found in a range of aquatic environments, from freshwater to seawater. This genus contains evolutionarily and ecologically unique species, such as those that have lost photosynthetic capacity or those that live symbiotically in dinoflagellates. Several Nitzschia species have been used as indicators of water pollution. Recently, Nitzschia species have attracted considerable attention in the field of biotechnology. In this study, a transformation method for the marine pennate diatom Nitzschia sp. strain NIES-4635, isolated from the coastal Seto Inland Sea, was established. Plasmids containing the promoter/terminator of the fucoxanthin chlorophyll a/c binding protein gene (fcp, or Lhcf) derived from Nitzschia palea were constructed and introduced into cells by multi-pulse electroporation, resulting in 500 µg/mL nourseothricin-resistant transformants with transformation frequencies of up to 365 colonies per 108 cells. In addition, when transformation was performed using a new plasmid containing a promoter derived from a diatom-infecting virus upstream of the green fluorescent protein gene (gfp), 44% of the nourseothricin-resistant clones exhibited GFP fluorescence. The integration of the genes introduced into the genomes of the transformants was confirmed by Southern blotting. The Nitzschia transformation method established in this study will enable the transformation this species, thus allowing the functional analysis of genes from the genus Nitzschia, which are important species for environmental and biotechnological development.

Morphology and phylogeny of Prorocentrum porosum sp. nov. (Dinophyceae): A new benthic toxic dinoflagellate from the Atlantic and Pacific Oceans.

A new marine benthic toxic Prorocentrum species is described from the tropical/subtropical regions of the Atlantic (Colombian Caribbean Sea and Northeast Brazil) and Pacific (Southern Japan) oceans. Morphological cell structures were examined using light (LM) and scanning electron (SEM) microscopy. Prorocentrum porosum sp. nov. was characterized by 35.9-50.2 µm long and 25.4-45.7 µm deep cells, covered by broadly ovoid symmetric thecal plates. The surface of both thecal plates is smooth and covered by randomly scattered kidney-shaped pores (n = 102-149), rounder towards the center, absent in the central part, and surrounded by a conspicuous marginal ring of about 69-92 evenly spaced pores. Broad V-shaped periflagellar area exhibiting flagellar and accessory pores. The molecular phylogenetic position of P. porosum sp. nov. was inferred using partial LSU rRNA gene (rDNA) and rDNA ITS sequences. This new species branched with high support in a Prorocentrum clade including P. caipirignum, P. hoffmannianum and P. cf. lima (P. lima morphotype 5 sensuZhang et al., 2015). Pairwise comparison of ITS1 and ITS2 transcripts with these closest relatives revealed the presence of compensatory base changes (CBCs), with the exception of P. cf. lima (P. lima morphotype 5), which only showed in ITS2 a hemi-CBC (HCBC) and two base changes that possibly induce a structural modification. Toxin analyses performed in two Colombian and Brazilian strains in the present study detected the presence of low amounts of okadaic acid.

Utilization of phosphonic acid compounds by marine bacteria of the genera Phaeobacter, Ruegeria, and Thalassospira (α-Proteobacteria).

Phosphonic acid (phosphonate) that possesses a carbon-phosphours bond is a chemically stable form of organic phosphorus. Various phosphonic acids are widely distributed in oceanic waters; in particular, methylphosphonic acid (namely methylphosphonate) is believed to be responsible for global methane production. To discuss the microbial degradation of phosphonic acids, we investigated the utilization of phosphonic acid compounds by cultures of marine bacteria, Phaeobacter sp., Ruegeria sp. (Rhodobacterales), and Thalassospira sp. (Rhodospirillales). These bacterial cultures were able to grow on methylphosphonic acid as well as on the tested alkyl-, carboxy-, aminoalkyl-, and hydroxyalkyl-phosphonic acid compounds. Cell yields and growth rates of Ruegeria and Thalassospira cultures grown on methyl-, ethyl-, propyl-, and butyl-phosphonic acid compounds tended to decrease with increasing alkyl chain length. In contrast, Phaeobacter sp. grew well on such alkyl-phosphonic acids. Our results suggest that these marine bacteria, which exhibit varied utilization, are involved in microbial degradation of various phosphonic acid compounds.

Toxicity and growth characteristics of epiphytic dinoflagellate Gambierdiscus silvae in Japan.

The genus Gambierdiscus is a marine benthic/epiphytic dinoflagellate that has been investigated worldwide as the causative agent of ciguatera poisoning (CP). In Japan, CP occurs mainly in the subtropical region and sporadically in the temperate region. To understand the mechanism of CP outbreaks in the coastal regions, identifying the species of Gambierdiscus occurring in the regions and determining their toxicity and growth characteristics, such as growth responses to temperature, salinity, and light intensity, are important. Recently, the occurrence of G. silvae in the Japanese temperate and subtropical regions has been revealed through metabarcoding. However, the toxicity and growth characteristics of G. silvae have not yet been investigated. In this study, three strains of Gambierdiscus were isolated from a depth of 30 m in subtropical waters in Japan and were identified as Gambierdiscus silvae based on morphological characteristics and phylogenetic positions. A dichloromethane soluble fraction (DSF) and aqueous methanol soluble fraction (MSF) of the three strains showed high mouse toxicity by intraperitoneal injection, but only the DSF of the three strains showed toxicity by gavage. All strains grew in the range of 17.5-30 °C and salinity range of 25-40, and grew well at 25 °C and salinity 30. The optimal light intensity for growth of the strains was 42.0-83.0 µmol photons/m2/s. These results suggest that G. silvae has the potential to be widely distributed from temperate to subtropical/ regions and in shallow to deep coastal waters of Japan. Understanding the growth characteristics of this species would be useful in predicting the occurrence of this species in Japanese coastal waters. Finally, the results obtained in this study suggest that G. silvae showing high toxicity is one of the causative agents of CP in Japan, and knowledge of this species would be useful in understanding the mechanism of CP outbreaks in Japan.

Effectiveness of blocking primers and a peptide nucleic acid (PNA) clamp for 18S metabarcoding dietary analysis of herbivorous fish.

The structure of food webs and carbon flow in aquatic ecosystems can be better understood by studying contributing factors such as the diets of herbivorous fish. Metabarcoding using a high-throughput sequencer has recently been used to clarify prey organisms of various fish except herbivorous fish. Since sequences of predator fish have dominated in sequences obtained by metabarcoding, we investigated a method for suppressing the amplification of fish DNA by using a blocking primer or peptide nucleic acid (PNA) clamp to determine the prey organisms of herbivorous fish. We designed three blocking primers and one PNA clamp that anneal to fish-specific sequences and examined how efficient they were in suppressing DNA amplification in various herbivorous fish. The results showed that the PNA clamp completely suppressed fish DNA amplification, and one of the blocking primers suppressed fish DNA amplification but less efficiently than the PNA clamp. Finally, we conducted metabarcoding using mock community samples as templates to determine whether the blocking primer or the PNA clamp was effective in suppressing fish DNA amplification. The results showed that the PNA clamp suppressed 99.3%-99.9% of fish DNA amplification, whereas the blocking primer suppressed 3.3%-32.9%. Therefore, we propose the application of the PNA clamp for clarifying the prey organisms and food preferences of various herbivorous fish.

Characterization of Chaetoceros lorenzianus-infecting DNA virus-derived promoters of genes from open reading frames of unknown function in Phaeodactylum tricornutum.

Promoters are key elements for the regulation of gene expression. Recently, we investigated the activity of promoters derived from marine diatom-infecting viruses (DIVs) in marine diatoms. Previously, we focused on potential promoter regions of the replication-associated protein gene and the capsid protein gene of the DIVs. In addition to these genes, two genes of unknown function (VP1 and VP4 genes) have been found in the DIV genomes. In this study, the promoter regions of the VP1 gene and VP4 gene derived from a Chaetoceros lorenzianus-infecting DNA virus (named ClP3 and ClP4, respectively) were newly isolated. ClP4 was found to be a constitutive promoter and displayed the highest activity. In particular, the 3' region of ClP4 (ClP4 3' region) showed a higher promoter activity than full-length ClP4. The ClP4 3' region might involve high-level promoter activity of ClP4. In addition, the ClP4 3' region may be useful for substance production and metabolic engineering of diatoms.

Horizontal and vertical distribution of Gambierdiscus spp. (Dinophyceae) including novel phylotypes in Japan identified by 18S rDNA metabarcoding.

The genus Gambierdiscus is a marine benthic/epiphytic dinoflagellate considered the causative agent of ciguatera poisoning (CP). Clarifying the geographical distribution of this genus to understand the potential risk of CP is important. Many studies have focused only on the species/phylotype composition of Gambierdiscus in shallow waters, but no study has investigated the species/phylotype composition of the genus in deep waters. In the present study, the distributions of Gambierdiscus species/phylotypes at two depths (2-8 and 30 m) and two sampling sites (temperate and subtropical) in Japan was investigated using high throughput sequencing (HTS) with a newly developed primer set that preferentially amplifies the 18S rDNA V8-V9 region of Alveolata. A phylogenetic analysis using 89 samples collected over three years revealed of ten Gambierdiscus species/phylotypes including not only two species that have not been reported in Japan (G. caribaeus and G. silvae) but also four novel phylotypes (Gambierdiscus spp. Clade II_1, Clade II_2, Clade II_3, and Clade VI_1). Uncorrected genetic distances also supported that these new phylotypes clearly diverged from other Gambierdiscus species. All four new phylotypes, G. caribaeus, and G. silvae were distributed in the subtropical region. Among them, Clade II_2, Clade VI_1, and G. silvae were also distributed in the temperate region. Four species/phylotypes previously reported from Japan showed a similar distribution as reported previously. Among the ten species/phylotypes, Gambierdiscus sp. type 3 and Clade VI_1 were found only in deep waters, whereas five species/phylotypes were observed only in shallow waters. The other three species/phylotypes were found in both deep and shallow waters. The results of the horizontal and vertical distribution suggest that the growth characteristics of each species/phylotypes found in Japan might adapt to the ambient environmental conditions. This study revealed an inclusive assemblage of Gambierdiscus species/phylotypes in Japan through metabarcoding using the Alveolata primer set. In the future, the abundance and toxicities/toxin productions of the newly reported species/phylotypes need to be clarified to understand the mechanism of CP outbreaks in Japan.

Determination of optimal culture conditions for toxin production by a Prorocentrum lima complex strain with high diarrhetic shellfish toxins yield.

Diarrhetic shellfish poisoning (DSP) is caused by the consumption of shellfish contaminated by diarrhetic shellfish toxins (DSTs) such as okadaic acid (OA) and dinophysistoxins (DTXs) produced by some species of dinoflagellates. To prevent the occurrence of human intoxication cases, inspection of DSTs (OA and DTXs) in shellfish is important. An instrumental method using liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been recently employed in Japan for the monitoring of OA and DTXs in shellfish. For such analysis, reference materials (RMs) of OA and DTXs are essential. Demand for the reference materials, especially dinophysistoxin-1 (DTX1), is recently increasing in Japan. Production of the materials has been performed by mass cultivation of a dinoflagellate (Prorocentrum lima) strain that produces DTXs and OA, which indicates that the efficiency of production depends on the toxin production of the strain used. In this study, P. lima complex subclade 1e strain MIO12P was determined to be a high DTX1 producer among the three Japanese strains of the P. lima complex (subclades 1e, 1f, and 1i). It was clarified that the culture medium suitable for toxin production by strain MIO12P was metals mix SWII medium, and the optimal temperature and salinity for toxin production were 25 °C and salinity 30, respectively. The DTX1 yield (1265.3 ng ml-1) of strain MIO12P cultured under the conditions described above was the highest reported worldwide. Prorocentrum lima complex subclade 1e strain MIO12P is expected to be useful for the sustainable production of DTX1 as a source of RMs for chemical and biochemical methods in the future.

First report of Alexandrium (Dinophyceae) associated with marine macroalgae off Japan: Diversity, distribution, and toxicity.

Macroalgal samples were collected from coastal waters in subboreal to subtropical zones in Japan (< 3-30 m depths) and 32 clonal strains of non-motile dinoflagellate-like protists were established. Molecular phylogenetic analyses of the LSU rDNA D1/D2, SSU rDNA, ITS region, and concatenated SSU rDNA + LSU rDNA D1/D2 sequences revealed that the strains nested within the genus Alexandrium. They were separated into three novel phylotypes: Alexandrium spp. type 1, type 2, and type 3. Analysis of the concatenated sequences revealed that the most closely related species for the three phylotypes was A. ostenfeldii. Most cells from strains of the three phylotypes were non-motile and hemispherical to spherical in shape. The average diameters of the non-motile cells were between 35 and 39 µm. Type 1 and type 2 were widely distributed in Japan from the temperate to subtropical zones, whereas type 3 was restricted to the temperate zone. Furthermore, type 2 was widespread from shallow to deep waters, whereas type 1 and type 3 were restricted to deep waters. Growth experiments in strains belonging to the three phylotypes revealed that the occurrence ratios of motile cells were very low (≤ 1.1% of the total cells). The production of paralytic shellfish poisoning toxins, tetrodotoxin, and cyclic imines was assessed in strains belonging to the three phylotypes by LC/MS/MS analysis. The strains did not produce any of the toxins tested. The strains of the three phylotypes showed lethal toxicity to mice by intraperitoneal administration. To the best of our knowledge, this is the first study to report the existence of Alexandrium associated with marine macroalgae from Japan.

Diel vertical movements and feeding behaviour of blue humphead parrotfish Scarus ovifrons in a temperate reef of Japan.

The feeding ecology of scarinine parrotfishes on tropical coral reefs has received considerable attention in the past few decades; nonetheless, relatively few studies have been conducted in high-latitude reefs. Among the Indo-Pacific Scarus species, Scarus ovifrons is unique, being largely restricted to the warm temperate waters of Japan. Nonetheless, there is very little information available on the feeding ecology of this species. In this study, the authors used acoustic telemetry to detect the diel vertical movement patterns of S. ovifrons, video survey to detect its feeding depths and substrata and focal follow survey and genetic analysis to identify algae composition on the feeding scars at Kashiwajima Island, southwestern Japan (32° 46' N, 132° 38' E). Acoustic telemetry revealed that S. ovifrons spent most of its time in shallow water (<10 m) during the day and slept in deeper water (10-15 m) at night. Video and focal follow surveys revealed that most fishes of various sizes regularly took bites on epilithic algae and detrital materials on rocky substrata at depths of <10 m, but large fishes (>40 cm total length) sometimes took bites directly on live corals (Acropora solitaryensis) at the 5 m depth zone where live tabular corals dominated the benthos. Molecular phylogenetic analyses revealed that epilithic algae collected from feeding scars were mainly composed of Rhodophyta, and coralline algae were less often targeted. Overall, this study revealed that S. ovifrons feeds mostly at depths <10 m, and the feeding algae substrata of the species are similar to those of tropical coral reef parrotfishes.

Morphological and phylogenetic data do not support the split of Alexandrium into four genera.

A recently published study analyzed the phylogenetic relationship between the genera Centrodinium and Alexandrium, confirming an earlier publication showing the genus Alexandrium as paraphyletic. This most recent manuscript retained the genus Alexandrium, introduced a new genus Episemicolon, resurrected two genera, Gessnerium and Protogonyaulax, and stated that: "The polyphyly [sic] of Alexandrium is solved with the split into four genera". However, these reintroduced taxa were not based on monophyletic groups. Therefore this work, if accepted, would result in replacing a single paraphyletic taxon with several non-monophyletic ones. The morphological data presented for genus characterization also do not convincingly support taxa delimitations. The combination of weak molecular phylogenetics and the lack of diagnostic traits (i.e., autapomorphies) render the applicability of the concept of limited use. The proposal to split the genus Alexandrium on the basis of our current knowledge is rejected herein. The aim here is not to present an alternative analysis and revision, but to maintain Alexandrium. A better constructed and more phylogenetically accurate revision can and should wait until more complete evidence becomes available and there is a strong reason to revise the genus Alexandrium. The reasons are explained in detail by a review of the available molecular and morphological data for species of the genera Alexandrium and Centrodinium. In addition, cyst morphology and chemotaxonomy are discussed, and the need for integrative taxonomy is highlighted.

Abundance of the benthic dinoflagellate Prorocentrum and the diversity, distribution, and diarrhetic shellfish toxin production of Prorocentrum lima complex and P. caipirignum in Japan.

In the present study, the abundance of Prorocentrum and the molecular phylogeny, distribution, and DST production of P. lima complex and P. caipirignum in Japan were investigated. First, the cell densities of Prorocentrum were assessed from the temperate to subtropical zones in Japan between 2014 and 2018. The cell density in the subtropical zone [19.0 ± 40.2 cells/g wet weight (ww) algae] was significantly higher than that in the temperate zone (1.4 ± 3.4 cells/g ww algae). A total of 244 clonal strains were established from the temperate and subtropical zones. Phylogenetic analyses based on the large-subunit ribosomal DNA D1/D2 revealed that the strains were separated into four species/species complex/phylotypes (P. lima complex, P. caipirignum, and new phylotypes Prorocentrum spp. types 1 and 2). The strains of P. lima complex could be separated into two clades (1 and 3). Furthermore, the strains of clades 1 and 3 could be separated into nine subclades (1a, 1c, 1d, 1e, 1f, 1g, 1h, 1i, and 1j) and three subclades (3a, 3b, and 3c), respectively. The strains of P. caipirignum were separated into two subclades (b and e). Each phylotype/subclade showed a unique distribution pattern in Japan: P. lima complex subclades 1a, 1c, and 3a and P. caipirignum subclades b and e were widespread from the temperate to subtropical zones. On the other hand, P. lima complex subclades 1e and 1i were restricted to the temperate zone, and P. lima complex subclades 1d, 1f, 1g, 1h, 1j, 3b, and 3c and Prorocentrum spp. types 1 and 2 were restricted to the subtropical zone. Furthermore, the DST production of the 243 clonal strains was assessed by LC/MS/MS analysis. The results revealed that all strains produced okadaic acid (OA) and that the OA contents of P. lima complex subclades 1d and 1f, P. caipirignum subclades b and e, and Prorocentrum sp. type 2 tended to be higher than those of the other subclades. While P. lima complex subclades 1a, 1e, 1f, and 1i produced DTX1, the other phylotype/subclades produced either no or low quantities of DTX1. A strain of P. lima complex subclade 1e showed the highest OA and DTX1 contents (55.27 and 70.73 pg/cell, respectively) in the world. These results suggest that there are potential risks for DST accumulation in benthic animals in Japan.

The possibility of using marine diatom-infecting viral promoters for the engineering of marine diatoms.

Marine diatoms constitute a major group of unicellular photosynthetic eukaryotes. Diatoms are widely applicable for both basic studies and applied studies. Molecular tools and techniques have been developed for diatom research. Among these tools, several endogenous gene promoters (e.g., the fucoxanthin chlorophyll a/c-binding protein gene promoter) have become available for expressing transgenes in diatoms. Gene promoters that drive transgene expression at a high level are very important for the metabolic engineering of diatoms. Various marine diatom-infecting viruses (DIVs), including both DNA viruses and RNA viruses, have recently been isolated, and their genome sequences have been characterized. Promoters from viruses that infect plants and mammals are widely used as constitutive promoters to achieve high expression of transgenes. Thus, we recently investigated the activity of promoters derived from marine DIVs in the marine diatom, Phaeodactylum tricornutum. We discuss novel viral promoters that will be useful for the future metabolic engineering of diatoms.

Development of endogenous promoters that drive high-level expression of introduced genes in the model diatom Phaeodactylum tricornutum.

The marine diatom Phaeodactylum tricornutum is attractive for basic and applied diatom research. We isolated putative endogenous gene promoters derived from genes that are highly expressed in P. tricornutum: the fucoxanthin chlorophyll a/c-binding protein (FCP) C gene, the vacuolar ATP synthase 16-kDa proteolipid subunit (V-ATPase C) gene, the clumping factor A gene and the solute carrier family 34 member 2 gene. Five putative promoter regions were isolated, linked to an antibiotic resistance gene (Sh ble) and transformed into P. tricornutum. Using quantitative RT-PCR, the promoter activities in the transformants were analyzed and compared to that of the diatom endogenous gene promoter, the FCP A gene promoter which has been used for the transformation of P. tricornutum. Among the five isolated potential promoters, the activity of the V-ATPase C gene promoter was approximately 2.73 times higher than that of the FCP A gene promoter. The V-ATPase C gene promoter drove the expression of Sh ble mRNA transcripts under both light and dark conditions at the stationary phase. These results suggest that the V-ATPase C gene promoter is a novel tool for the genetic engineering of P. tricornutum.

Examination of prezygotic and postzygotic isolating barriers in tropical Ulva (Ulvophyceae, Chlorophyta): evidence for ongoing speciation.

Phylogenetic clades based on DNA sequences such as the chloroplast rbcL gene and the nuclear ITS region are frequently used to delimit algal species. However, these molecular markers cannot accurately delimit boundaries among some Ulva species. Although Ulva reticulata and Ulva ohnoi occasionally bloom in tropical to warm-temperate regions and are clearly distinguishable by their reticulate or plain blade morphology, they have few or no sequence divergences in these molecular markers and form a monophyletic clade. In this study, to clarify the speciation and species delimitation in the U. reticulata-ohnoi complex clade, reproductive relationships among several sexual strains from the Philippines and Japan including offspring that originated from the type specimen of U. ohnoi were examined by culturing and hybridization in addition to the ITS-based analysis. As a result, both prezygotic and postzygotic reproductive isolation were revealed to occur between genetically perforated U. reticulata and imperforate U. ohnoi. They were also separated on the basis of sequence analysis of the ITS region. That strongly supports that the two taxa are independent biological species. Although no prezygotic barrier among the Philippine and Japanese strains of U. reticulata was observed, unexpectedly zoospores produced by hybrid sporophytes in some of their combinations mostly failed to develop, indicating partial formation of a postzygotic barrier despite a 0.2% divergence in the ITS sequence. These findings suggest speciation is still ongoing in U. reticulata.

An exception among diatoms: unique organization of genes involved in isoprenoid biosynthesis in Rhizosolenia setigera CCMP 1694.

The marine diatom Rhizosolenia setigera is unique among this group of microalgae given that it is only one of a handful of diatom species that can produce highly branched isoprenoid (HBI) hydrocarbons. In our efforts to determine distinguishing molecular characteristics in R. setigera CCMP 1694 that could help elucidate the underlying mechanisms for its ability to biosynthesize HBIs, we discovered the occurrence of independent genes encoding for two isopentenyl diphosphate isomerases (RsIDI1 and RsIDI2) and one squalene synthase (RsSQS), enzymes that catalyze non-consecutive steps in isoprenoid biosynthesis. These genes are peculiarly fused in all other genome-sequenced diatoms to date, making their organization in R. setigera CCMP 1694 a clear distinguishing molecular feature. Phylogenetic and sequence analysis of RsIDI1, RsIDI2, and RsSQS revealed that such an arrangement of individually transcribed genes involved in isoprenoid biosynthesis could have arisen through a secondary gene fission event. We further demonstrate that inhibition of squalene synthase (SQS) shifts the flux of exogenous isoprenoid precursors towards HBI biosynthesis suggesting the competition for isoprenoid substrates in the form of farnesyl diphosphate between the sterol and HBI biosynthetic pathways in this diatom.

LSU rDNA based RFLP assays for the routine identification of Gambierdiscus species.

The Gambierdiscus genus is a group of benthic dinoflagellates commonly associated with ciguatera fish poisoning (CFP), which is generally found in tropical or sub-tropical regions around the world. Morphologically similar species within the genus can vary in toxicity; however, species identifications are difficult or sometimes impossible using light microscopy. DNA sequencing of ribosomal RNA genes (rDNA) is thus often used to identify and describe Gambierdiscus species and ribotypes, but the expense and time can be prohibitive for routine culture screening and/or large-scale monitoring programs. This study describes a restriction fragment length polymorphism (RFLP) typing method based on analysis of the large subunit rDNA that can successfully identify at least nine of the described Gambierdiscus species and two Fukuyoa species. The software programs DNAMAN 6.0 and Restriction Enzyme Picker were used to identify a set of restriction enzymes (SpeI, HpyCH4IV, and TaqαI) capable of distinguishing most of the known Gambierdiscus species for which DNA sequences were available. This assay was tested using in silico analysis and cultured isolates, and species identifications of isolates assigned by RFLP typing were confirmed by DNA sequencing. To verify the assay and assess intra-specific heterogeneity in RFLP patterns, identifications of 63 Gambierdiscus isolates comprising ten Gambierdiscus species, one ribotype, and two Fukuyoa species were confirmed using RFLP typing, and this method was subsequently employed in the routine identification of isolates collected from the Caribbean Sea. The RFLP assay presented here reduces the time and cost associated with morphological identification via scanning electron microscopy and/or DNA sequencing, and provides a phylogenetically sensitive method for routine Gambierdiscus species assignment.