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Culture-Independent Study of the Late-Stage of a Bloom of the Toxic Dinoflagellate Ostreopsis cf. ovata: Preliminary Findings Suggest Genetic Differences at the Sub-Species Level and Allow ITS2 Structure Characterization.

Toxins (Basel). 2015;7(7):2514-2533

Authors: Ramos V, Salvi D, Machado JP, Vale M, Azevedo J, Vasconcelos V

Abstract
Available genomic data for the toxic, bloom-forming, benthic Ostreopsis spp. are traditionally obtained from isolates rather than from individuals originally present in environmental samples. Samples from the final phase of the first reported Ostreopsis bloom in European North Atlantic waters (Algarve, south coast of Portugal) were studied and characterized, using a culture-independent approach. In the first instance, a microscopy-based analysis revealed the intricate complexity of the samples. Then, we evaluated the adequacy of commonly used molecular tools (i.e., primers and nuclear ribosomal markers) for the study of Ostreopsis diversity in natural samples. A PCR-based methodology previously developed to identify/detect common Ostreopsis species was tested, including one new combination of existing PCR primers. Two sets of environmental rRNA sequences were obtained, one of them (1052 bp) with the newly tested primer set. These latter sequences encompass both the ITS1-5.8S-ITS2 region and the D1/D2 domain of the LSU rRNA gene, leading us to an accurate identification of ITS2. In turn, this allowed us to predict and show for the first time the ITS2 secondary structure of Ostreopsis. With 92 bp in length and a two-helix structure, the ITS2 of this genus revealed to be unique among the dinoflagellates. Both the PCR approach as the phylogenetic analyses allowed to place the Ostreopsis cells observed in the samples within the O. cf. ovata phylospecies’ complex, discarding the presence of O. cf. siamensis. The (phylo)genetic results point out a certain level of nucleotide sequence divergence, but were inconclusive in relation to a possible geographic origin of the O. cf. ovata population from the Algarve’s bloom.

PMID: 26134259 [PubMed – as supplied by publisher]

Related Articles

Paralytic shellfish toxin content is related to genomic sxtA4 copy number in Alexandrium minutum strains.

Front Microbiol. 2015;6:404

Authors: Stüken A, Riobó P, Franco J, Jakobsen KS, Guillou L, Figueroa RI

Abstract
Dinoflagellates are microscopic aquatic eukaryotes with huge genomes and an unusual cell regulation. For example, most genes are present in numerous copies and all copies seem to be obligatorily transcribed. The consequence of the gene copy number (CPN) for final protein synthesis is, however, not clear. One such gene is sxtA, the starting gene of paralytic shellfish toxin (PST) synthesis. PSTs are small neurotoxic compounds that can accumulate in the food chain and cause serious poisoning incidences when ingested. They are produced by dinoflagellates of the genera Alexandrium, Gymnodium, and Pyrodinium. Here we investigated if the genomic CPN of sxtA4 is related to PST content in Alexandrium minutum cells. SxtA4 is the 4th domain of the sxtA gene and its presence is essential for PST synthesis in dinoflagellates. We used PST and genome size measurements as well as quantitative PCR to analyze sxtA4 CPN and toxin content in 15 A. minutum strains. Our results show a strong positive correlation between the sxtA4 CPN and the total amount of PST produced in actively growing A. minutum cells. This correlation was independent of the toxin profile produced, as long as the strain contained the genomic domains sxtA1 and sxtA4.

PMID: 25983733 [PubMed]

Dinoflagellate Gene Structure and Intron Splice Sites in a Genomic Tandem Array.

J Eukaryot Microbiol. 2015 May 12;

Authors: Mendez GS, Delwiche CF, Apt KE, Lippmeier JC

Abstract
Dinoflagellates are one of the last major lineages of eukaryotes for which little is known about genome structure and organization. We report here the sequence and gene structure of a clone isolated from a cosmid library which, to our knowledge, represents the largest contiguously sequenced dinoflagellate genomic tandem gene array. These data, combined with information from a large transcriptomic library, allowed a high level of confidence of every base pair call. This degree of confidence is not possible with PCR-based contigs. The sequence contains an intron-rich set of five highly-expressed gene repeats arranged in tandem. One of the tandem repeat gene members contains an intron 26,372 bp long. This study characterizes a splice-site consensus sequence for dinoflagellate introns. Two to nine base pairs around the 3′ splice site are repeated by an identical two to nine base pairs around the 5′ splice site. The 5′ and 3′ splice sites are in the same locations within each repeat so that the repeat is found only once in the mature mRNA. This identically repeated intron boundary (IRIB) sequence might be useful in gene modeling and annotation of genomes. This article is protected by copyright. All rights reserved.

PMID: 25963315 [PubMed – as supplied by publisher]

Endosymbiosis undone by stepwise elimination of the plastid in a parasitic dinoflagellate.

Proc Natl Acad Sci U S A. 2015 Apr 20;

Authors: Gornik SG, Febrimarsa, Cassin AM, MacRae JI, Ramaprasad A, Rchiad Z, McConville MJ, Bacic A, McFadden GI, Pain A, Waller RF

Abstract
Organelle gain through endosymbiosis has been integral to the origin and diversification of eukaryotes, and, once gained, plastids and mitochondria seem seldom lost. Indeed, discovery of nonphotosynthetic plastids in many eukaryotes-notably, the apicoplast in apicomplexan parasites such as the malaria pathogen Plasmodium-highlights the essential metabolic functions performed by plastids beyond photosynthesis. Once a cell becomes reliant on these ancillary functions, organelle dependence is apparently difficult to overcome. Previous examples of endosymbiotic organelle loss (either mitochondria or plastids), which have been invoked to explain the origin of eukaryotic diversity, have subsequently been recognized as organelle reduction to cryptic forms, such as mitosomes and apicoplasts. Integration of these ancient symbionts with their hosts has been too well developed to reverse. Here, we provide evidence that the dinoflagellate Hematodinium sp., a marine parasite of crustaceans, represents a rare case of endosymbiotic organelle loss by the elimination of the plastid. Extensive RNA and genomic sequencing data provide no evidence for a plastid organelle, but, rather, reveal a metabolic decoupling from known plastid functions that typically impede organelle loss. This independence has been achieved through retention of ancestral anabolic pathways, enzyme relocation from the plastid to the cytosol, and metabolic scavenging from the parasite’s host. Hematodinium sp. thus represents a further dimension of endosymbiosis-life after the organelle.

PMID: 25902514 [PubMed – as supplied by publisher]

Intra-genomic variation in symbiotic dinoflagellates: recent divergence or recombination between lineages?

BMC Evol Biol. 2015;15(1):46

Authors: Wilkinson SP, Fisher PL, van Oppen MJ, Davy SK

Abstract
BACKGROUND: The symbiosis between corals and the dinoflagellate alga Symbiodinium is essential for the development and survival of coral reefs. Yet this fragile association is highly vulnerable to environmental disturbance. A coral’s ability to tolerate temperature stress depends on the fitness of its resident symbionts, whose thermal optima vary extensively between lineages. However, the in hospite population genetic structure of Symbiodinium is poorly understood and mostly based on analysis of bulk DNA extracted from thousands to millions of cells. Using quantitative single-cell PCR, we enumerated DNA polymorphisms in the symbionts of the reef-building coral Pocillopora damicornis, and applied a model selection approach to explore the potential for recombination between coexisting Symbiodinium populations.
RESULTS: Two distinct Symbiodinium ITS2 sequences (denoted C100 and C109) were retrieved from all P. damicornis colonies analysed. However, the symbiont assemblage consisted of three distinct Symbiodinium populations: cells featuring pure arrays of ITS2 type C109, near-homogeneous cells of type C100 (with trace ITS2 copies of type C109), and those with co-dominant C100 and C109 ITS2 repeats. The symbiont consortia of some colonies consisted almost entirely of these putative C100 × C109 recombinants.
CONCLUSIONS: Our results are consistent with the occurrence of sexual recombination between Symbiodinium types C100 and C109. While the multiple-copy nature of the ITS2 dictates that the observed pattern of intra-genomic co-dominance may be a result of incomplete concerted evolution of intra-genomic polymorphisms, this is a less likely explanation given the occurrence of homogeneous cells of the C109 type. Conclusive evidence for inter-lineage recombination and introgression in this genus will require either direct observational evidence or a single-cell genotyping approach targeting multiple, single-copy loci.

PMID: 25887753 [PubMed – as supplied by publisher]