Characterization of Dinophysis spp. (Dinophyceae, Dinophysiales) from the mid-Atlantic region of the United States1.

Due to the increasing prevalence of Dinophysis spp. and their toxins on every US coast in recent years, the need to identify and monitor for problematic Dinophysis populations has become apparent. Here, we present morphological analyses, using light and scanning electron microscopy, and rDNA sequence analysis, using a ~2-kb sequence of ribosomal ITS1, 5.8S, ITS2, and LSU DNA, of Dinophysis collected in mid-Atlantic estuarine and coastal waters from Virginia to New Jersey to better characterize local populations. In addition, we analyzed for diarrhetic shellfish poisoning (DSP) toxins in water and shellfish samples collected during blooms using liquid-chromatography tandem mass spectrometry and an in vitro protein phosphatase inhibition assay and compared this data to a toxin profile generated from a mid-Atlantic Dinophysis culture. Three distinct morphospecies were documented in mid-Atlantic surface waters: D. acuminata, D. norvegica, and a "small Dinophysis sp." that was morphologically distinct based on multivariate analysis of morphometric data but was genetically consistent with D. acuminata. While mid-Atlantic D. acuminata could not be distinguished from the other species in the D. acuminata-complex (D. ovum from the Gulf of Mexico and D. sacculus from the western Mediterranean Sea) using the molecular markers chosen, it could be distinguished based on morphometrics. Okadaic acid, dinophysistoxin 1, and pectenotoxin 2 were found in filtered water and shellfish samples during Dinophysis blooms in the mid-Atlantic region, as well as in a locally isolated D. acuminata culture. However, DSP toxins exceeded regulatory guidance concentrations only a few times during the study period and only in noncommercial shellfish samples.

Major transitions in dinoflagellate evolution unveiled by phylotranscriptomics.

Dinoflagellates are key species in marine environments, but they remain poorly understood in part because of their large, complex genomes, unique molecular biology, and unresolved in-group relationships. We created a taxonomically representative dataset of dinoflagellate transcriptomes and used this to infer a strongly supported phylogeny to map major morphological and molecular transitions in dinoflagellate evolution. Our results show an early-branching position of Noctiluca, monophyly of thecate (plate-bearing) dinoflagellates, and paraphyly of athecate ones. This represents unambiguous phylogenetic evidence for a single origin of the group's cellulosic theca, which we show coincided with a radiation of cellulases implicated in cell division. By integrating dinoflagellate molecular, fossil, and biogeochemical evidence, we propose a revised model for the evolution of thecal tabulations and suggest that the late acquisition of dinosterol in the group is inconsistent with dinoflagellates being the source of this biomarker in pre-Mesozoic strata. Three distantly related, fundamentally nonphotosynthetic dinoflagellates, Noctiluca, Oxyrrhis, and Dinophysis, contain cryptic plastidial metabolisms and lack alternative cytosolic pathways, suggesting that all free-living dinoflagellates are metabolically dependent on plastids. This finding led us to propose general mechanisms of dependency on plastid organelles in eukaryotes that have lost photosynthesis; it also suggests that the evolutionary origin of bioluminescence in nonphotosynthetic dinoflagellates may be linked to plastidic tetrapyrrole biosynthesis. Finally, we use our phylogenetic framework to show that dinoflagellate nuclei have recruited DNA-binding proteins in three distinct evolutionary waves, which included two independent acquisitions of bacterial histone-like proteins.

Lacking catalase, a protistan parasite draws on its photosynthetic ancestry to complete an antioxidant repertoire with ascorbate peroxidase.

BACKGROUND: Antioxidative enzymes contribute to a parasite's ability to counteract the host's intracellular killing mechanisms. The facultative intracellular oyster parasite, Perkinsus marinus, a sister taxon to dinoflagellates and apicomplexans, is responsible for mortalities of oysters along the Atlantic coast of North America. Parasite trophozoites enter molluscan hemocytes by subverting the phagocytic response while inhibiting the typical respiratory burst. Because P. marinus lacks catalase, the mechanism(s) by which the parasite evade the toxic effects of hydrogen peroxide had remained unclear. We previously found that P. marinus displays an ascorbate-dependent peroxidase (APX) activity typical of photosynthetic eukaryotes. Like other alveolates, the evolutionary history of P. marinus includes multiple endosymbiotic events. The discovery of APX in P. marinus raised the questions: From which ancestral lineage is this APX derived, and what role does it play in the parasite's life history? RESULTS: Purification of P. marinus cytosolic APX activity identified a 32 kDa protein. Amplification of parasite cDNA with oligonucleotides corresponding to peptides of the purified protein revealed two putative APX-encoding genes, designated PmAPX1 and PmAPX2. The predicted proteins are 93% identical, and PmAPX2 carries a 30 amino acid N-terminal extension relative to PmAPX1. The P. marinus APX proteins are similar to predicted APX proteins of dinoflagellates, and they more closely resemble chloroplastic than cytosolic APX enzymes of plants. Immunofluorescence for PmAPX1 and PmAPX2 shows that PmAPX1 is cytoplasmic, while PmAPX2 is localized to the periphery of the central vacuole. Three-dimensional modeling of the predicted proteins shows pronounced differences in surface charge of PmAPX1 and PmAPX2 in the vicinity of the aperture that provides access to the heme and active site. CONCLUSIONS: PmAPX1 and PmAPX2 phylogenetic analysis suggests that they are derived from a plant ancestor. Plant ancestry is further supported by the presence of ascorbate synthesis genes in the P. marinus genome that are similar to those in plants. The localizations and 3D structures of the two APX isoforms suggest that APX fulfills multiple functions in P. marinus within two compartments. The possible role of APX in free-living and parasitic stages of the life history of P. marinus is discussed.

Characterization of Acetyl-CoA Carboxylases in the Basal Dinoflagellate Amphidinium carterae.

Dinoflagellates make up a diverse array of fatty acids and polyketides. A necessary precursor for their synthesis is malonyl-CoA formed by carboxylating acetyl CoA using the enzyme acetyl-CoA carboxylase (ACC). To date, information on dinoflagellate ACC is limited. Through transcriptome analysis in Amphidinium carterae, we found three full-length homomeric type ACC sequences; no heteromeric type ACC sequences were found. We assigned the putative cellular location for these ACCs based on transit peptide predictions. Using streptavidin Western blotting along with mass spectrometry proteomics, we validated the presence of ACC proteins. Additional bands showing other biotinylated proteins were also observed. Transcript abundance for these ACCs follow the global pattern of expression for dinoflagellate mRNA messages over a diel cycle. This is one of the few descriptions at the transcriptomic and protein level of ACCs in dinoflagellates. This work provides insight into the enzymes which make the CoA precursors needed for fatty acid and toxin synthesis in dinoflagellates.

Complex Ancestries of Isoprenoid Synthesis in Dinoflagellates.

Isoprenoid metabolism occupies a central position in the anabolic metabolism of all living cells. In plastid-bearing organisms, two pathways may be present for de novo isoprenoid synthesis, the cytosolic mevalonate pathway (MVA) and nuclear-encoded, plastid-targeted nonmevalonate pathway (DOXP). Using transcriptomic data we find that dinoflagellates apparently make exclusive use of the DOXP pathway. Using phylogenetic analyses of all DOXP genes we inferred the evolutionary origins of DOXP genes in dinoflagellates. Plastid replacements led to a DOXP pathway of multiple evolutionary origins. Dinoflagellates commonly referred to as dinotoms due to their relatively recent acquisition of a diatom plastid, express two completely redundant DOXP pathways. Dinoflagellates with a tertiary plastid of haptophyte origin, by contrast, express a hybrid pathway of dual evolutionary origin. Here, changes in the targeting motif of signal/transit peptide likely allow for targeting the new plastid by the proteins of core isoprenoid metabolism proteins. Parasitic dinoflagellates of the Amoebophyra species complex appear to have lost the DOXP pathway, suggesting that they may rely on their host for sterol synthesis.

Does a blue crab putative insulin-like peptide binding protein (ILPBP) play a role in a virus infection?

Insulin-like peptides (ILPs) have regulatory roles in reproduction, development and metabolism in invertebrates. The mode of ILP actions has not been well studied in invertebrates in regard to the role of binding partners, i.e., ILP binding protein (ILPBP). In this study, the full-length cDNA of Callinectes sapidus ILPBP (Cas-ILPBP, 960 bp) has been isolated using RACE cloning, having short 5' and 3' UTRs of 30 and 162 bp, respectively. The predicted precursor of Cas-ILPBP (255 aa) contains, in order a signal peptide (23 aa), an insulin-like growth factor (IGF) binding (IB) domain (79 aa), a kazal-type serine protease inhibitor (KI) domain (36 aa) and an immunoglobulin (Ig) domain (101 aa). Phylogenetic analysis shows that Cas-ILPBP is grouped with the ILPBPs of other crustacean species, and it shares the closest relationship with the ILPBP from another crab species, Scylla paramamosain. Transcripts of Cas-ILPBP are found in all examined tissues, with the highest levels in the nervous tissues (eyestalk ganglia, brain and thoracic ganglia complex) and followed by midgut, the pericardial organ, abdominal muscle and the heart. As Cas-ILPBP contains a putative Ig domain, it is hypothesized that this protein may be involved in immunity, particularly in the adult females infected with a reo-like virus (CsRV1). The expression levels of Cas-ILPBP are examined in several tissues (hemocytes, midgut, eyestalk ganglia) from the animals carrying varying levels of CsRV1 at 17 and 23 °C water temperatures. Cas-ILPBP levels in the midgut are most significantly affected by high levels of CsRV1 infection. Reduction in Cas-ILPBP levels in the midguts is noted from the animals infected with high levels of CsRV1 that show reduced or stop feeding activity, indicating that it may play an important role in midgut functions such as digestion and nutrient absorption.

Galectin CvGal2 from the Eastern Oyster (Crassostrea virginica) Displays Unique Specificity for ABH Blood Group Oligosaccharides and Differentially Recognizes Sympatric Perkinsus Species.

Galectins are highly conserved lectins that are key to multiple biological functions, including pathogen recognition and regulation of immune responses. We previously reported that CvGal1, a galectin expressed in phagocytic cells (hemocytes) of the eastern oyster (Crassostrea virginica), is hijacked by the parasite Perkinsus marinus to enter the host, where it causes systemic infection and death. Screening of an oyster hemocyte cDNA library revealed a novel galectin, which we designated CvGal2, with four tandemly arrayed carbohydrate recognition domains (CRDs). Phylogentic analysis of the CvGal2 CRDs suggests close relationships with homologous CRDs from CvGal1. Glycan array analysis, however, revealed that, unlike CvGal1 which preferentially binds to the blood group A tetrasaccharide, CvGal2 recognizes both blood group A and B tetrasaccharides and related structures, suggesting that CvGal2 has broader binding specificity. Furthermore, SPR analysis demonstrated significant differences in the binding kinetics of CvGal1 and CvGal2, and structural modeling revealed substantial differences in their interactions with the oligosaccharide ligands. CvGal2 is homogeneously distributed in the hemocyte cytoplasm, is released to the extracellular space, and binds to the hemocyte surface. CvGal2 binds to P. marinus trophozoites in a dose-dependent and ß-galactoside-specific manner. Strikingly, negligible binding of CvGal2 was observed for Perkinsus chesapeaki, a sympatric parasite species mostly prevalent in the clams Mya arenaria and Macoma balthica. The differential recognition of Perkinsus species by the oyster galectins is consistent with their relative prevalence in oyster and clam species and supports their role in facilitating parasite entry and infectivity in a host-preferential manner.

The alveolate translation initiation factor 4E family reveals a custom toolkit for translational control in core dinoflagellates.

BACKGROUND: Dinoflagellates are eukaryotes with unusual cell biology and appear to rely on translational rather than transcriptional control of gene expression. The eukaryotic translation initiation factor 4E (eIF4E) plays an important role in regulating gene expression because eIF4E binding to the mRNA cap is a control point for translation. eIF4E is part of an extended, eukaryote-specific family with different members having specific functions, based on studies of model organisms. Dinoflagellate eIF4E diversity could provide a mechanism for dinoflagellates to regulate gene expression in a post-transcriptional manner. Accordingly, eIF4E family members from eleven core dinoflagellate transcriptomes were surveyed to determine the diversity and phylogeny of the eIF4E family in dinoflagellates and related lineages including apicomplexans, ciliates and heterokonts. RESULTS: The survey uncovered eight to fifteen (on average eleven) different eIF4E family members in each core dinoflagellate species. The eIF4E family members from heterokonts and dinoflagellates segregated into three clades, suggesting at least three eIF4E cognates were present in their common ancestor. However, these three clades are distinct from the three previously described eIF4E classes, reflecting diverse approaches to a central eukaryotic function. Heterokonts contain four clades, ciliates two and apicomplexans only a single recognizable eIF4E clade. In the core dinoflagellates, the three clades were further divided into nine sub-clades based on the phylogenetic analysis and species representation. Six of the sub-clades included at least one member from all eleven core dinoflagellate species, suggesting duplication in their shared ancestor. Conservation within sub-clades varied, suggesting different selection pressures. CONCLUSIONS: Phylogenetic analysis of eIF4E in core dinoflagellates revealed complex layering of duplication and conservation when compared to other eukaryotes. Our results suggest that the diverse eIF4E family in core dinoflagellates may provide a toolkit to enable selective translation as a strategy for controlling gene expression in these enigmatic eukaryotes.

Ichthyotoxic Karlodinium veneficum (Ballantine) J Larsen in the Upper Swan River Estuary (Western Australia): Ecological conditions leading to a fish kill.

Ichthyotoxic Karlodinium veneficum has become a persistent problem in the eutrophic Swan River Estuary (SRE) near Perth, Western Australia. Karlotoxin (KmTx) concentrations and K. veneficum were sampled from March to July 2005, spanning a bloom confirmed by microscopy and genetics (ITS sequence), and a fish kill coincident with end of the bloom. The objective of this study was to investigate K. veneficum cell and toxin dynamics, and water quality conditions, leading up to the bloom and fish kill in this estuarine system. Abundance of K. veneficum increased as diatom abundance decreased over a 3-month period (Jan-Mar) preceding the bloom. Low freshwater flow to the SRE characterized the bloom initiation period, while elevated seasonal flows altered water quality and preceded the end of the bloom and fish kill. The bloom of K. veneficum was localized over a bottom layer of hypoxic water in a stratified water column. Low nitrate levels, DIN:DIP (mol) near unity, and particulate C:N:P of K. veneficum-rich water samples were consistent with nitrogen limitation of phytoplankton. A KmTx 2 congener was present in the concentration range 0-1052 ng KmTx mL-1, levels that were sufficient to kill larval fish in the laboratory within 4 h. A KmTx cell quota of 2.8 pg KmTx cell-1 was estimated for the bloom, which is moderately high for the species. Gill histopathology of fish from this fish kill showed signs of damage similar to those caused by KmTx in the lab. Results from this study suggest that conditions in the SRE, including elevated K. veneficum abundance and KmTx cell quotas, as well as hypoxia in the upper SRE, likely contribute to seasonal fish kills observed in this system.

Dinoflagellate phylogeny revisited: using ribosomal proteins to resolve deep branching dinoflagellate clades.

The alveolates are composed of three major lineages, the ciliates, dinoflagellates, and apicomplexans. Together these 'protist' taxa play key roles in primary production and ecology, as well as in illness of humans and other animals. The interface between the dinoflagellate and apicomplexan clades has been an area of recent discovery, blurring the distinction between these two clades. Moreover, phylogenetic analysis has yet to determine the position of basal dinoflagellate clades hence the deepest branches of the dinoflagellate tree currently remain unresolved. Large-scale mRNA sequencing was applied to 11 species of dinoflagellates, including strains of the syndinean genera Hematodinium and Amoebophrya, parasites of crustaceans and dinoflagellates, respectively, to optimize and update the dinoflagellate tree. From the transcriptome-scale data a total of 73 ribosomal protein-coding genes were selected for phylogeny. After individual gene orthology assessment, the genes were concatenated into a >15,000 amino acid alignment with 76 taxa from dinoflagellates, apicomplexans, ciliates, and the outgroup heterokonts. Overall the tree was well resolved and supported, when the data was subsampled with gblocks or constraint trees were tested with the approximately unbiased test. The deepest branches of the dinoflagellate tree can now be resolved with strong support, and provides a clearer view of the evolution of the distinctive traits of dinoflagellates.

Metatranscriptome profiling of a harmful algal bloom.

Metagenomic methods provide a powerful means to investigate complex ecological phenomena. Developed originally for study of Bacteria and Archaea, the application of these methods to eukaryotic microorganisms is yet to be fully realized. Most prior environmental molecular studies of eukaryotes have relied heavily on PCR amplification with eukaryote-specific primers. Here we apply high throughput short-read sequencing of poly-A selected RNA to capture the metatranscriptome of an estuarine dinoflagellate bloom. To validate the metatranscriptome assembly process we simulated metatranscriptomic datasets using short-read sequencing data from clonal cultures of four algae of varying phylogenetic distance. We find that the proportion of chimeric transcripts reconstructed from community transcriptome sequencing is low, suggesting that metatranscriptomic sequencing can be used to accurately reconstruct the transcripts expressed by bloom-forming communities of eukaryotes. To further validate the bloom metatransciptome assembly we compared it to a transcriptomic assembly from a cultured, clonal isolate of the dominant bloom-causing alga and found that the two assemblies are highly similar. Eukaryote-wide phylogenetic analyses reveal the taxonomic composition of the bloom community, which is comprised of several dinoflagellates, ciliates, animals, and fungi. The assembled metatranscriptome reveals the functional genomic composition of a metabolically active community. Highlighting the potential power of these methods, we found that relative transcript abundance patterns suggest that the dominant dinoflagellate might be expressing toxin biosynthesis related genes at a higher level in the presence of competitors, predators and prey compared to it growing in monoculture.

A mobile genetic element with unknown function found in distantly related viruses.

BACKGROUND: The genetic element s2m seems to represent one of very few examples of mobile genetic elements in viruses. The function remains obscure and a scattered taxonomical distribution has been reported by numerous groups. METHODS: We have searched GenBank in order to identify all viral accessions that have s2m(-like) sequence motifs. Rigorous phylogenetic analyses and constrained tree topology testing were also performed in order to investigate the apparently mobile nature of s2m. RESULTS: The stem-loop s2m structure can be found in four families of + ssRNA viruses; Astroviridae, Caliciviridae, Picornaviridae and Coronaviridae. In all of these virus families, with the possible exception of Caliciviridae, multiple gains and/or losses of s2m would have to be postulated in order to explain the distribution of this character. CONCLUSIONS: s2m appears to be a mobile genetic element with a unique evolutionary history in all of the four virus families where it can be found. Based on our findings and a review of the current literature on s2m, a hypothesis implying an RNAi-like function for the s2m element is also outlined.

Dynamics of actin evolution in dinoflagellates.

Dinoflagellates have unique nuclei and intriguing genome characteristics with very high DNA content making complete genome sequencing difficult. In dinoflagellates, many genes are found in multicopy gene families, but the processes involved in the establishment and maintenance of these gene families are poorly understood. Understanding the dynamics of gene family evolution in dinoflagellates requires comparisons at different evolutionary scales. Studies of closely related species provide fine-scale information relative to species divergence, whereas comparisons of more distantly related species provides broad context. We selected the actin gene family as a highly expressed conserved gene previously studied in dinoflagellates. Of the 142 sequences determined in this study, 103 were from the two closely related species, Dinophysis acuminata and D. caudata, including full length and partial cDNA sequences as well as partial genomic amplicons. For these two Dinophysis species, at least three types of sequences could be identified. Most copies (79%) were relatively similar and in nucleotide trees, the sequences formed two bushy clades corresponding to the two species. In comparisons within species, only eight to ten nucleotide differences were found between these copies. The two remaining types formed clades containing sequences from both species. One type included the most similar sequences in between-species comparisons with as few as 12 nucleotide differences between species. The second type included the most divergent sequences in comparisons between and within species with up to 93 nucleotide differences between sequences. In all the sequences, most variation occurred in synonymous sites or the 5' UnTranslated Region (UTR), although there was still limited amino acid variation between most sequences. Several potential pseudogenes were found (approximately 10% of all sequences depending on species) with incomplete open reading frames due to frameshifts or early stop codons. Overall, variation in the actin gene family fits best with the "birth and death" model of evolution based on recent duplications, pseudogenes, and incomplete lineage sorting. Divergence between species was similar to variation within species, so that actin may be too conserved to be useful for phylogenetic estimation of closely related species.

Molecular diversity of the syndinean genus Euduboscquella based on single-cell PCR analysis.

The genus Euduboscquella is one of a few described genera within the syndinean dinoflagellates, an enigmatic lineage with abundant diversity in marine environmental clone libraries based on small subunit (SSU) rRNA. The region composed of the SSU through to the partial large subunit (LSU) rRNA was determined from 40 individual tintinnid ciliate loricae infected with Euduboscquella sampled from eight surface water sites in the Northern Hemisphere, producing seven distinct SSU sequences. The corresponding host SSU rRNA region was also amplified from eight host species. The SSU tree of Euduboscquella and syndinean group I sequences from environmental clones had seven well-supported clades and one poorly supported clade across data sets from 57 to 692 total sequences. The genus Euduboscquella consistently formed a supported monophyletic clade within a single subclade of group I sequences. For most parasites with identical SSU sequences, the more variable internal transcribed spacer (ITS) to LSU rRNA regions were polymorphic at 3 to 10 sites. However, in E. cachoni there was variation between ITS to LSU copies at up to 20 sites within an individual, while in a parasite of Tintinnopsis spp., variation between different individuals ranged up to 19 polymorphic sites. However, applying the compensatory base change model to the ITS2 sequences suggested no compensatory changes within or between individuals with the same SSU sequence, while one to four compensatory changes between individuals with similar but not identical SSU sequences were found. Comparisons between host and parasite phylogenies do not suggest a simple pattern of host or parasite specificity.

Revision of the family Duboscquellidae with description of Euduboscquella crenulata n. gen., n. sp. (Dinoflagellata, Syndinea), an intracellular parasite of the ciliate Favella panamensis Kofoid & Campbell.

Recent recognition that tintinnids are infected by dinophycean as well as syndinean parasites prompts taxonomic revision of dinoflagellate species that parasitize these ciliates. Long overlooked features of the type species Duboscquella tintinnicola are used to emend the genus and family Duboscquellidae, resulting in both taxa being moved from the Syndinea to the Dinophyceae. Syndinean species previously classified as Duboscquella are relocated to Euduboscquella n. gen., with Euduboscquella crenulata n. sp. as the type. As an endoparasitic species, E. crenulata shares with its congeners processes associated with intracellular development and sporogenesis, but differs from closely related species in nuclear and cortical morphology of the trophont, including a distinctively grooved shield (= episome) that imparts a crenulated appearance in optical section. In addition, E. crenulata produces three morphologically distinct spore types, two of which undergo syngamy to form a uninucleate zygote. The zygote undergoes successive division to produce four daughter cells of unequal size, but that resemble the nonmating spore type.

Diversity of Eukaryotic Translational Initiation Factor eIF4E in Protists.

The greatest diversity of eukaryotic species is within the microbial eukaryotes, the protists, with plants and fungi/metazoa representing just two of the estimated seventy five lineages of eukaryotes. Protists are a diverse group characterized by unusual genome features and a wide range of genome sizes from 8.2 Mb in the apicomplexan parasite Babesia bovis to 112,000-220,050 Mb in the dinoflagellate Prorocentrum micans. Protists possess numerous cellular, molecular and biochemical traits not observed in "text-book" model organisms. These features challenge some of the concepts and assumptions about the regulation of gene expression in eukaryotes. Like multicellular eukaryotes, many protists encode multiple eIF4Es, but few functional studies have been undertaken except in parasitic species. An earlier phylogenetic analysis of protist eIF4Es indicated that they cannot be grouped within the three classes that describe eIF4E family members from multicellular organisms. Many more protist sequences are now available from which three clades can be recognized that are distinct from the plant/fungi/metazoan classes. Understanding of the protist eIF4Es will be facilitated as more sequences become available particularly for the under-represented opisthokonts and amoebozoa. Similarly, a better understanding of eIF4Es within each clade will develop as more functional studies of protist eIF4Es are completed.

Alveolate phylogeny inferred using concatenated ribosomal proteins.

Dinoflagellates and apicomplexans are a strongly supported monophyletic group in rDNA phylogenies, although this phylogeny is not without controversy, particularly between the two groups. Here we use concatenated protein-coding genes from expressed sequence tags or genomic data to construct phylogenies including "typical" dinophycean dinoflagellates, a parasitic syndinian dinoflagellate, Amoebophrya sp., and two related species, Oxyrrhis marina, and Perkinsus marinus. Seventeen genes encoding proteins associated with the ribosome were selected for phylogenetic analysis. The dataset was limited for the most part by data availability from the dinoflagellates. Forty-five taxa from four major lineages were used: the heterokont outgroup, ciliates, dinoflagellates, and apicomplexans. Amoebophrya sp. was included in this phylogeny as a sole representative of the enigmatic marine alveolate or syndinian lineage. The atypical dinoflagellate O. marina, usually excluded from rDNA analyses due to long branches, was also included. The resulting phylogenies were well supported in concatenated analyses with only a few unstable or weakly supported branches; most features were consistent when different lineages were pruned from the tree or different genes were concatenated. The least stable branches involved the placement of Cryptosporidium spp. within the Apicomplexa and the relationships between P. marinus, Amoebophrya sp., and O. marina. Both bootstrap and approximately unbiased test results confirmed that P. marinus, Amoebophrya sp., O. marina, and the remaining dinoflagellates form a monophyletic lineage to the exclusion of Apicomplexa.

A Global Approach to Estimating the Abundance and Duplication of Polyketide Synthase Domains in Dinoflagellates.

Many dinoflagellate species make toxins in a myriad of different molecular configurations but the underlying chemistry in all cases is presumably via modular synthases, primarily polyketide synthases. In many organisms modular synthases occur as discrete synthetic genes or domains within a gene that act in coordination thus forming a module that produces a particular fragment of a natural product. The modules usually occur in tandem as gene clusters with a syntenic arrangement that is often predictive of the resultant structure. Dinoflagellate genomes however are notoriously complex with individual genes present in many tandem repeats and very few synthetic modules occurring as gene clusters, unlike what has been seen in bacteria and fungi. However, modular synthesis in all organisms requires a free thiol group that acts as a carrier for sequential synthesis called a thiolation domain. We scanned 47 dinoflagellate transcriptomes for 23 modular synthase domain models and compared their abundance among 10 orders of dinoflagellates as well as their co-occurrence with thiolation domains. The total count of domain types was quite large with over thirty-thousand identified, 29 000 of which were in the core dinoflagellates. Although there were no specific trends in domain abundance associated with types of toxins, there were readily observable lineage specific differences. The Gymnodiniales, makers of long polyketide toxins such as brevetoxin and karlotoxin had a high relative abundance of thiolation domains as well as multiple thiolation domains within a single transcript. Orders such as the Gonyaulacales, makers of small polyketides such as spirolides, had fewer thiolation domains but a relative increase in the number of acyl transferases. Unique to the core dinoflagellates, however, were thiolation domains occurring alongside tetratricopeptide repeats that facilitate protein-protein interactions, especially hexa and hepta-repeats, that may explain the scaffolding required for synthetic complexes capable of making large toxins. Clustering analysis for each type of domain was also used to discern possible origins of duplication for the multitude of single domain transcripts. Single domain transcripts frequently clustered with synonymous domains from multi-domain transcripts such as the BurA and ZmaK like genes as well as the multi-ketosynthase genes, sometimes with a large degree of apparent gene duplication, while fatty acid synthesis genes formed distinct clusters. Surprisingly the acyl-transferases and ketoreductases involved in fatty acid synthesis (FabD and FabG, respectively) were found in very large clusters indicating an unprecedented degree of gene duplication for these genes. These results demonstrate a complex evolutionary history of core dinoflagellate modular synthases with domain specific duplications throughout the lineage as well as clues to how large protein complexes can be assembled to synthesize the largest natural products known.

Chromosome-level genome assembly of the blue crab, Callinectes sapidus.

The blue crab, Callinectes sapidus (Rathbun, 1896) is an economically, culturally, and ecologically important species found across the temperate and tropical North and South American Atlantic coast. A reference genome will enable research for this high-value species. Initial assembly combined 200× coverage Illumina paired-end reads, a 60× 8 kb mate-paired library, and 50× PacBio data using the MaSuRCA assembler resulting in a 985 Mb assembly with a scaffold N50 of 153 kb. Dovetail Chicago and HiC sequencing with the 3d DNA assembler and Juicebox assembly tools were then used for chromosome scaffolding. The 50 largest scaffolds span 810 Mb are 1.5-37 Mb long and have a repeat content of 36%. The 190 Mb unplaced sequence is in 3921 sequences over 10 kb with a repeat content of 68%. The final assembly N50 is 18.9 Mb for scaffolds and 9317 bases for contigs. Of arthropod BUSCO, ∼88% (888/1013) were complete and single copies. Using 309 million RNAseq read pairs from 12 different tissues and developmental stages, 25,249 protein-coding genes were predicted. Between C. sapidus and Portunus trituberculatus genomes, 41 of 50 large scaffolds had high nucleotide identity and protein-coding synteny, but 9 scaffolds in both assemblies were not clear matches. The protein-coding genes included 9423 one-to-one putative orthologs, of which 7165 were syntenic between the two crab species. Overall, the two crab genome assemblies show strong similarities at the nucleotide, protein, and chromosome level and verify the blue crab genome as an excellent reference for this important seafood species.

Tintinnophagus acutus n. g., n. sp. (Phylum Dinoflagellata), an ectoparasite of the ciliate Tintinnopsis cylindrica Daday 1887, and its relationship to Duboscquodinium collini Grassé 1952.

The dinoflagellate Tintinnophagus acutus n. g., n. sp., an ectoparasite of the ciliate Tintinnopsis cylindrica Daday, superficially resembles Duboscquodinium collini Grassé, a parasite of Eutintinnus fraknoii Daday. Dinospores of T. acutus are small transparent cells having a sharply pointed episome, conspicuous eyespot, posteriorly positioned nucleus with condensed chromosomes, and rigid form that may be supported by delicate thecal plates. Dinospores attach to the host via a feeding tube, losing their flagella, sulcus, and girdle to become spherical or ovoid cells. The trophont of T. acutus feeds on the host for several days, increasing dramatically in size before undergoing sporogenesis. Successive generations of daughter sporocytes are encompassed in an outer membrane or cyst wall, a feature not evident in trophonts. Tintinnophagus acutus differs from D. collini in host species, absence of a second membrane surrounding pre-sporogenic stages, and failure to differentiate into a gonocyte and a trophocyte at the first sporogenic division. Phylogenetic analyses based on small subunit (SSU) ribosomal DNA (rDNA) sequences placed T. acutus and D. collini in the class Dinophyceae, with T. acutus aligned loosely with Pfiesteria piscicida and related species, including Amyloodinium ocellatum, a parasite of fish, and Paulsenella vonstoschii, a parasite of diatoms. Dubosquodinium collini nested in a clade composed of several Scrippsiella species and Peridinium polonicum. Tree construction using longer rDNA sequences (i.e. SSU through partial large subunit) strengthened the placement of T. acutus and D. collini within the Dinophyceae.