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Dinoflagellate Biology: Using Molecular Approaches to Unlock Their Ecology and Evolution

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A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Environmental Microbiology".

Deadline for manuscript submissions: closed (30 April 2022) | Viewed by 23065

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Special Issue Editor

Prof. Dr. Shauna Murray

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Guest Editor

Climate Change Cluster, University of Technology Sydney, Ultimo, NSW 2007, Australia
Interests: harmful algal blooms; qPCR; phylogenetics; evolution; molecular ecology; benthic dinoflagellates; systematics
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Dinoflagellates are an important group of aquatic microbial eukaryotes, showing great diversity in life histories, ecological niches, and morphology and pigment composition. They include species with photosynthetic, heterotrophic, symbiotic, mixotrophic and parasitic lifestyles, and encompass coral symbionts, harmful algal bloom forming species, and important fish parasites. They have a presence in fossil records that date back several hundred million years. Dinoflagellates include the majority of species that produce marine biotoxins, impacting aquaculture. In recent years, molecular approaches have been applied to understand dinoflagellate biology, including techniques for studying dinoflagellate ecology, physiology, basic genetics and evolution. This special issue is dedicated to the application and development of molecular approaches for enhancing our understanding of dinoflagellate biology.

Prof. Dr. Shauna Murray
Guest Editor

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Keywords

harmful algal bloom Symbiodinium
Alexandrium
protist
amplicon sequencing
qPCR
genomics
phylogenetics
molecular ecology
transcriptomics
gene expression
molecular barcoding

Published Papers (10 papers)

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20 pages, 40809 KiB

Open AccessArticle

Improved Cladocopium goreaui Genome Assembly Reveals Features of a Facultative Coral Symbiont and the Complex Evolutionary History of Dinoflagellate Genes

by Yibi Chen, Sarah Shah, Katherine E. Dougan, Madeleine J. H. van Oppen, Debashish Bhattacharya and Cheong Xin Chan

Microorganisms 2022, 10(8), 1662; https://doi.org/10.3390/microorganisms10081662 - 17 Aug 2022

Cited by 9 | Viewed by 2930

Abstract

Dinoflagellates of the family Symbiodiniaceae are crucial photosymbionts in corals and other marine organisms. Of these, Cladocopium goreaui is one of the most dominant symbiont species in the Indo-Pacific. Here, we present an improved genome assembly of C. goreaui combining new long-read sequence data with previously generated short-read data. Incorporating new full-length transcripts to guide gene prediction, the C. goreaui genome (1.2 Gb) exhibits a high extent of completeness (82.4% based on BUSCO protein recovery) and better resolution of repetitive sequence regions; 45,322 gene models were predicted, and 327 putative, topologically associated domains of the chromosomes were identified. Comparison with other Symbiodiniaceae genomes revealed a prevalence of repeats and duplicated genes in C. goreaui, and lineage-specific genes indicating functional innovation. Incorporating 2,841,408 protein sequences from 96 taxonomically diverse eukaryotes and representative prokaryotes in a phylogenomic approach, we assessed the evolutionary history of C. goreaui genes. Of the 5246 phylogenetic trees inferred from homologous protein sets containing two or more phyla, 35–36% have putatively originated via horizontal gene transfer (HGT), predominantly (19–23%) via an ancestral Archaeplastida lineage implicated in the endosymbiotic origin of plastids: 10–11% are of green algal origin, including genes encoding photosynthetic functions. Our results demonstrate the utility of long-read sequence data in resolving structural features of a dinoflagellate genome, and highlight how genetic transfer has shaped genome evolution of a facultative symbiont, and more broadly of dinoflagellates. Full article

(This article belongs to the Special Issue Dinoflagellate Biology: Using Molecular Approaches to Unlock Their Ecology and Evolution

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14 pages, 2046 KiB

Open AccessArticle

Metagenomic Analysis of the Species Composition and Seasonal Distribution of Marine Dinoflagellate Communities in Four Korean Coastal Regions

by Jinik Hwang, Hee Woong Kang, Seung Joo Moon, Jun-Ho Hyung, Eun Sun Lee and Jaeyeon Park

Microorganisms 2022, 10(7), 1459; https://doi.org/10.3390/microorganisms10071459 - 19 Jul 2022

Cited by 1 | Viewed by 1682

Abstract

Biomonitoring of dinoflagellate communities in marine ecosystems is essential for efficient water quality management and limiting ecosystem disturbances. Current identification and monitoring of toxic dinoflagellates, which cause harmful algal blooms, primarily involves light or scanning electron microscopy; however, these techniques are limited in their ability to monitor dinoflagellates and plankton, leaving an incomplete analysis. In this study, we analyzed the species composition and seasonal distribution of the dinoflagellate communities in four Korean coastal regions using 18S rRNA amplicon sequencing. The results showed significantly high diversity in the dinoflagellate communities in all regions and seasons. Furthermore, we found seasonally dominant species and causative species of harmful algal blooms (Cochlodinium sp., Alexandrium sp., Dinophysis sp., and Gymnodinium sp.). Moreover, dominant species were classified by region and season according to the difference in geographical and environmental parameters. The molecular analysis of the dinoflagellate community based on metagenomics revealed more diverse species compositions that could not be identified by microscopy and revealed potentially harmful or recently introduced dinoflagellate species. In conclusion, metagenomic analysis of dinoflagellate communities was more precise and obtained results faster than microscopic analysis, and could improve the existing monitoring techniques for community analysis. Full article

(This article belongs to the Special Issue Dinoflagellate Biology: Using Molecular Approaches to Unlock Their Ecology and Evolution

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13 pages, 1696 KiB

Open AccessArticle

A Strategy for Gene Knockdown in Dinoflagellates

by Miranda Judd and Allen R. Place

Microorganisms 2022, 10(6), 1131; https://doi.org/10.3390/microorganisms10061131 - 31 May 2022

Cited by 1 | Viewed by 1954

Abstract

Dinoflagellates are unicellular protists that display unusual nuclear features such as large genomes, condensed chromosomes and multiple gene copies organized as tandem gene arrays. Genetic regulation is believed to be controlled at the translational rather than transcriptional level. An important player in this process is initiation factor eIF4E which binds the 7-methylguanosine cap structure (m7G) at the 5′-end of mRNA. Transcriptome analysis of eleven dinoflagellate species has established that each species encodes between eight to fifteen eIF4E family members. Determining the role of eIF4E family members in gene expression requires a method of knocking down their expression. In other eukaryotes this can be accomplished using translational blocking morpholinos that bind to complementary strands of RNA, therefore inhibiting the mRNA processing. Previously, unmodified morpholinos lacked the ability to pass through cell membranes, however peptide-based reagents have been used to deliver substances into the cytosol of cells by an endocytosis-mediated process without damaging the cell membrane. We have successfully delivered fluorescently-tagged morpholinos to the cytosol of Amphidinium carterae by using a specific cell penetrating peptide with the goal to target an eIF4e-1a sequence to inhibit translation. Specific eIF4e knockdown success (up to 42%) has been characterized via microscopy and western blot analysis. Full article

(This article belongs to the Special Issue Dinoflagellate Biology: Using Molecular Approaches to Unlock Their Ecology and Evolution

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16 pages, 4232 KiB

Open AccessArticle

Dinoflagellate Phosphopantetheinyl Transferase (PPTase) and Thiolation Domain Interactions Characterized Using a Modified Indigoidine Synthesizing Reporter

by Ernest Williams, Tsvetan Bachvaroff and Allen Place

Microorganisms 2022, 10(4), 687; https://doi.org/10.3390/microorganisms10040687 - 23 Mar 2022

Cited by 1 | Viewed by 1899

Abstract

Photosynthetic dinoflagellates synthesize many toxic but also potential therapeutic compounds therapeutics via polyketide/non-ribosomal peptide synthesis, a common means of producing natural products in bacteria and fungi. Although canonical genes are identifiable in dinoflagellate transcriptomes, the biosynthetic pathways are obfuscated by high copy numbers and fractured synteny. This study focuses on the carrier domains that scaffold natural product synthesis (thiolation domains) and the phosphopantetheinyl transferases (PPTases) that thiolate these carriers. We replaced the thiolation domain of the indigoidine producing BpsA gene from Streptomyces lavendulae with those of three multidomain dinoflagellate transcripts and coexpressed these constructs with each of three dinoflagellate PPTases looking for specific pairings that would identify distinct pathways. Surprisingly, all three PPTases were able to activate all the thiolation domains from one transcript, although with differing levels of indigoidine produced, demonstrating an unusual lack of specificity. Unfortunately, constructs with the remaining thiolation domains produced almost no indigoidine and the thiolation domain for lipid synthesis could not be expressed in E. coli. These results combined with inconsistent protein expression for different PPTase/thiolation domain pairings present technical hurdles for future work. Despite these challenges, expression of catalytically active dinoflagellate proteins in E. coli is a novel and useful tool going forward. Full article

(This article belongs to the Special Issue Dinoflagellate Biology: Using Molecular Approaches to Unlock Their Ecology and Evolution

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7 pages, 1393 KiB

Open AccessCommunication

Transcriptome Analysis of Durusdinium Associated with the Transition from Free-Living to Symbiotic

by Ikuko Yuyama, Naoto Ugawa and Tetsuo Hashimoto

Microorganisms 2021, 9(8), 1560; https://doi.org/10.3390/microorganisms9081560 - 22 Jul 2021

Cited by 3 | Viewed by 1945

Abstract

To detect the change during coral–dinoflagellate endosymbiosis establishment, we compared transcriptome data derived from free-living and symbiotic Durusdinium, a coral symbiont genus. We detected differentially expressed genes (DEGs) using two statistical methods (edgeR using raw read data and the Student’s t-test using bootstrap resampling read data) and detected 1214 DEGs between the symbiotic and free-living states, which we subjected to gene ontology (GO) analysis. Based on the representative GO terms and 50 DEGs with low false discovery rates, changes in Durusdinium during endosymbiosis were predicted. The expression of genes related to heat-shock proteins and microtubule-related proteins tended to decrease, and those of photosynthesis genes tended to increase. In addition, a phylogenetic analysis of dapdiamide A (antibiotics) synthase, which was upregulated among the 50 DEGs, confirmed that two genera in the Symbiodiniaceae family, Durusdinium and Symbiodinium, retain dapdiamide A synthase. This antibiotic synthase-related gene may contribute to the high stress tolerance documented in Durusdinium species, and its increased expression during endosymbiosis suggests increased antibacterial activity within the symbiotic complex. Full article

(This article belongs to the Special Issue Dinoflagellate Biology: Using Molecular Approaches to Unlock Their Ecology and Evolution

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15 pages, 2076 KiB

Open AccessArticle

sxtA4+ and sxtA4- Genotypes Occur Together within Natural Pyrodinium bahamense Sub-Populations from the Western Atlantic

by Kathleen Cusick and Gabriel Duran

Microorganisms 2021, 9(6), 1128; https://doi.org/10.3390/microorganisms9061128 - 23 May 2021

Cited by 2 | Viewed by 2651

Abstract

Saxitoxin (STX) is a secondary metabolite and potent neurotoxin produced by several genera of harmful algal bloom (HAB) marine dinoflagellates. The basis for variability in STX production within natural bloom populations is undefined as both toxic and non-toxic strains (of the same species) have been isolated from the same geographic locations. Pyrodinium bahamense is a STX-producing bioluminescent dinoflagellate that blooms along the east coast of Florida as well as the bioluminescent bays in Puerto Rico (PR), though no toxicity reports exist for PR populations. The core genes in the dinoflagellate STX biosynthetic pathway have been identified, and the sxtA4 gene is essential for toxin production. Using sxtA4 as a molecular proxy for the genetic capacity of STX production, we examined sxtA4+ and sxtA4- genotype frequency at the single cell level in P. bahamense populations from different locations in the Indian River Lagoon (IRL), FL, and Mosquito Bay (MB), a bioluminescent bay in PR. Multiplex PCR was performed on individual cells with Pyrodinium-specific primers targeting the 18S rRNA gene and sxtA4. The results reveal that within discrete natural populations of P. bahamense, both sxtA4+ and sxtA4- genotypes occur, and the sxtA4+ genotype dominates. In the IRL, the frequency of the sxtA4+ genotype ranged from ca. 80–100%. In MB, sxtA4+ genotype frequency ranged from ca 40–66%. To assess the extent of sxtA4 variation within individual cells, sxtA4 amplicons from single cells representative of the different sampling sites were cloned and sequenced. Overall, two variants were consistently obtained, one of which is likely a pseudogene based on alignment with cDNA sequences. These are the first data demonstrating the existence of both genotypes in natural P. bahamense sub-populations, as well as sxtA4 presence in P. bahamense from PR. These results provide insights on underlying genetic factors influencing the potential for toxin variability among natural sub-populations of HAB species and highlight the need to study the genetic diversity within HAB sub-populations at a fine level in order to identify the molecular mechanisms driving HAB evolution. Full article

(This article belongs to the Special Issue Dinoflagellate Biology: Using Molecular Approaches to Unlock Their Ecology and Evolution

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14 pages, 1844 KiB

Open AccessArticle

Screening a Spliced Leader-Based Symbiodinium microadriaticum cDNA Library Using the Yeast-Two Hybrid System Reveals a Hemerythrin-Like Protein as a Putative SmicRACK1 Ligand

by Tania Islas-Flores, Edgardo Galán-Vásquez and Marco A. Villanueva

Microorganisms 2021, 9(4), 791; https://doi.org/10.3390/microorganisms9040791 - 09 Apr 2021

Cited by 6 | Viewed by 1938

Abstract

The dinoflagellate Symbiodiniaceae family plays a central role in the health of the coral reef ecosystem via the symbiosis that establishes with its inhabiting cnidarians and supports the host metabolism. In the last few decades, coral reefs have been threatened by pollution and rising temperatures which have led to coral loss. These events have raised interest in studying Symbiodiniaceae and their hosts; however, progress in understanding their metabolism, signal transduction pathways, and physiology in general, has been slow because dinoflagellates present peculiar characteristics. We took advantage of one of these peculiarities; namely, the post-transcriptional addition of a Dino Spliced Leader (Dino-SL) to the 5′ end of the nuclear mRNAs, and used it to generate cDNA libraries from Symbiodinium microadriaticum. We compared sequences from two Yeast-Two Hybrid System cDNA Libraries, one based on the Dino-SL sequence, and the other based on the SMART technology (Switching Mechanism at 5′ end of RNA Transcript) which exploits the template switching function of the reverse transcriptase. Upon comparison of the performance of both libraries, we obtained a significantly higher yield, number and length of sequences, number of transcripts, and better 5′ representation from the Dino-SL based library than from the SMART library. In addition, we confirmed that the cDNAs from the Dino-SL library were adequately expressed in the yeast cells used for the Yeast-Two Hybrid System which resulted in successful screening for putative SmicRACK1 ligands, which yielded a putative hemerythrin-like protein. Full article

(This article belongs to the Special Issue Dinoflagellate Biology: Using Molecular Approaches to Unlock Their Ecology and Evolution

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20 pages, 2932 KiB

Open AccessArticle

Assessing the Use of Molecular Barcoding and qPCR for Investigating the Ecology of Prorocentrum minimum (Dinophyceae), a Harmful Algal Species

by Kate McLennan, Rendy Ruvindy, Martin Ostrowski and Shauna Murray

Microorganisms 2021, 9(3), 510; https://doi.org/10.3390/microorganisms9030510 - 28 Feb 2021

Cited by 7 | Viewed by 2622 | Correction

Abstract

Prorocentrum minimum is a species of marine dinoflagellate that occurs worldwide and can be responsible for harmful algal blooms (HABs). Some studies have reported it to produce tetrodotoxin; however, results have been inconsistent. qPCR and molecular barcoding (amplicon sequencing) using high-throughput sequencing have been increasingly applied to quantify HAB species for ecological analyses and monitoring. Here, we isolated a strain of P. minimum from eastern Australian waters, where it commonly occurs, and developed and validated a qPCR assay for this species based on a region of ITS rRNA in relation to abundance estimates from the cultured strain as determined using light microscopy. We used this tool to quantify and examine ecological drivers of P. minimum in Botany Bay, an estuary in southeast Australia, for over ~14 months in 2016–2017. We compared abundance estimates using qPCR with those obtained using molecular barcoding based on an 18S rRNA amplicon. There was a significant correlation between the abundance estimates from amplicon sequencing and qPCR, but the estimates from light microscopy were not significantly correlated, likely due to the counting method applied. Using amplicon sequencing, ~600 unique actual sequence variants (ASVs) were found, much larger than the known phytoplankton diversity from this region. P. minimum abundance in Botany Bay was found to be significantly associated with lower salinities and higher dissolved CO₂ levels. Full article

(This article belongs to the Special Issue Dinoflagellate Biology: Using Molecular Approaches to Unlock Their Ecology and Evolution

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13 pages, 3131 KiB

Open AccessCommunication

Response of Coral Reef Dinoflagellates to Nanoplastics under Experimental Conditions Suggests Downregulation of Cellular Metabolism

by Christina Ripken, Konstantin Khalturin and Eiichi Shoguchi

Microorganisms 2020, 8(11), 1759; https://doi.org/10.3390/microorganisms8111759 - 09 Nov 2020

Cited by 23 | Viewed by 3438

Abstract

Plastic products contribute heavily to anthropogenic pollution of the oceans. Small plastic particles in the microscale and nanoscale ranges have been found in all marine ecosystems, but little is known about their effects upon marine organisms. In this study, we examine changes in cell growth, aggregation, and gene expression of two symbiotic dinoflagellates of the family Symbiodiniaceae, Symbiodinium tridacnidorum (clade A3), and Cladocopium sp. (clade C) under exposure to 42-nm polystyrene beads. In laboratory experiments, the cell number and aggregation were reduced after 10 days of nanoplastic exposure at 0.01, 0.1, and 10 mg/L concentrations, but no clear correlation with plastic concentration was observed. Genes involved in dynein motor function were upregulated when compared to control conditions, while genes related to photosynthesis, mitosis, and intracellular degradation were downregulated. Overall, nanoplastic exposure led to more genes being downregulated than upregulated and the number of genes with altered expression was larger in Cladocopium sp. than in S. tridacnidorum, suggesting different sensitivity to nano-plastics between species. Our data show that nano-plastic inhibits growth and alters aggregation properties of microalgae, which may negatively affect the uptake of these indispensable symbionts by coral reef organisms. Full article

(This article belongs to the Special Issue Dinoflagellate Biology: Using Molecular Approaches to Unlock Their Ecology and Evolution

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