Natural Dinoflagellate Research Articles

The occurrence patterns and alternative host use of the generalist parasitoid Parvilucifera infectans (Alveolata, Perkinsozoa) of marine dinoflagellates revealed by a high-frequency time series microscopic data at a temperate coastal site

Species belonging to the genus Parvilucifera (Alveolata, Perkinsozoa) are known as generalist parasitoids that can infect a variety of marine dinoflagellates and ultimately kill the host cell. Despite the accumulation of knowledge on Parvilucifera parasitoids over the last twenty years, the occurrence pattern and use of alternative hosts for persistent transmission within fluctuating natural dinoflagellate host communities have received relatively little attention. To address this, high-frequency (i.e., daily) time series protist monitoring was conducted for 411 days, from April 2020 to May 2021, at a temperate coastal site in Jinhae Bay, Republic of Korea. This site was characterized by frequent blooms of mixed dinoflagellates throughout most of the year. Nonetheless, Parvilucifera infections were detected only in the spring seasons, and its occurrence pattern was primarily governed by the presence and cell density of its primary dinoflagellate host, Akashiwo sanguinea. As long as the primary host was present at high densities (greater than 104 cells L-1), this primary host alone could support the transmission and persistence of the parasitoid. However, if the cell density of the primary host started to decline, then Parvilucifera searched for and exploited alternative hosts for its continued survival. Taken together, this study suggests that the presence and cell density of its preferred primary host species and the duration for which the density remains above the threshold level are key factors that regulate the timing of Parvilucifera occurrence and alternative host use.

Preservation of Dinoflagellate Cysts in Different Oxygen Regimes: Differences in Cyst Survival between Oxic and Anoxic Natural Environments

This quantitative dinoflagellate cyst study reveals an enormous difference in survival rates in oxygenated versus anoxic sediments. Replicate samples of concentrated natural dinoflagellate cysts with the same initial species composition (1.4 × 104 resting cysts·cm−3 sediment, 61% filled with live-appearing contents) were placed in bags of 20 µm plankton screen. Replicate bags containing 10.0 cm−3 concentrated cyst samples were placed on the seafloor in different environments in Long Island Sound, USA (anoxic and oxygenated), as well as refrigerated in test tubes in the laboratory. Three sets of 15 bags were placed in each environment. Once every year for four consecutive years, three bags were recovered from each set, and the contents were analyzed by cyst counting and germination experiments. An enormous difference in preservation potential between samples in oxygenated versus anoxic environments was revealed. The number of dinoflagellate cysts decreased abruptly within the first year in the oxygen-rich environment; living cysts became very rare (only 5% remained) and also empty walls of cysts disappeared (20% of total cysts remained). In anoxic sediment samples, living cysts also decreased significantly with time, but less quickly. After 1 year, 35% of the living cysts in the anoxic environment and 70% of the living cysts refrigerated in test tubes remained intact. After 4 years, 21% of the cysts with contents in the anoxic environment remained, and 31% in test tubes. The empty cyst walls remained intact for a longer time under anoxic conditions, especially of species known to fossilize well. Germination experiments showed that cysts with live-appearing contents were likely alive, because species with identifiable live-appearing cysts were also identified as vegetative cells in corresponding slurry cultures. The cyst assemblage was dominated by Protoperidinaceae, Dipolopsalidaceae, and Gonyaulacaceae. Of special interest is the ichthyotoxic Margalefodinium polykrikoides, the bloom-forming Peridinium quinquecorne, which has an undescribed resting cyst, and a previously undescribed Krypoperidinium species. The results show greater preservation of dinoflagellate cysts in “dead-zone sea bottoms” and may also provide an answer to the question of the absence of cyst beds in an area despite observed sedimentation of dense blooms.

Temporal heterogeneity of microbial communities and metabolic activities during a natural algal bloom

Elucidating the interactions between algae and associated microbial communities is critical for understanding the mechanisms that mediate the dynamic of harmful algal blooms (HABs) in marine environment. However, the microbial functional profiles and their biogeochemical potential in HABs process remains elusive, especially during a complete natural HAB cycle. Here, we used pyrosequencing and functional gene array (GeoChip) to investigate microbial community dynamics and metabolic potential during a natural dinoflagellate (Noctiluca scintillans) bloom. The results shown that bacterioplankton exhibited significant temporal heterogeneity over the course of the bloom stages. Microbial succession was co-driven by environmental parameters and biotic interactions. The functional analysis revealed significant variations in microbial metabolism during matter cycling. At bloom onset-stage, metabolic potential associated with iron oxidation and transport was elevated. Carbon fixation and degradation, denitrification, phosphorus acquisition, and sulfur transfer/oxidation were significantly enhanced at the plateau stage. During the decline and terminal stages, oxidative stress, lysis of compounds, and toxin degradation & protease synthesis increased. This work reveal phycosphere microorganisms can enhanced organic C decomposition capacity, altered N assimilation rate and S/P turnover efficiency, and balancing of the Fe budget during HAB process. The ecological linkage analysis has further shown that microbial composition and functional potential were significantly linked to algal blooms occurrence. It suggest that structural variability and functional plasticity of microbial communities influence HAB trajectory.

Harmful algal blooms significantly reduce the resource use efficiency in a coastal plankton community

Harmful algal blooms (HABs) have been investigated for their catastrophic effects on public health and aquaculture intensively, but the research about HABs effects on the diversity patterns and intrinsic functions of the plankton community based on a species identification with high resolution and accuracy has been scarce. We therefore investigated the shifts of plankton diversity via pyrosequencing during and around a natural dinoflagellate (Prorocentrum donghaiense) bloom and analyzed the effect of P. donghaiense abundance on the operationally-defined resource use efficiency (RUE) of plankton community to test our hypothesis that outbreaks of HABs will reduce RUE of the plankton community via shifting the plankton community structure, species composition in particular. We found that the species diversity of eukaryotic plankton community was significantly decreased during the bloom, as reflected in OTU (operational taxonomic unit) richness, and Pielou’s evenness index. Principal coordinates analysis indicated significant difference in plankton community structure between blooming and non-blooming periods. As hypothesized, the species richness was positively correlated to RUE (defined as the ratio of phytoplankton biomass to total phosphorus), and more importantly, the cell density of P. donghaiense exhibited significant negative correlation with RUE. Our results explicitly demonstrated HABs reduce RUE via reducing species richness (corresponding to a less occupancy of the trophic niches), which supports the previously documented notion that niche partitioning enhances RUE (a key ecosystem function). Also, our work provides striking evidence for the relationship between plankton species richness (or diversity) and community function (resource use efficiency) via studying on HABs, a natural but exceptional phenomenon, in addition to revealing a profound consequence of HABs.

Molecular and biochemical basis for the loss of bioluminescence in the dinoflagellate Noctiluca scintillans along the west coast of the USA.

The globally distributed heterotrophic dinoflagellate Noctiluca scintillans (Macartney) Kofoid & Swezy is well known for its dense blooms and prominent displays of bioluminescence. Intriguingly, along the west coast of the USA its blooms are not bioluminescent. We investigated the basis for the regional loss of bioluminescence using molecular, cellular and biochemical analyses of isolates from different geographic regions. Prominent differences of the non-bioluminescent strains were: (1) the fused luciferase and luciferin binding protein gene (lcf/lbp) was present but its transcripts were undetectable; (2) lcf/lbp contained multiple potentially deleterious mutations; (3) the substrate luciferin was absent, based on the lack of luciferin blue autofluorescence and the absence of luciferin derived metabolites; (4) although the cells possessed scintillons, the vesicles that contain the luminescent chemistry, electron microscopy revealed additional scintillon-like vesicles with an atypical internal structure; (5) cells isolated from the California coast were 43% smaller in size than bioluminescent cells from the Gulf of Mexico. Phylogenetic analyses based on the large subunit of rDNA did not show divergence of the non-bioluminescent population in relation to other bioluminescent N. scintillans from the Pacific Ocean and Arabian Sea. Our study demonstrates that gene silencing and the lack of the luciferin substrate have resulted in the loss of a significant dinoflagellate functional trait over large spatial scales in the ocean. As the bioluminescence system of dinoflagellates is well characterized, non-bioluminescent N. scintillans is an ideal model to explore the evolutionary and ecological mechanisms that lead to intraspecific functional divergence in natural dinoflagellate populations.

Comparative proteomic studies of a Scrippsiella acuminata bloom with its laboratory-grown culture using a 15N-metabolic labeling approach

Comparative proteomic analysis was carried out using cells isolated from a natural bloom of Scrippsiella acuminata (formerly Scrippsiella trochoidea) in the early bloom (EB) and late bloom (LB) stages as well as with laboratory-grown cultures of cells isolated from the bloom in early growth (EG) and late growth (LG) stages. For quantitative proteomics, LG cells were grown for 20 generations in the presence of 15N as a reference (i.e. common denominator) for all comparison. In comparisons with early growth laboratory grown cells (EG/LG), nearly 64% of proteins identified had similar abundance levels, with the remaining 36% mostly more abundant in EG cells. Calvin cycle, amino acid metabolism, chlorophyll biosynthesis and transcription/translation were among the up-regulated processes. Cells from the early bloom (EB/LG) had a greater abundance of transporters and enzymes related to light harvesting and oxidative phosphorylation, while the abundance of these proteins decreased in late bloom cells (LB/LG). All natural bloom samples showed either constant or lower abundance levels of enzymes involved in sugar synthesis and glycolytic pathways compared to laboratory grown cells. Our results represent the first examination of the proteomic changes in the development of a natural dinoflagellate bloom. Importantly, our results demonstrate that the proteome of cells grown in the laboratory is distinctively different from cells in a natural bloom.

Effects of a bacterial algicide, IRI-160AA, on dinoflagellates and the microbial community in microcosm experiments

Filtrates from the bacterium Shewanella sp. IRI-160 (termed IRI-160AA) have been shown to inhibit population growth and kill a variety of dinoflagellates grown in culture. Here we test the immediate efficacy of IRI-160AA in laboratory microcosms initiated from three natural dinoflagellate blooms (Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum). We measured target dinoflagellate abundance, total chlorophyll-a, photosystem II (PSII) photochemistry, and changes to the prokaryotic and eukaryotic community composition over 2–3 days of IRI-160AA incubation. Naked dinoflagellates were impacted more, while abundance of the thecate P. minimum was not affected. However, dinoflagellate growth inhibition was generally lower than that observed in uni-algal cultures, and took longer to occur. Eukaryotic community composition in IRI-160AA treated microcosms was significantly different from control incubations, and was driven predominantly by increases in heterotrophic protists (e.g. Euplotes sp. and Paraphysomonas sp.). Similarly, significant changes to the prokaryotic community structure were evident. Microcosms of G. instriatum with higher algicide concentrations indicated that algicidal activity was enhanced in a dose dependent manner. Furthermore, total ciliate abundance as well as a bactivorous chyrsophyte (Paraphysomonas sp.) increased in a dose dependent manner. Total diatom abundance increased at lower IRI-160AA concentrations, but increased less with increasing dose. Overall, the bio-activity of IRI-160AA on naturally occurring dinoflagellates in mixed natural microbial communities is encouraging from the applied perspective of using the active compound(s) in IRI-160AA as natural agent(s) to manage harmful dinoflagellate blooms.

Characterization of mycosporine-serine-glycine methyl ester, a major mycosporine-like amino acid from dinoflagellates: a mass spectrometry study.

Several unknown mycosporine-like amino acids (MAAs) have been previously isolated from some cultured species of toxic dinoflagellates of the Alexandrium genus (Dinophyceae). One of them, originally called M-333, was tentatively identified as a shinorine methyl ester, but the precise nature of this compound is still unknown. Using a high-resolution reversed-phase liquid chromatography mass spectrometry analyses (HPLC/MS), we found that natural populations of the red tide dinoflagellate Prorocentrum micans Ehrenberg showed a net dominance of M-333 together with lesser amounts of other MAAs. We also documented the isolation and characterization of this MAA from natural dinoflagellate populations and from Alexandrium tamarense (Lebour) Balech cultures. Using a comparative fragmentation study in electrospray mass spectrometry between deuterated and non-deuterated M-333 compounds and synthesized mono and dimethyl esters of shinorine, this novel compound was characterized as mycosporine-serine-glycine methyl ester, a structure confirmed by nuclear magnetic resonance. These isobaric compounds can be differentiated by their fragmentation patterns in MS(3) experiments because the extension and the specific site of the methylation changed the fragmentation pathway.

Diurnal variations of dinoflagellate bioluminescence within the open-ocean north-east Atlantic

In regions where dinoflagellates dominate bioluminescent emissions, diurnal variations in bioluminescence potential (BPOT) can be influenced by both exogenous and endogenous factors. In summer 2009, measurements were made in the north-east Atlantic to examine the diurnal variations in BPOT in natural dinoflagellate communities and determine the influence of circadian regulation and light exposure. The maximum night BPOT was >23 times greater than the daytime levels for the same populations. Photosynthetic species were responsible for 55–75% of measured BPOT based on calculated light budgets. Under continual darkness, diurnal variability of BPOT was retained over a 48-h period, demonstrating a degree of circadian control. Results suggest that both photosynthetic and heterotrophic dinoflagellates exhibit circadian regulation of their bioluminescent capacity and light strongly influences the diurnal variation of BPOT. Circadian rhythms were photo-entrained to the phase of the natural photoperiod and light further inhibited daytime bioluminescence. Maximum night BPOT was significantly correlated with the previous day integrated photosynthetically active radiation (PAR) suggesting photo-enhancement within natural populations. A 21% decrease in integrated PAR was associated with a 26–29% decrease in maximum night BPOT for constant populations. Maximum night BPOT was damped by up to 73% when organisms were kept in constant darkness. Findings further quantify diurnal variations in BPOT in natural dinoflagellate populations and their relation to a number of taxonomic, cellular and environmental factors. Results emphasize the importance of considering the recent light history of bioluminescent communities when analysing or predicting in situ BPOT.

Grazing on a natural assemblage of ciliate and dinoflagellate cysts by the eastern oyster Crassostrea virginica

A natural dinoflagellate- and ciliate-cyst community from anoxic sediment collected from New Haven, Long Island Sound, was concentrated with a particle sorter and fed to oysters Cras- sostrea virginica. The total number of cysts and the species composition of cysts in beakers contain- ing live oysters were measured before and after feeding by the oysters. The oysters significantly reduced the numbers of both dinoflagellate and ciliate cysts. Both empty cyst walls and filled (live) cysts were consumed. The oysters decreased the number of total cysts to less than half compared to control beakers containing empty oyster shells. Results from the experiment show that natural assem- blages of cysts in sediment are degraded by the feeding activities of oysters. If toxic cysts were pre- sent in natural sediments resuspended from the bottom, digestion of resting cysts could lead to toxin accumulation in oysters. There was no major shift in species composition of cysts; cysts known as fossilizable were destroyed, as well as cysts not known to be very resistant or preservable.

Mitochondrial cob and cox1 Genes and Editing of the Corresponding mRNAs in Dinophysis acuminata from Narragansett Bay, with Special Reference to the Phylogenetic Position of the Genus Dinophysis

Dinophysis acuminata cells were isolated from Narragansett Bay water samples in June 2005 using flow cytometry. Dinoflagellate-specific PCR primers were used to isolate small-subunit rRNA (18S rRNA), mitochondrial cytochrome b (cob), and cytochrome c oxidase I (cox1) genes and the encoded cDNAs. Maximum-likelihood analysis of a concatenated data set of ribosomal DNA and cDNA sequences of cob and cox1 showed that D. acuminata was sister to Gonyaulacoids, but without strong bootstrap support. The approximately unbiased test could not reject alternative positions of D. acuminata. To gain better resolution, mRNA editing of cob and cox1 was inferred for D. acuminata and 13 other dinoflagellate species. The location and type of editing as well as the distribution pattern in D. acuminata were generally similar to those in other dinoflagellates except for two edited sites that are unique to this species. Bayesian analyses of a matrix that recorded the location and type of editing, and of a matrix that included the protein sequences of COB and COX1 with the editing data yielded tree topologies similar to the three-gene tree but again failed to resolve the phylogenetic position of D. acuminata. However, the density of edited sites in the D. acuminata mitochondrial genes, consistent with phylogenetic trees, indicated that Dinophysis is a derived dinoflagellate lineage, diverging after other lineages such as Oxyrrhis, Amphidinium, and Symbiodinium. We demonstrate that dinoflagellate-specific PCR coupled with flow cytometry can be a useful tool to analyze genes and their transcripts from a natural dinoflagellate population.

Use of intracellular amino acid analysis as an indicator of the physiological status of natural dinoflagellate populations

Use of intracellular amino acid analysis as an indicator of the physiological status of natural dinoflagellate populations

Use of intracellular amino acid analysis as an indicator of the physiological status of natural dinoflagellate populations

The physiological status of 2 dinoflagellate populations in Bantry Bay, Ireland, was examined by use of amino acid analys~s. Observations were made durlng two 24 h periods. In one, during an upwelling event, the phytoplankton population was of low density (ca 1 yg chl a I ' ) and dominated mainly by Ceratium, Prorocentrum and Scrippsiella species. In the second (1 wk later), following relaxation of the upwelling and an incursion of surface water from the adjacent open coastal region, the phytoplankton community was dominated by Ceratium spp. and Gyrodiniurn aureolum with chl a concentrations up to 10 times higher. During the first sampling period, there was evidence of slight N-stress at the surface; the ratio of intracellular glutamine/glutamate (Gln/Glu) was 0.35, increasing with ammonium spiking There was evidence of C-stress at the chl maximum, where Gln/Glu was high (1.2) and decreased on exposure to increased irradiance. During the second samphng period, Gln/Glu was low (0.1) suggesting a poor N-status. However, there was little or even a negative response of Gln/Glu to ammonium splking; glutamate responded to the spiklng rather than glutamine. The general composition of the intracellular amino acid pool was similar to that at the first period, with high proportions of N-rich amino acids such as arginine. This second population appeared to be physiologically damaged in some way; this would not appear to have been simply a result of N-stress, but perhaps reflected the consequences of the associated advective process and exposure of the algae to high photon flux densities. The concentration of dissolved free a m n o acids (DFAA) during the second period was up to 4 times higher than at the first, but still dominated by serine and glycine. DFAA appeared to relate more to the presence of zooplankton than to the phytoplankton.

Changes in dinoflagellate intracellular amino acids in response to diurnal changes in light and N supply

Seven species of dinoflagellates were examined with respect to their intracellular amino acid (InAA) content under different conditions of N-nutrition and light, and for release of dissolved free amino acids (DFAA). Alexandnum spp. did not contain significant amounts of a neutral nonprotein amine present in all other phototrophic dinoflagellates tested but, in contrast, it always maintained high levels of glutamine and of the ratio of intracellular glutamine/glutamate (Gln/Glu). Dinoflagellates responded to a decreased availability of N with a fall in Gln/Glu but, unlike other microalgae, arginine was always a significant component of InAA. Refeeding of N-limited cultures with nitrate during the dark phase of growth, simulating refeeding of a natural population following a diurnal migration to the nutricline, resulted in an increased Gln/Glu during the dark phase followed by a decline during the Light phase. N-refeeding of N-deprived cells led a rapid rise in the ratio, except during N-refeeding of stationary phase Gymnodinium catenatum (which were of abnormal shape and motility) when Glu rather than Gln increased. The sensitivity of the analytical method would enable sampling from natural dinoflagellate populations in order to assess their physiological status; extracts from 200 to 2000 cells, depending on cell size, are sufficient. A significant quantity of an unidentified amino acid accumulated in the growth media of Gymnodinjum catenatum but otherwise DFAA in dinoflagellate culture media were similar to that from other microalgae.

PIGMENTS, FATTY ACIDS, AND STEROLS OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM1

ABSTRACTCultures and field samples of the toxic dinoflagellate Gymnodinium catenatum Graham from Tasmania, Australia, were analyzed for pigment, fatty acid, and sterol composition. Gymnodinium catenatum contained the characteristic pigments of photosynthetic dinoflagellates, including chlorophyll a, chlorophyll c2, and the carotenoids peridinin, dinoxanthin, diadinoxanthin, diatoxanthin, and β,β‐carotene. In midlogarithmic and early stationary phase cultures, the chlorophyll a content ranged 50–72 pg · cell−1, total lipids 956–2084 pg · cell−1, total fatty acids 426–804 pg · cell−1, and total sterols 8–20 pg · cell−1. The major fatty acids (in order of decreasing abundance) were 16:0, 22:6(n‐3), and 20:5(n‐3) (collectively 65–70% of the total fatty acids), followed by 16:1(n‐7), 18:2(n‐6), and 14:0. This distribution is characteristic of most dinoflagellates, except for the low abundance (<3%) of the fatty acid 18:5(n‐3), considered by some authors to be a marker for dinoflagellates. The three major sterols were 4α‐methyl‐5α‐cholest‐7‐en‐3β‐ol, 4α,23,24‐trimethyl‐5α‐cholest‐22E‐en‐3β‐ol (the dinoflagellate sterol, dinosterol), and 4α,23,24‐trimethyl‐5α‐cholest‐7‐en‐3β‐ol. These three sterols comprised about 75% of the total sterols in both logarithmic and early stationary phase cultures, and they were also found in high proportions (22–25%) in natural dinoflagellate bloom samples. 4‐Desmethyl sterols, which are common in most microalgae, were only present in trace amounts in G. catenatum. The chemotaxonomic affinities of G. catenatum and the potential for using specific signature lipids for monitoring toxic dinoflagellate blooms are discussed.

Green light-mediated photomorphogenesis in a dinoflagellate resting cyst

Developmental responses to light are well known and ubiquitous among higher plants1, but examples of such responses among the algae are less common. Those responses which have been reported in this group are generally photoperiodic or require prolonged light exposures; most involve macrophytes2. We now report a non-photosynthetic, low-threshold, photomorphogenic response in a unicellular alga, the dinoflagellate Scrippsiella trochoidea (Stein) Loeblich. In contrast to the reported behaviour of most other dinoflagellate species, resting cysts of S. trochoidea require light to germinate. This requirement is satisfied to a large extent by low photon fluences delivered in exposures as short as 1 second. Green light is most effective in eliciting the response. Given the importance of dinoflagellates as primary producers in many aquatic ecosystems and the potential role of resting cysts in controlling the dynamics of natural dinoflagellate populations, the present observations are of obvious ecological significance. The primitive phylogenetic standing of dinoflagellates3, and the relative rarity of green light-mediated photomorphogenic responses in eukaryotes generally4,5,suggest that this phenomenon may also hold considerable evolutionary and photophysiological interest.