Developing model systems for dinoflagellates in the post‐genomic era

Dinoflagellates are a diverse group of eukaryotic microbes that are ubiquitous in aquatic environments. Largely photosynthetic, they encompass symbiotic, parasitic, and free-living lineages with a broad spectrum of trophism. Many free-living taxa can produce bioactive secondary metabolites such as biotox- ins, some of which cause harmful algal blooms. In contrast, most symbiotic species are crucial for sustaining coral reef health. The year 2023 marked a decade since the first genome data of dinoflagellates became available. The growing genome-scale resources for these taxa are highlighting their remarkable evolutionary and genomic complexities. Here, we discuss the prospect of developing dinoflagellate models using the criteria of accessibil- ity, tractability, resources, research support, and promise. Moving forward in the post-genomic era, we argue for the development of fit-to-purpose models that tailor to specific biological contexts, and that a one-size-fits-all model is inadequate for encapsulating the complex biology, ecology, and evolutionary history of dinoflagellates

The taxonomic significance of species that have only been observed once : the genus Gymnodinium (Dinoflagellata) as an example

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS ONE 7 (2012): e44015, doi:10.1371/journal.pone.0044015.Taxonomists have been tasked with cataloguing and quantifying the Earth’s biodiversity. Their progress is measured in code-compliant species descriptions that include text, images, type material and molecular sequences. It is from this material that other researchers are to identify individuals of the same species in future observations. It has been estimated that 13% to 22% (depending on taxonomic group) of described species have only ever been observed once. Species that have only been observed at the time and place of their original description are referred to as oncers. Oncers are important to our current understanding of biodiversity. They may be validly described species that are members of a rare biosphere, or they may indicate endemism, or that these species are limited to very constrained niches. Alternatively, they may reflect that taxonomic practices are too poor to allow the organism to be re-identified or that the descriptions are unknown to other researchers. If the latter are true, our current tally of species will not be an accurate indication of what we know. In order to investigate this phenomenon and its potential causes, we examined the microbial eukaryote genus Gymnodinium. This genus contains 268 extant species, 103 (38%) of which have not been observed since their original description. We report traits of the original descriptions and interpret them in respect to the status of the species. We conclude that the majority of oncers were poorly described and their identity is ambiguous. As a result, we argue that the genus Gymnodinium contains only 234 identifiable species. Species that have been observed multiple times tend to have longer descriptions, written in English. The styles of individual authors have a major effect, with a few authors describing a disproportionate number of oncers. The information about the taxonomy of Gymnodinium that is available via the internet is incomplete, and reliance on it will not give access to all necessary knowledge. Six new names are presented – Gymnodinium campbelli for the homonymous name Gymnodinium translucens Campbell 1973, Gymnodinium antarcticum for the homonymous name Gymnodinium frigidum Balech 1965, Gymnodinium manchuriensis for the homonymous name Gymnodinium autumnale Skvortzov 1968, Gymnodinium christenum for the homonymous name Gymnodinium irregulare Christen 1959, Gymnodinium conkufferi for the homonymous name Gymnodinium irregulare Conrad & Kufferath 1954 and Gymnodinium chinensis for the homonymous name Gymnodinium frigidum Skvortzov 1968.This work was funded by grants from the John D and Catherine T MacArthur Foundation and the Alfred P Sloan Foundation to the Encyclopedia of Life and the National Science Foundation Data Net Program 0830976 and Global Names Project DBI-1062387

Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Murray, S. A., Ruvindy, R., Kohli, G. S., Anderson, D. M., & Brosnahan, M. L. Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1. Scientific Reports, 9(1), (2019): 14532, doi:10.1038/s41598-019-51074-3.Alexandrium catenella (formerly A. tamarense Group 1, or A. fundyense) is the leading cause of Paralytic Shellfish Poisoning in North and South America, Europe, Africa, Australia and Asia. The quantification of A.catenella via sxtA, a gene involved in Paralytic Shellfish Toxin synthesis, may be a promising approach, but has not been evaluated in situ on blooms of A. catenella, in which cell abundances may vary from not detectable to in the order of 106 cells L−1. In this study, we compared sxtA assay performance to a qPCR assay targeted to a species-specific region of ribosomal DNA (rDNA) and an established fluorescent in situ hybridization (FISH) microscopy method. Passing-Bablok regression analyses revealed the sxtA assay to overestimate abundances when <5 cell equivalents A. catenella DNA were analysed, but otherwise was closer to microscopy estimates than the rDNA assay, which overestimated abundance across the full range of concentrations analysed, indicative of a copy number difference between the bloom population and a culture used for assay calibration a priori. In contrast, the sxtA assay performed more consistently, indicating less copy number variation. The sxtA assay was generally reliable, fast and effective in quantifying A. catenella and was predictive of PST contamination of shellfish.We thank the Australian Research Council for Funding (FT120100704). We thank Chowdhury Sarowar for the toxicity measurements, at the Sydney Institute of Marine Science. Support to MB and DA was provided by MIT Sea Grant (NA14OAR4170077) and the Woods Hole Center for Oceans and Human Health (National Science Foundation award OCE-1840381 and National Institute of Environmental Health Sciences award 1-P01-ES028938–01). We are grateful for assistance from David Kulis, Claire Mullen, and Isaac Rosenthal for assistance in the collection and processing of Salt Pond samples

De novo assembly and characterisation of transcriptomes from Amphidinium species.

Amphidinium Claparede et Lachmann is one of the most abundant and diverse dinoflagellate genera found in marine benthic, sand dwelling and pelagic habitats. Species of Amphidinium have been widely studied for their potential to produce natural products, in particular, long chain and linear polyketide and macrolide compounds. More than 40 compounds produced by different species of Amphidinium have been isolated. These compounds with varying bioactivities are being investigated for their efficacy as antimicrobial and/or therapeutic agents. Some compounds produced by Amphidinium species have bioactivities against fish gill cells, and may lead to fish kills. Structural elucidation of many toxic compounds produced by dinoflagellates suggest that they may be based on a polyether ladder backbone, and that polyketide synthase (PKS) enzymes may have a role in their biosynthesis. The aim of our study was to screen Amphidinium species for the production of polyketide compounds and test their toxicity. We conducted a transcriptomic sequencing study to determine the presence of PKS genes (both mono-functionally expressed catalytic domains and genes encoding multimodular PKS enzyme complexes) in Amphidinium. The ketosynthase domain encoding transcripts were found to form distinct clades in the phylogenetic analysis, in comparison to similar genes from other organisms. We also show the presence of genes encoding six key enzymes essential for fatty acid production in Amphidinium. The results presented here are a step forward towards recognising the genes encoding critical steps in toxin biosynthesis and designing tools to monitor for and mitigate human illnesses due to harmful algal blooms

Investigating the biological properties of carbohydrate derived fulvic acid (CHD-FA) as a potential novel therapy for the management of oral biofilm infections.

Background: A number of oral diseases, including periodontitis, derive from microbial biofilms and are associated with increased antimicrobial resistance. Despite the widespread use of mouthwashes being used as adjunctive measures to control these biofilms, their prolonged use is not recommended due to various side effects. Therefore, alternative broad-spectrum antimicrobials that minimise these effects are highly sought after. Carbohydrate derived fulvic acid (CHD-FA) is an organic acid which has previously demonstrated to be microbiocidal against Candida albicans biofilms, therefore, the aims of this study were to evaluate the antibacterial activity of CHD-FA against orally derived biofilms and to investigate adjunctive biological effects.&lt;p&gt;&lt;/p&gt; Methods: Minimum inhibitory concentrations were evaluated for CHD-FA and chlorhexidine (CHX) against a range of oral bacteria using standardised microdilution testing for planktonic and sessile. Scanning electron microscopy was also employed to visualise changes in oral biofilms after antimicrobial treatment. Cytotoxicity of these compounds was assessed against oral epithelial cells, and the effect of CHD-FA on host inflammatory markers was assessed by measuring mRNA and protein expression.&lt;p&gt;&lt;/p&gt; Results: CHD-FA was highly active against all of the oral bacteria tested, including Porphyromonas gingivalis, with a sessile minimum inhibitory concentration of 0.5%. This concentration was shown to kill multi-species biofilms by approximately 90%, levels comparable to that of chlorhexidine (CHX). In a mammalian cell culture model, pretreatment of epithelial cells with buffered CHD-FA was shown to significantly down-regulate key inflammatory mediators, including interleukin-8 (IL-8), after stimulation with a multi-species biofilm.&lt;p&gt;&lt;/p&gt; Conclusions: Overall, CHD-FA was shown to possess broad-spectrum antibacterial activity, with a supplementary function of being able to down-regulate inflammation. These properties offer an attractive spectrum of function from a naturally derived compound, which could be used as an alternative topical treatment strategy for oral biofilm diseases. Further studies in vitro and in vivo are required to determine the precise mechanism by which CHD-FA modulates the host immune response.&lt;p&gt;&lt;/p&gt

Abiotic and biotic factors controlling sexual reproduction in populations of Pseudo-nitzschia pungens (Bacillariophyceae)

Pseudo-nitzschia pungens is a widely distributed marine pennate diatom. Hybrid zones, regions in which two different genotypes may interbreed, are important areas for speciation and ecology, and have been reported across the globe for this species. However, sexual reproduction between differing clades in the natural environment is yet to be observed and is difficult to predict. Here we carried out experiments using two mono-clonal cultures of P. pungens from different genotypes to measure the frequency and timing of sexual reproduction across varying biotic (growth phases and cell activity potential) and abiotic conditions (nutrients, light, turbulence). We found the mating rates and number of zygotes gradually decreased from exponential to late stationary growth phases. The maximum zygote abundance observed was 1,390 cells mL− 1 and the maximum mating rate was 7.1%, both which occurred during the exponential growth phase. Conversely, only 9 cells mL− 1 and a maximum mating rate of 0.1% was observed during the late stationary phase. We also found the higher the relative potential cell activity (rPCA) in parent cells, as determined by the concentration of chlorophyll a per cell and the ratio of colony formation during parent cultivations, revealed higher mating rates. Furthermore, sexual events were reduced under nutrient enrichment conditions, and mating pairs and zygotes were not formed under aphotic (dark) or shaking culture conditions (150 rpm). In order to understand the sexual reproduction of Pseudonitzschia in the natural environment, our results highlight that it is most likely the combination of both biotic (growth phase, Chl. a content) and abiotic factors (nutrients, light, turbulence) that will determine the successful union of intraspecific populations of P. pungens in any given region

Evolutionary distinctiveness of fatty acid and polyketide synthesis in eukaryotes

© 2016 International Society for Microbial Ecology All rights reserved. Fatty acids, which are essential cell membrane constituents and fuel storage molecules, are thought to share a common evolutionary origin with polyketide toxins in eukaryotes. While fatty acids are primary metabolic products, polyketide toxins are secondary metabolites that are involved in ecologically relevant processes, such as chemical defence, and produce the adverse effects of harmful algal blooms. Selection pressures on such compounds may be different, resulting in differing evolutionary histories. Surprisingly, some studies of dinoflagellates have suggested that the same enzymes may catalyse these processes. Here we show the presence and evolutionary distinctiveness of genes encoding six key enzymes essential for fatty acid production in 13 eukaryotic lineages for which no previous sequence data were available (alveolates: dinoflagellates, Vitrella, Chromera; stramenopiles: bolidophytes, chrysophytes, pelagophytes, raphidophytes, dictyochophytes, pinguiophytes, xanthophytes; Rhizaria: chlorarachniophytes, haplosporida; euglenids) and 8 other lineages (apicomplexans, bacillariophytes, synurophytes, cryptophytes, haptophytes, chlorophyceans, prasinophytes, trebouxiophytes). The phylogeny of fatty acid synthase genes reflects the evolutionary history of the organism, indicating selection to maintain conserved functionality. In contrast, polyketide synthase gene families are highly expanded in dinoflagellates and haptophytes, suggesting relaxed constraints in their evolutionary history, while completely absent from some protist lineages. This demonstrates a vast potential for the production of bioactive polyketide compounds in some lineages of microbial eukaryotes, indicating that the evolution of these compounds may have played an important role in their ecological success

Transient Receptor Potential Ion Channels Control Thermoregulatory Behaviour in Reptiles

Biological functions are governed by thermodynamics, and animals regulate their body temperature to optimise cellular performance and to avoid harmful extremes. The capacity to sense environmental and internal temperatures is a prerequisite for the evolution of thermoregulation. However, the mechanisms that enable ectothermic vertebrates to sense heat remain unknown. The recently discovered thermal characteristics of transient receptor potential ion channels (TRP) render these proteins suitable to act as temperature sensors. Here we test the hypothesis that TRPs are present in reptiles and function to control thermoregulatory behaviour. We show that the hot-sensing TRPV1 is expressed in a crocodile (Crocodylus porosus), an agamid (Amphibolurus muricatus) and a scincid (Pseudemoia entrecasteauxii) lizard, as well as in the quail and zebrafinch (Coturnix chinensis and Poephila guttata). The TRPV1 genes from all reptiles form a unique clade that is delineated from the mammalian and the ancestral Xenopus sequences by an insertion of two amino acids. TRPV1 and the cool-sensing TRPM8 are expressed in liver, muscle (transversospinalis complex), and heart tissues of the crocodile, and have the potential to act as internal thermometer and as external temperatures sensors. Inhibition of TRPV1 and TRPM8 in C. porosus abolishes the typically reptilian shuttling behaviour between cooling and heating environments, and leads to significantly altered body temperature patterns. Our results provide the proximate mechanism of thermal selection in terrestrial ectotherms, which heralds a fundamental change in interpretation, because TRPs provide the mechanism for a tissue-specific input into the animals' thermoregulatory response

Genetic Diversity, Morphological Uniformity and Polyketide Production in Dinoflagellates (Amphidinium, Dinoflagellata)

Dinoflagellates are an intriguing group of eukaryotes, showing many unusual morphological and genetic features. Some groups of dinoflagellates are morphologically highly uniform, despite indications of genetic diversity. The species Amphidinium carterae is abundant and cosmopolitan in marine environments, grows easily in culture, and has therefore been used as a ‘model’ dinoflagellate in research into dinoflagellate genetics, polyketide production and photosynthesis. We have investigated the diversity of ‘cryptic’ species of Amphidinium that are morphologically similar to A. carterae, including the very similar species Amphidinium massartii, based on light and electron microscopy, two nuclear gene regions (LSU rDNA and ITS rDNA) and one mitochondrial gene region (cytochrome b). We found that six genetically distinct cryptic species (clades) exist within the species A. massartii and four within A. carterae, and that these clades differ from one another in molecular sequences at levels comparable to other dinoflagellate species, genera or even families. Using primers based on an alignment of alveolate ketosynthase sequences, we isolated partial ketosynthase genes from several Amphidinium species. We compared these genes to known dinoflagellate ketosynthase genes and investigated the evolution and diversity of the strains of Amphidinium that produce them