Species and genetic diversity of notorious dinoflagellates Pfiesteria piscicida, Luciella masanensis, and relatives in marine sediments of China.

The dinoflagellate genus Pfiesteria, encompassing Pfiesteria piscicida and P. shumwayae, became a hot topic in HABs research in the early 2000s due to numerous but controversial reports regarding life cycle and toxicity. While Pfiesteria presence has been reported from all continents, surprisingly, there has been no documented presence in China to date.

The resting cyst of dinoflagellate host bacterial microbiomes with more diverse trophic strategies under conditions typically observed in marine sediments.

Variation in the condition of marine sediments provides selective preservation milieus, which act as a key determinant for the abundance and distribution of dinoflagellate resting cysts in natural sediments. Microbial degradation is an understudied biological factor of potential importance in the processes. However, gaps remain in our knowledge about the fundamental information of the bacterial consortia associated with dinoflagellate resting cysts both in laboratory cultures and in the field.

Evidence for the production of asexual resting cysts in a free-living species of Symbiodiniaceae (Dinophyceae).

Coral reef ecosystems are the most productive and biodiverse marine ecosystems, with their productivity levels highly dependent on the symbiotic dinoflagellates belonging to the family Symbiodiniaceae. As a unique life history strategy, resting cyst production is of great significance in the ecology of many dinoflagellate species, those HABs-causing species in particular, however, there has been no confirmative evidence for the resting cyst production in any species of the family Symbiodiniaceae. Based on morphological and life history observations of cultures in the laboratory and morpho-molecular detections of cysts from the marine sediments via fluorescence in situ hybridization (FISH), cyst photography, and subsequent singe-cyst PCR sequencing, here we provide evidences for the asexual production of resting cysts by Effrenium voratum, the free-living, red tide-forming, and the type species of the genus Effrenium in Symbiodiniaceae.

Transoceanic ships as a source of alien dinoflagellate invasions of inland freshwater ecosystems.

Ships' ballast water and sediments have long been linked to the global transport and expansion of invasive species and thus have become a hot research topic and administrative challenge in the past decades. The relevant concerns, however, have been mainly about the ocean-to-ocean invasion and sampling practices have been almost exclusively conducted onboard. We examined and compared the dinoflagellate cysts assemblages in 49 sediment samples collected from ballast tanks of international and domestic routes ships, washing basins associated with a ship-repair yard, Jiangyin Port (PS), and the nearby area of Yangtze River (YR) during 2017-2018.

DNA and RNA Stability of Marine Microalgae in Cold-Stored Sediments and Its Implications in Metabarcoding Analyses.

The ever-increasing applications of metabarcoding analyses for environmental samples demand a well-designed assessment of the stability of DNA and RNA contained in cells that are deposited or buried in marine sediments. We thus conducted a qPCR quantification of the DNA and RNA in the vegetative cells of three microalgae entrapped in facsimile marine sediments and found that >90% of DNA and up to 99% of RNA for all microalgal species were degraded within 60 days at 4 °C. A further examination of the potential interference of the relic DNA of the vegetative cells with resting cyst detection in sediments was performed via a metabarcoding analysis in artificial marine sediments spiked with the vegetative cells of two Kareniaceae dinoflagellates and the resting cysts of another three dinoflagellates.

A combined approach detected novel species diversity and distribution of dinoflagellate cysts in the Yellow Sea, China.

Resting cysts of dinoflagellates seed harmful algal blooms (HABs) and their geographic expansion, which makes it fundamentally important to obtain comprehensive inventories of dinoflagellate resting cysts in HABs-prone regions. The Yellow Sea (YS) of China has observed numerous outbreaks of dinoflagellate HABs with some novel species recorded recently indicating an underestimated HABs-causing species diversity. We report our investigation of dinoflagellate cysts of YS via an approach combining metabarcoding sequencing and single-cyst morpho-molecular identification, which identified many novel cyst species and a significant controlling effect of the Yellow Sea Cold Water Mass on cyst composition.

Characterizing the Status of Energetic Metabolism of Dinoflagellate Resting Cysts under Mock Conditions of Marine Sediments via Physiological and Transcriptional Measurements.

Similar to the seeds of higher plants, resting cysts, a non-motile, benthic, and dormant stage in the life history of many dinoflagellate species, play vital roles via germination in the seasonal dynamics and particularly the initiation of harmful algal blooms (HABs) of dinoflagellates. It is thus crucial for resting cysts to balance between the energetic catabolism for viability maintenance and the energy preservation for germination during their dormancy. Despite this importance, studies on how resting cysts of dinoflagellates accomplish energetic metabolism in marine sediment have been virtually absent.

Deficiency of nitrogen but not phosphorus triggers the life cycle transition of the dinoflagellate Scrippsiella acuminata from vegetative growth to resting cyst formation.

Nitrogen (N) and phosphorus (P) are essential elements for algal growth. When N and P are deficient, dinoflagellates will take a series of measures to achieve population continuation including formation of resting cysts, an important ecological strategy of dinoflagellates that plays a key role in the initiation and termination of harmful algal blooms (HABs). How the deficiency of N and P affects algal growth and cyst formation has been investigated in some dinoflagellate species, but how it affects the life cycle transition in dinoflagellates has been poorly understood.

Identification and implications of a core bacterial microbiome in 19 clonal cultures laboratory-reared for months to years of the cosmopolitan dinoflagellate .

Identification of a core microbiome (a group of taxa commonly present and consistently abundant in most samples of host populations) is important to capture the key microbes closely associated with a host population, as this process may potentially contribute to further revealing their spatial distribution, temporal stability, ecological influence, and even impacts on their host's functions and fitness. The naked dinoflagellate is a cosmopolitan and toxic species, which is also notorious in forming harmful algal blooms (HABs) and causing massive fish-kills. Here we reported the core microbiome tightly associated with 19 strains of that were originally isolated from 6 geographic locations along the coast of China and from an estuary of Chesapeake Bay, United States, and have been maintained in the laboratory for several months to over 14 years.

Possible functions of CobW domain-containing (CBWD) genes in dinoflagellates using Karlodinium veneficum as a representative.

Since > 91% of dinoflagellates are proven auxotrophs of vitamin B and the cobalamin synthetase W (CobW) is a key gene involved in vitamin B synthesis pathway, a number of CobW domain-containing (CBWD) genes in dinoflagellates (DinoCBWDs) were surprisedly found from our transcriptomic and meta-transcriptomic studies. A total of 88 DinoCBWD genes were identified from the genomes and transcriptomes of four dinoflagellates, with five being cloned for full-lengths and characterized using the cosmopolitan and ecologically-important dinoflagellates Karlodinium veneficum and Scrippsiella trochoidea (synonym of Scrippsiella acuminata). DinoCBWDs were verified being irrelevant to vitamin B biosynthesis due to their transcriptions irresponsive to vitamin B levels and their phylogenetic positions.

Measuring viability of dinoflagellate cysts and diatoms with stains to test the efficiency of facsimile treatments possibly applicable to ships' ballast water and sediment.

Expansion of harmful algal bloom (HAB) species through ships' ballast water and sediment has been an increasing concern. Determining whether a microalgal cell, particularly for the toxic and HAB-forming species, is "viable" or "dead" is fundamental to understanding the effectiveness of the many ballast-water treatments that have been considered. To this end, we screened a variety of stains to assess the viability of dinoflagellate (Gymnodinium catenatum, GC) cysts and diatom (Corethron hystrix) vegetative cells to test the efficiency of ballast water treatments.

Abundant Species Diversity and Essential Functions of Bacterial Communities Associated with Dinoflagellates as Revealed from Metabarcoding Sequencing for Laboratory-Raised Clonal Cultures.

Interactions between algae and bacteria represent an important inter-organism association in aquatic environments, which often have cascading bottom-up influences on ecosystem-scale processes. Despite the increasing recognition of linkages between bacterioplankton and dynamics of dinoflagellate blooms in the field, knowledge about the forms and functions of dinoflagellate-bacteria associations remains elusive, mainly due to the ephemeral and variable conditions in the field. In this study, we characterized the bacterial community associated with laboratory cultures of 144 harmful algal strains, including 130 dinoflagellates (covering all major taxonomic orders of dinoflagellates) and 14 non-dinoflagellates, via high-throughput sequencing for 16S rRNA gene amplicons.

Vitamin B-auxotrophy in dinoflagellates caused by incomplete or absent cobalamin-independent methionine synthase genes ().

Dinoflagellates are responsible for most marine harmful algal blooms (HABs) and play vital roles in many ocean processes. More than 90% of dinoflagellates are vitamin B auxotrophs and that B availability can control dinoflagellate HABs, yet the genetic basis of B auxotrophy in dinoflagellates in the framework of the ecology of dinoflagellates and particularly HABs, which was the objective of this work. Here, we investigated the presence, phylogeny, and transcription of two methionine synthase genes (B-dependent and B-independent ) via searching and assembling transcripts and genes from transcriptomic and genomic databases, cloning 38 cDNA isoforms of the two genes from 14 strains of dinoflagellates, measuring the expression at different scenarios of B, and comprehensive phylogenetic analyses of more than 100 organisms.

Emerging harmful algal bloom species over the last four decades in China.

The first recorded micro-algae bloom in Chinese coastal waters dates back to 1933 and was caused by a mixture of Noctiluca scintillans and Skeletonema costatum sensu lato along the Zhejiang coast (the East China Sea). While well-documented harmful algal blooms (HABs) appeared to be extremely scarce from the 1950s to 1990, both the frequency and intensity have been reportedly increasing since 1990. Among them, the fish-killing HABs, mainly caused by Karenia mikimotoi, Karlodinium digitatum, Karlodinium veneficum, Margalefidinium polykrikoides, and Heterocapsa spp.

Resting Cysts Detected in the Ballast Tank Sediment of Ships Arriving in the Ports of China and North America and the Implications in the Species' Geographic Distribution and Possible Invasion.

Over the past several decades, much attention has been focused on the dispersal of aquatic nonindigenous species via ballast tanks of shipping vessels worldwide. The recently reclassified dinoflagellate (previously identified as sp., , or ) was not reported in China until 2006.

Dependence of genome size and copy number of rRNA gene on cell volume in dinoflagellates.

Dinoflagellates are an ecologically important group of protists in aquatic environment and have evolved many unusual and enigmatic genomic features such as immense genome sizes, high repeated genes, and a large portion of hydroxymethyluracil in DNA. Although previous studies have observed positive correlations between the large subunit (LSU) rRNA gene copy number and genome size of a variety of eukaryotic organisms (e.g.

Geographic distribution and historical presence of the resting cysts of Karenia mikimotoi in the seas of China.

The toxic dinoflagellate Karenia mikimotoi frequently forms harmful algal blooms (HABs) and thus causes massive kills of fish and shellfish in worldwide coastal waters, which has led to intensive investigations on multiple facets of the species. Following our recent discovery of K. mikimotoi forming resting cyst, a very possible mechanism for the inoculation of blooms and geographic expansion for this and many other HABs-causing species, here we report our detection of K.

Expression Patterns of the Heat Shock Protein 90 (Hsp90) Gene Suggest Its Possible Involvement in Maintaining the Dormancy of Dinoflagellate Resting Cysts.

Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone functioning in cellular structural folding and conformational integrity maintenance and thus plays vital roles in a variety of biological processes. However, many aspects of these functions and processes remain to be fully elucidated, particularly for non-model organisms. Dinoflagellates are a group of eukaryotes that are exceedingly important in primary production and are responsible for the most harmful algal blooms (HABs) in aquatic ecosystems.

Morpho-molecular description of a new HAB species, Pseudocochlodinium profundisulcus gen. et sp. nov., and its LSU rRNA gene based genetic diversity and geographical distribution.

Harmful algal blooms (HABs) caused by an unknown dinoflagellate species have frequently occurred in the Pearl River Estuary, China Since 2006. These blooms were associated with severe water discoloration and economic losses, ranging from several km to 300 km with the maximum recorded cell density being 2.77 × 10 cells⋅L.

Exploration of resting cysts (stages) and their relevance for possibly HABs-causing species in China.

The studies on the species diversity, distribution, environmental implications, and molecular basis of resting cysts (stages) of dinoflagellates and a few species of other groups conducted in China during the last three decades are reviewed. The major achievements are summarized as the following five aspects: 1) The continual efforts in detecting the species diversity of resting cysts (spores) in dinoflagellates and other classes using either morphological or molecular approaches, or both, in the four seas of China, which led to identifications of 106 species of dinoflagellate resting cysts and 4 species of resting stages from other groups of microalgae, with a total of 64 species of dinoflagellate cysts and the resting stage of the brown tide-causing Aureococcus anophagefferens being unequivocally identified via molecular approaches from the sediments of Chinese coastal waters; 2) The well-known toxic and HABs-causing dinoflagellates Karenia mikimotoi, Karlodinium veneficum, Akashiwo sanguinea and the pelagophyte A. anophagefferens were proven to be resting cyst (stage) producers via laboratory studies on their life cycles and field detections of resting cysts (resting stage cells).

Probing the Energetic Metabolism of Resting Cysts under Different Conditions from Molecular and Physiological Perspectives in the Harmful Algal Blooms-Forming Dinoflagellate .

Energetic metabolism is essential in maintaining the viability of all organisms. Resting cysts play important roles in the ecology of dinoflagellates, particularly for harmful algal blooms (HABs)-causative species. However, the energetic metabolism underlying the germination potency maintenance of resting cysts of dinoflagellate have been extremely scarce in studies from physiological and, particularly, molecular perspectives.

Potent allelopathy and non-PSTs, non-spirolides toxicity of the dinoflagellate Alexandrium leei to phytoplankton, finfish and zooplankton observed from laboratory bioassays.

The dinoflagellate genus Alexandrium has been well known for causing paralytic shellfish poisoning (PSP) worldwide. Several non-PSP toxin-producing species, however, have shown to exhibit fish-killing toxicity. Here, we report the allelopathic activity of Alexandrium leei from Malaysia to other algal species, and its toxicity to finfish and zooplankton, via laboratory bioassays.

Transcriptional Responses of the Heat Shock Protein 20 (Hsp20) and 40 (Hsp40) Genes to Temperature Stress and Alteration of Life Cycle Stages in the Harmful Alga (Dinophyceae).

The small heat shock protein (sHsp) and Hsp40 are Hsp members that have not been intensively investigated but are functionally important in most organisms. In this study, the potential roles of a () and a () in dinoflagellates during adaptation to temperature fluctuation and alteration of different life stages were explored using the representative harmful algal blooms (HABs)-causative dinoflagellate species, . We isolated the full-length cDNAs of the two genes via rapid amplification of cDNA ends (RACE) and tracked their differential transcriptions via real-time qPCR.

Contact micropredation may play a more important role than exotoxicity does in the lethal effects of Karlodinium australe blooms: Evidence from laboratory bioassays.

Multiple dinoflagellate species from the genus Karlodinium have been well known to form massive and toxic blooms that consequently cause fish kills in many coastal waters around the world. Karlodinium australe is a mixotrophic and potentially ichthyotoxic species associated with fish kills. Here, we investigated phagotrophy of K.

An assessment on the intrapopulational and intraindividual genetic diversity in LSU rDNA in the harmful algal blooms-forming dinoflagellate Margalefidinium (= Cochlodinium) fulvescens based on clonal cultures and bloom samples from Jiaozhou Bay, China.

Large subunit ribosomal DNA (LSU rDNA) sequences have been increasingly used to infer the phylogeny and species identity of organisms, a few previous studies, however, have observed high intraspecific and even intraindividual variability in LSU rDNA in some dinoflagellate species due to, assumably, large copy numbers of rDNA in dinoflagellates. Since the copy number of LSU rDNA varies tremendously among dinoflagellate species, the intraspecific and intraindividual diversity for a species of particular interest thus needs to be investigated individually. As a toxic and HABs-forming dinoflagellate, Margalefidinium (= Cochlodinium) fulvescens has been observed to approach blooming density in Jiaozhou Bay, China since 2015 after numerous blooms having been reported from other countries.