Role of turbulence in Dinophysis spp. growth, feeding, and toxin leakage in culture.

Dinophysis, a mixotrophic dinoflagellate that is known to prey on the ciliate Mesodinium rubrum, and retain its chloroplasts, is responsible for diarrhetic shellfish poisoning (DSP) in humans and has been identified on all U.S. coasts. Monocultures of Dinophysis have been used to investigate the growth of Dinophysis species in response to variations in environmental conditions, however, little is known about the roles of system stability (turbulence) and mixotrophy in the growth and toxicity of Dinophysis species in the U.S.. To begin to address this gap in knowledge, culturing experiments were conducted with three species (four strains) of Dinophysis, that included predator-prey co-incubation (Dinophysis spp.+ M. rubrum) and prey-only (M. rubrum) flasks. Cultures were investigated for effects of low or high turbulence on Dinophysis spp. growth, feeding, and amounts of intra- and extracellular toxins: okadaic acid and derivatives (diarrhetic shellfish toxins, DSTs) and pectenotoxins (PTXs). Turbulence did not have a measurable effect on the rates of ingestion of M. rubrum prey by Dinophysis spp. for any of the four strains, however, effects on growth and particulate and dissolved toxins were observed. High turbulence (ε = 10-2 m2s-3) significantly slowed growth of both D. acuminata and D. ovum relative to still controls, but significantly stimulated growth of the D. caudata strain. Increasing turbulence also resulted in significantly higher intracellular toxin content in D. acuminata cultures (DSTs and PTXs), but significantly reduced intracellular toxin content (PTXs) in those of D. caudata. An increase in turbulence appeared to promote toxin leakage, as D. ovum had significantly more extracellular DSTs found in the medium under high turbulence when compared to the still control. Overall, significant responses to turbulence were observed, whereby the three strains from the "Dinophysis acuminata complex" displayed a stress response to turbulence, i.e., decreasing growth, increasing intracellular toxin content and/or increasing toxin leakage, while the D. caudata strain had an opposite response, appearing stimulated by, or more tolerant of, high turbulence.

Naturally and Anthropogenically Induced Lingulodinium polyedra Dinoflagellate Red Tides in the Galician Rias (NW Iberian Peninsula).

Despite the fact that the first red tide reported on the coasts of the Iberian Peninsula was due to Lingulodinium polyedra, knowledge about their frequency and, particularly, about the environmental conditions contributing to bloom initiation is still scarce. For this reason, L. polyedra bloom episodes were observed and studied in three Galician rias during the summer season based on the 1993-2008 record database period; additionally, samples were collected in summer 2008. Proliferations of L. polyedra occurred in the rias of Ares and Barqueiro in June and August, respectively, while in the Ria of Coruña, they persisted from the end of June to early September. Red tides developed when the surface temperature reached 17 °C, with "seasonal thermal window" conditions, and when salinities were ≥30, i.e., an "optimal salinity window"; when these parameters were lower than these thresholds, cyst germination decreased. A cyst transport mechanism from sediments to the surface must also exist; this mechanism was found to be natural (tidal currents) in the ria of Barqueiro or anthropogenic (dredging) in the rias of Ares and Coruña. Surface temperatures during summer were usually favorable for cyst germination (85 to 100%) during the 1993-2008 period; however, water temperatures below 10 m depth only rarely reached the 17 °C threshold (2 to 18%). During this 16-year period, dredging activities could explain 71% (Coruña) and 44% (Ares) of the recorded bloom events. When a bloom episode developed in early summer, favorable conditions did not lead to a new red tide, probably due to the lag period required by cysts for germination. Moreover, blooms did not develop when high densities of diatoms (>1,000,000 cells·L-1) remained in the water column as a result of summer upwelling pulses occurring in specific years. The temperature-sediment disturbance pattern found in this study provides a useful tool for the prevention of eventual risks resulting from red tides of this dinoflagellate.

Ecophysiological responses of Ostreopsis towards temperature: A case study of benthic HAB facing ocean warming.

Reports of the benthic dinoflagellate Ostreopsis spp. have been increasing in the last decades, especially in temperate areas. In a context of global warming, evidences of the effects of increasing sea temperatures on its physiology and its distribution are still lacking and need to be investigated. In this study, the influence of temperature on growth, ecophysiology and toxicity was assessed for several strains of O. cf. siamensis from the Bay of Biscay (NE Atlantic) and O. cf. ovata from NW Mediterranean Sea. Cultures were acclimated to temperatures ranging from 14.5 °C to 32 °C in order to study the whole range of each strain-specific thermal niche. Acclimation was successful for temperatures ranging from 14.5 °C to 25 °C for O. cf. siamensis and from 19 °C to 32 °C for O. cf. ovata, with the highest growth rates measured at 22 °C (0.54-1.06 d-1) and 28 °C (0.52-0.75 d-1), respectively. The analysis of cellular content of pigments and lipids revealed some aspects of thermal acclimation processes in Ostreopsis cells. Specific capacities of O. cf. siamensis to cope with stress of cold temperatures were linked with the activation of a xanthophyll cycle based on diadinoxanthin. Lipids (neutral reserve lipids and polar ones) also revealed species-specific variations, with increases in cellular content noted under extreme temperature conditions. Variations in toxicity were assessed through the Artemia franciscana bioassay. For both species, a decrease in toxicity was observed when temperature dropped under the optimal temperature for growth. No PLTX-like compounds were detected in O. cf. siamensis strains. Thus, the main part of the lethal effect observed on A. franciscana was dependent on currently unknown compounds. From a multiclonal approach, this work allowed for defining specificities in the thermal niche and acclimation strategies of O. cf. siamensis and O. cf. ovata towards temperature. Potential impacts of climate change on the toxic risk associated with Ostreopsis blooms in both NW Mediterranean Sea and NE Atlantic coast is further discussed, taking into account variations in the geographic distribution, growth abilities and toxicity of each species.

Margalefidinium polykrikoides dinoflagellate blooms increase mortality of Acartia tonsa copepods.

Previous research on phytoplankton blooms has often focused on the initiation of blooms, while studies on the mechanisms underlying bloom decline and termination have been more limited. This study aimed to explore the extent of which Acartia tonsa (copepod) grazing does or does not contribute to Margalefidinium polykrikoides (dinoflagellate) bloom decline. M. polykrikoides is a prominent harmful algal bloom (HAB) species that forms dense blooms in coastal and estuarine systems around the world with known ichthyotoxic effects. Sampling occurred in the lower York River Estuary, Virginia, USA in 2021 and 2022 during two M. polykrikoides blooms. Prey removal experiments were conducted using organisms collected from the field to estimate A. tonsa ingestion rates on M. polykrikoides. While A. tonsa was capable of ingesting M. polykrikoides at low abundance, when M. polykrikoides abundance exceeded 2000 cells mL-1, A. tonsa experienced nearly 100% mortality in the 24-hour prey removal experiments. This suggests that A. tonsa likely cannot exert any top-down control on M. polykrikoides blooms, rather, at high concentrations, M. polykrikoides may act as its own grazing deterrent. Extensive M. polykrikoides blooms could therefore continue to persist due to a reduction in grazing pressure, rather than an increase. This would suggest that the decline of M. polykrikoides blooms is likely caused by another factor. As the frequency, duration, and magnitude of HABs are expected to increase in the future, these findings provide key insights to the trophic interactions that may be influencing the duration of M. polykrikoides blooms.

From micro to mesoscale: Understanding the influence of macroalgal communities on Ostreopsis Schmidt blooms.

Harmful Algal Blooms (HABs) are increasing in temperate areas, and the growth rates of benthic harmful dinoflagellates may be favoured in the context of global climate change. Benthic dinoflagellates, including species belonging to the Ostreopsis Schmidt genus, are known to develop on the surface of macroalgae and different macroalgal morphotypes and communities could host higher or lower cell abundances. The physical structure of the macroalgal substrate at the small scale (cm, microhabitat scale) and the structural complexity of the macroalgal community at the medium scale (few m, mesohabitat scale) could play a relevant role in bloom facilitation: the hypothesis that Ostreopsis species could be associated with macroalgal turfs and shrubs, structurally less complex communities than canopy-forming macroalgae, is especially under discussion and, if confirmed, could link bloom occurrence to regime shifts in temperate ecosystems. The present study, performed in two locations of the Ligurian Sea (Rochambeau, France and Vernazzola, Italy) aimed at understanding marine vegetation's role at the micro and mesohabitat scales in controlling the distribution and abundance of Ostreopsis. The abundance of the microalgal cells was quantified at different spatial scales, from cm to a few m, on different macroalgal species and communities, including artificial substrates, to tease apart the micro and mesohabitat effects. The results obtained show a high spatio-temporal variability, potentially hiding habitat-related patterns. The substrate's preferences diminish when cell abundances are very high, as in the case of Rochambeau, while in presence of moderate cell abundances as in Vernazzola or the first phases of blooms, it is possible to appreciate differences in abundances among substrates (in our study, Dictyota fasciola (Roth) Lamouroux supporting higher abundances). Our results open new research topics such as the study of blooms at a larger scale (macrohabitat) and testing different sampling methods to standardise the cells' abundances independently on the substrate.

Population dynamics of dominant dinoflagellate species in the North Sea: in situ growth rates, photosynthetic potential, and losses due to parasitism.

In the North Sea, Tripos and Dinophysis are commonly occurring mixotrophic planktonic dinoflagellate genera. In order to understand their bloom dynamics, an occurring bloom dominated by T. furca and D. norvegica was followed for several days. High cell abundances of these species were located to estimate: in situ growth rates from cell cycle analyses, depth distributions, growth rates sustained by photosynthesis, and parasite infection prevalence in all T. furca, T. fusus, D. norvegica and D. acuminata. Cell abundances were over 10000 cells L-1 for T. furca and up to 18000 cells L-1 for D. norvegica. Cells accumulated between 15-25 m depth and presented low specific in situ growth rates of 0.04-0.15 d-1 for T. furca and 0.02-0.16 d-1 for D. norvegica. Photosynthesis could sustain growth rates of 0.01-0.18 d-1 for T. furca and 0.02 to 0.14 d-1 for D. norvegica, suggesting that these species were relying mainly on photosynthesis. Parasite infections where generally low, with occasional high prevalence in D. norvegica (by Parvilucifera sp.) and T. fusus (by Amoebophrya sp.), while both parasites showed comparable prevalence in D. acuminata, which could offset in situ growth rates by parasite-induced host mortality. The restructuring effect of parasites on dinoflagellate blooms is often overlooked and this study elucidates their effect to cell abundances and their growth at the final stages of a bloom.

Effects of temperature and nutrients on growth and toxicity of Alexandrium affine from southeastern Gulf of California.

An Alexandrium affine strain (AAJQ-1) from San José Island, Gulf of California was characterized for growth and toxicology. Fivefold of f/2 + Se cultures were incubated for 34 days in a temperature gradient (21-29 °C). Aliquots were collected every third day for cell counting, toxin determination, and nutrient analyses. In this study ELISA method was used to evaluate the PSP toxin production due to the lower detection limit than the HPLC method. The highest cell density (6724 cells mL-1) and growth rate (0.22 day-1) were obtained at 27 °C and they were related to temperature in all treatments. Cell density showed negative correlation with nitrate at temperatures ≥23 °C, and with orthophosphate 27 °C, furthermore, these correlations promote the toxin production (0.05-0.45 fmol STX cell-1); beyond that nitrite at high temperature seems to promote toxin production, which has not been sufficiently documented.

A comprehensive review on the heterotrophic production of bioactive compounds by microalgae.

Bioactive compounds derived from microalgae have garnered considerable attention as valuable resources for drugs, functional foods, and cosmetics. Among these compounds, photosynthetic pigments and polyunsaturated fatty acids (PUFAs) have gained increasing interest due to their numerous beneficial properties, including anti-oxidant, anti-viral, anti-bacterial, anti-fungal, anti-inflammatory, and anti-tumor effects. Several microalgae species have been identified as rich sources of bioactive compounds, including the Chlorophyceae Dunaliella and Haematococcus, the Bacillariophyta Phaeodactylum and Nitzschia, and the dinoflagellate Crypthecodinium cohnii. However, most of the reported microalgae species primarily grow through autotrophic mechanisms, resulting in low yields and high production costs of bioactive compounds. Consequently, the utilization of heterotrophic microalgae, such as Chromochloris zofingiensis and Nitzschia laevis, has shown significant advantages in the production of astaxanthin and eicosapentaenoic acid (EPA), respectively. These heterotrophic microalgae exhibit superior capabilities in synthesizing target compounds. This comprehensive review provides a thorough examination of the heterotrophic production of bioactive compounds by microalgae. It covers key aspects, including the metabolic pathways involved, the impact of cultivation conditions, and the practical applications of these compounds. The review discusses how heterotrophic cultivation strategies can be optimized to enhance bioactive compound yields, shedding light on the potential of microalgae as a valuable resource for high-value product development.

Preliminary investigation on the causes of red tides in Qinhuangdao coastal areas in 2022.

To explore the causes of red tides in Qinhuangdao coastal water, we conducted surveys on both water quality and red tides during April to September of 2022 and analyzed the relationships between main environmental factors and red tide organisms through the factor analysis and canonical correspondence analysis. The results showed that there were eight red tides along the coast of Qinhuangdao in 2022, with a cumulative blooming area of 716.1 km2. The red tides could be divided into three kinds based on the major blooming organisms and occurrence time, Noctiluca scintillans bloom, diatom-euglena (Skeletonema costatum, Eutreptiella gymnastica, Pseudo-nitzschia spp.) bloom, and dinoflagellate (Scrippsiella trochoidea and Ceratium furca) bloom. Seasonal factor played roles mainly during July to September, while inorganic nutrients including nitrogen and phosphorus influenced the blooms mainly in April and July. The canonical correspondence analysis suggested that N. scintillans preferred low temperature, and often bloomed with high concentrations of ammonium nitrogen and dissolved inorganic phosphorus. S. costatum, E. gymnastica, and Pseudo-nitzschia spp. could tolerate broad ranges of various environmental factors, but favored high temperature and nitrogen-rich seawater. C. furca and S. trochoidea had higher survival rate and competitiveness in phosphate-poor waters. Combined the results from both analyses, we concluded that the causes for the three kinds of red tide processes in Qinhuangdao coastal areas in 2022 were different. Adequate diet algae and appropriate water temperature were important factors triggering and maintaining the N. scintillans bloom. Suitable temperature, salinity and eutrophication were the main reasons for the diatom-euglena bloom. The abundant nutrients and seawater disturbance promoted the germination of S. trochoidea cysts, while phosphorus limitation caused the blooming organism switched to C. furca and maintained the bloom hereafter.

Immediate and delayed effects of a heatwave and Prorocentrum lima ((Ehrenberg) Stein 1878) bloom on the toxin accumulation, physiology, and survival of the oyster Magallana gigas (Thunberg, 1793).

Warming could facilitate the intensification of toxic algal blooms, two important stressors for marine organisms that are predicted to co-occur more frequently in the future. We investigated the immediate and delayed effects of a heatwave and a simulated bloom (3 × 106 cells L-1) of the diarrhetic shellfish toxin (DST)-producing benthic dinoflagellate Prorocentrum lima on the survival, physiology (oxygen consumption rate, condition index, immune parameters), and toxin accumulation in the Pacific rock oyster Magallana (Crassostrea) gigas. Oysters exposed to both stressors contained higher mean DST concentrations (mean ± 1 SE: 173.3 ± 19.78 µg kg-1 soft tissue) than those exposed to P. lima bloom alone (120.4 ± 20.90 µg kg-1) and exceeded the maximum permitted levels for human consumption. Exposure to individual stressors and their combination modified the physiology of M. gigas. Oysters exposed to heatwave alone had significantly higher oxygen consumption rates (0.7 ± 0.06 mg O2 h-1 g-1) than the control (0.3 ± 0.06 mg O2 h-1 g-1). However, this was not observed in oysters exposed to both heatwave and P. lima (0.5 ± 0.06 mg O2 h-1 g-1). This alteration of the metabolic response to warming in the presence of P. lima may affect the ability of rock oysters to adapt to environmental stressors (i.e., a heatwave) to ensure survival. Immunomodulation, through changes in total hemocyte count, was observed in oysters exposed to P. lima alone and in combination with warming. Individual stressors and their combination did not influence the condition index, but one mortality was recorded in oysters exposed to both stressors. The findings of this study highlight the vulnerability of rock oysters to the predicted increased frequency of heatwaves and toxic algal blooms, and the increased likelihood of shellfish containing higher than regulatory levels of DST in warming coasts.

Quantifying Karenia brevis bloom severity and respiratory irritation impact along the shoreline of Southwest Florida.

Nearly all annual blooms of the toxic dinoflagellate Karenia brevis (K. brevis) pose a serious threat to coastal Southwest Florida. These blooms discolor water, kill fish and marine mammals, contaminate shellfish, cause mild to severe respiratory irritation, and discourage tourism and recreational activities, leading to significant health and economic impacts in affected communities. Despite these issues, we still lack standard measures suitable for assessing bloom severity or for evaluating the efficacy of modeling efforts simulating bloom initiation and intensity. In this study, historical cell count observations along the southwest Florida shoreline from 1953 to 2019 were used to develop monthly and annual bloom severity indices (BSI). Similarly, respiratory irritation observations routinely reported in Sarasota and Manatee Counties from 2006 to 2019 were used to construct a respiratory irritation index (RI). Both BSI and RI consider spatial extent and temporal evolution of the bloom, and can be updated routinely and used as objective criteria to aid future socioeconomic and scientific studies of K. brevis. These indices can also be used to help managers and decision makers both evaluate the risks along the coast during events and design systems to better respond to and mitigate bloom impacts. Before 1995, sampling was done largely in response to reports of discolored water, fish kills, or respiratory irritation. During this timeframe, lack of sampling during the fall, when blooms typically occur, generally coincided with periods of more frequent-than-usual offshore winds. Consequently, some blooms may have been undetected or under-sampled. As a result, the BSIs before 1995 were likely underestimated and cannot be viewed as accurately as those after 1995. Anomalies in the frequency of onshore wind can also largely account for the discrepancies between BSI and RI during the period from 2006 to 2019. These findings highlighted the importance of onshore wind anomalies when predicting respiratory irritation impacts along beaches.

Antibacterial Activity and Amphidinol Profiling of the Marine Dinoflagellate Amphidinium carterae (Subclade III).

Microalgae have received growing interest for their capacity to produce bioactive metabolites. This study aimed at characterising the antimicrobial potential of the marine dinoflagellate Amphidinium carterae strain LACW11, isolated from the west of Ireland. Amphidinolides have been identified as cytotoxic polyoxygenated polyketides produced by several Amphidinium species. Phylogenetic inference assigned our strain to Amphidinium carterae subclade III, along with isolates interspersed in different geographic regions. A two-stage extraction and fractionation process of the biomass was carried out. Extracts obtained after stage-1 were tested for bioactivity against bacterial ATCC strains of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa. The stage-2 solid phase extraction provided 16 fractions, which were tested against S. aureus and E. faecalis. Fractions I, J and K yielded minimum inhibitory concentrations between 16 µg/mL and 256 µg/mL for both Gram-positive. A targeted metabolomic approach using UHPLC-HRMS/MS analysis applied on fractions G to J evidenced the presence of amphidinol type compounds AM-A, AM-B, AM-22 and a new derivative dehydroAM-A, with characteristic masses of m/z 1361, 1463, 1667 and 1343, respectively. Combining the results of the biological assays with the targeted metabolomic approach, we could conclude that AM-A and the new derivative dehydroAM-A are responsible for the detected antimicrobial bioactivity.

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. The success of dinoflagellates in dominating the plankton community is undoubtedly pertinent to their remarkable adaptive strategies, characteristic of resting cyst production and broad tolerance to stresses of temperature and others. Therefore, this study was conducted to examine the putative roles of Hsp90 in the acclimation to temperature stress and life stage alterations of dinoflagellates. Firstly, we isolated the full-length cDNA of an Hsp90 gene (StHsp90) via RACE from the cosmopolitan HAB species Scrippsiella trochoidea and tracked its transcriptions in response to varied scenarios via real-time qPCR. The results indicated that StHsp90 displayed significant mRNA augment patterns, escalating during 180-min treatments, when the cells were exposed to elevated and lowered temperatures. Secondly, we observed prominently elevated StHsp90 transcriptions in the cysts that were stored at the cold and dark conditions compared to those in newly formed resting cysts and vegetative cells. Finally, and perhaps most importantly, we identified 29 entries of Hsp90-encoding genes with complete coding regions from a dinoflagellate-specific environmental cDNA library generated from marine sediment assemblages. The observed active transcription of these genes in sediment-buried resting cysts was fully supported by the qPCR results for the cold-stored resting cysts of S. trochoidea. Hsp90s expressions in both laboratory-raised and field-collected cysts collectively highlighted the possible involvement and engagement of Hsp90 chaperones in the resting stage persistence of dinoflagellates.

Salinity Affects Saxitoxins (STXs) Toxicity in the Dinoflagellate Alexandrium pacificum, with Low Transcription of SXT-Biosynthesis Genes sxtA4 and sxtG.

Salinity is an important factor for regulating metabolic processes in aquatic organisms; however, its effects on toxicity and STX biosynthesis gene responses in dinoflagellates require further elucidation. Herein, we evaluated the physiological responses, toxin production, and expression levels of two STX synthesis core genes, sxtA4 and sxtG, in the dinoflagellate Alexandrium pacificum Alex05 under different salinities (20, 25, 30, 35, and 40 psu). Optimal growth was observed at 30 psu (0.12 cell division/d), but cell growth significantly decreased at 20 psu and was irregular at 25 and 40 psu. The cell size increased at lower salinities, with the highest size of 31.5 µm at 20 psu. STXs eq was highest (35.8 fmol/cell) in the exponential phase at 30 psu. GTX4 and C2 were predominant at that time but were replaced by GTX1 and NeoSTX in the stationary phase. However, sxtA4 and sxtG mRNAs were induced, and their patterns were similar in all tested conditions. PCA showed that gene transcriptional levels were not correlated with toxin contents and salinity. These results suggest that A. pacificum may produce the highest amount of toxins at optimal salinity, but sxtA4 and sxtG may be only minimally affected by salinity, even under high salinity stress.

Evidence for massive and recurrent toxic blooms of Alexandrium catenella in the Alaskan Arctic.

Among the organisms that spread into and flourish in Arctic waters with rising temperatures and sea ice loss are toxic algae, a group of harmful algal bloom species that produce potent biotoxins. Alexandrium catenella, a cyst-forming dinoflagellate that causes paralytic shellfish poisoning worldwide, has been a significant threat to human health in southeastern Alaska for centuries. It is known to be transported into Arctic regions in waters transiting northward through the Bering Strait, yet there is little recognition of this organism as a human health concern north of the Strait. Here, we describe an exceptionally large A. catenella benthic cyst bed and hydrographic conditions across the Chukchi Sea that support germination and development of recurrent, locally originating and self-seeding blooms. Two prominent cyst accumulation zones result from deposition promoted by weak circulation. Cyst concentrations are among the highest reported globally for this species, and the cyst bed is at least 6× larger in area than any other. These extraordinary accumulations are attributed to repeated inputs from advected southern blooms and to localized cyst formation and deposition. Over the past two decades, warming has likely increased the magnitude of the germination flux twofold and advanced the timing of cell inoculation into the euphotic zone by 20 d. Conditions are also now favorable for bloom development in surface waters. The region is poised to support annually recurrent A. catenella blooms that are massive in scale, posing a significant and worrisome threat to public and ecosystem health in Alaskan Arctic communities where economies are subsistence based.

Interactive effects of light, CO2 and temperature on growth and resource partitioning by the mixotrophic dinoflagellate, Karlodinium veneficum.

There is little information on the impacts of climate change on resource partitioning for mixotrophic phytoplankton. Here, we investigated the hypothesis that light interacts with temperature and CO2 to affect changes in growth and cellular carbon and nitrogen content of the mixotrophic dinoflagellate, Karlodinium veneficum, with increasing cellular carbon and nitrogen content under low light conditions and increased growth under high light conditions. Using a multifactorial design, the interactive effects of light, temperature and CO2 were investigated on K. veneficum at ambient temperature and CO2 levels (25°C, 375 ppm), high temperature (30°C, 375 ppm CO2), high CO2 (30°C, 750 ppm CO2), or a combination of both high temperature and CO2 (30°C, 750 ppm CO2) at low light intensities (LL: 70 µmol photons m-2 s-2) and light-saturated conditions (HL: 140 µmol photons m-2 s-2). Results revealed significant interactions between light and temperature for all parameters. Growth rates were not significantly different among LL treatments, but increased significantly with temperature or a combination of elevated temperature and CO2 under HL compared to ambient conditions. Particulate carbon and nitrogen content increased in response to temperature or a combination of elevated temperature and CO2 under LL conditions, but significantly decreased in HL cultures exposed to elevated temperature and/or CO2 compared to ambient conditions at HL. Significant increases in C:N ratios were observed only in the combined treatment under LL, suggesting a synergistic effect of temperature and CO2 on carbon assimilation, while increases in C:N under HL were driven only by an increase in CO2. Results indicate light-driven variations in growth and nutrient acquisition strategies for K. veneficum that may benefit this species under anticipated climate change conditions (elevated light, temperature and pCO2) while also affecting trophic transfer efficiency during blooms of this species.

The Bloom-Forming Dinoflagellate Karenia mikimotoi Adopts Different Growth Modes When Exposed to Short or Long Period of Seawater Acidification.

Impacts of ocean acidification (OA) on noncalcifying organisms and the possibly responsible mechanism have aroused great research interests with the intensification of global warming. The present study focused on a noxious, noncalcifying, bloom-forming dinoflagellate, Karenia mikimotoi (K. mikimotoi), and its variation of growth patterns exposed to different periods of seawater acidification with stressing gradients was discussed. The dinoflagellates under short-time acidifying stress (2d) with different levels of CO2 presented significant growth inhibition (p < 0.05). The cell cycle was obviously inhibited at S phase, and the photosynthetic carbon fixation was also greatly suppressed (p < 0.05). Apoptosis was observed and the apoptotic rate increased with the increment of pCO2. Similar tendencies were observed in the key components of mitochondrial apoptotic pathway (the mitochondrial membrane potential (MMP), Caspase-3 and -9, and Bax/Bcl-2 ratio). However, under prolonged stressing time (8 d and 15 d), the growth of dinoflagellates was recovered or even stimulated, the photosynthetic carbon fixation was significantly increased (p < 0.05), the cell cycle of division presented little difference with those in the control, and no apoptosis was observed (p > 0.05). Besides, acidification adjusted by HCl addition and CO2 enrichment resulted in different growth performances, while the latter had a more negative impact. The results of present study indicated that (1) the short-time exposure to acidified seawater led to reduced growth performance via inducing apoptosis, blocking of cell cycle, and the alteration in photosynthetic carbon fixation. (2) K. mikimotoi had undergone adaptive changes under long-term exposure to CO2 induced seawater acidification. This further demonstrated that K. mikimotoi has strong adaptability in the face of seawater acidification, and this may be one of the reasons for the frequent outbreak of red tide. (3) Ions that dissociated by the dissolved CO2, instead of H+ itself, were more important for the impacts induced by the acidification. This work thus provides a new perspective and a possible explanation for the dominance of K. mikimotoi during the occurrence of HABs.

Nutrient Deficiency and an Algicidal Bacterium Improved the Lipid Profiles of a Novel Promising Oleaginous Dinoflagellate, Prorocentrum donghaiense, for Biodiesel Production.

The lipid production potentials of 8 microalgal species were investigated. Among these 8 species, the best strain was a dominant bloom-causing dinoflagellate, Prorocentrum donghaiense; this species had a lipid content of 49.32% ± 1.99% and exhibited a lipid productivity of 95.47 ± 0.99 mg liter-1 day-1, which was 2-fold higher than the corresponding values obtained for the oleaginous microalgae Nannochloropsis gaditana and Phaeodactylum tricornutum. P. donghaiense, which is enriched in C16:0 and C22:6, is appropriate for commercial docosahexaenoic acid (DHA) production. Nitrogen or phosphorus stress markedly induced lipid accumulation to levels surpassing 75% of the dry weight, increased the C18:0 and C17:1 contents, and decreased the C18:5 and C22:6 contents, and these effects resulted in decreases in the unsaturated fatty acid levels and changes in the lipid properties of P. donghaiense such that the species met the biodiesel specification standards. Compared with the results obtained under N-deficient conditions, the enhancement in the activity of alkaline phosphatase of P. donghaiense observed under P-deficient conditions partly alleviated the adverse effects on the photosynthetic system exerted by P deficiency to induce the production of more carbohydrates for lipogenesis. The supernatant of the algicidal bacterium Paracoccus sp. strain Y42 culture lysed P. donghaiense without decreasing its lipid content, which resulted in facilitation of the downstream oil extraction process and energy savings through the lysis of algal cells. The Y42 supernatant treatment improved the lipid profiles of algal cells by increasing their C16:0, C18:0, and C18:1 contents and decreasing their C18:5 and C22:6 contents, which is favorable for biodiesel production. IMPORTANCE This study demonstrates the high potential of Prorocentrum donghaiense, a dominant bloom-causing dinoflagellate, for lipid production. Compared with previously studied oleaginous microalgae, P. donghaiense exhibit greater potential for practical application due to its higher biomass and lipid contents. Nutrient deficiency and the algicidal bacterium Paracoccus sp. strain Y42 improved the suitability of the lipid profile of P. donghaiense for biodiesel production. Furthermore, Paracoccus sp. Y42 effectively lysed algal cells, which facilitates the downstream oil extraction process for biodiesel production and results in energy savings through the lysing of algal cells. This study provides a more promising candidate for the production of docosahexaenoic acid (DHA) for human nutritional products and of microalgal biofuel as well as a more cost-effective method for breaking algal cells. The high lipid productivity of P. donghaiense and algal cell lysis by algicidal bacteria contribute to reductions in the production cost of microalgal oil.

Low Temperature and Cold Stress Significantly Increase Saxitoxins (STXs) and Expression of STX Biosynthesis Genes sxtA4 and sxtG in the Dinoflagellate Alexandrium catenella.

Toxic dinoflagellate Alexandrium spp. produce saxitoxins (STXs), whose biosynthesis pathway is affected by temperature. However, the link between the regulation of the relevant genes and STXs' accumulation and temperature is insufficiently understood. In the present study, we evaluated the effects of temperature on cellular STXs and the expression of two core STX biosynthesis genes (sxtA4 and sxtG) in the toxic dinoflagellate Alexandrium catenella Alex03 isolated from Korean waters. We analyzed the growth rate, toxin profiles, and gene responses in cells exposed to different temperatures, including long-term adaptation (12, 16, and 20 °C) and cold and heat stresses. Temperature significantly affected the growth of A. catenella, with optimal growth (0.49 division/day) at 16 °C and the largest cell size (30.5 µm) at 12 °C. High concentration of STXs eq were detected in cells cultured at 16 °C (86.3 fmol/cell) and exposed to cold stress at 20→12 °C (96.6 fmol/cell) compared to those at 20 °C and exposed to heat stress. Quantitative real-time PCR (qRT-PCR) revealed significant gene expression changes of sxtA4 in cells cultured at 16 °C (1.8-fold) and cold shock at 20→16 °C (9.9-fold). In addition, sxtG was significantly induced in cells exposed to cold shocks (20→16 °C; 19.5-fold) and heat stress (12→20 °C; 25.6-fold). Principal component analysis (PCA) revealed that low temperature (12 and 16 °C) and cold stress were positively related with STXs' production and gene expression levels. These results suggest that temperature may affect the toxicity and regulation of STX biosynthesis genes in dinoflagellates.

Effects of Polystyrene Microplastics on Growth and Toxin Production of Alexandrium pacificum.

Microplastics (MP) widely distributed in aquatic environments have adverse effects on aquatic organisms. Currently, the impact of MP on toxigenic red tide microalgae is poorly understood. In this study, the strain of Alexandrium pacificum ATHK, typically producing paralytic shellfish toxins (PST), was selected as the target. Effects of 1 and 0.1 µm polystyrene MP with three concentration gradients (5 mg L-1, 25 mg L-1 and 100 mg L-1) on the growth, chlorophyll a (Chl a), photosynthetic activity (Fv/Fm) and PST production of ATHK were explored. Results showed that the high concentration (100 mg L-1) of 1 µm and 0.1 µm MP significantly inhibited the growth of ATHK, and the inhibition depended on the size and concentration of MP. Contents of Chl a showed an increase with various degrees after MP exposure in all cases. The photosynthesis indicator Fv/Fm of ATHK was significantly inhibited in the first 11 days, then gradually returned to the level of control group at day 13, and finally was gradually inhibited in the 1 µm MP treatments, and promotion or inhibition to some degree also occurred at different periods after exposure to 0.1 µm MP. Overall, both particle sizes of MP at 5 and 25 mg L-1 had no significant effect on cell toxin quota, and the high concentration 100 mg L-1 significantly promoted the PST biosynthesis on the day 7, 11 and 15. No significant difference occurred in the cell toxin quota and the total toxin content in all treatments at the end of the experiment (day 21). All MP treatments did not change the toxin profiles of ATHK, nor did the relative molar percentage of main PST components. The growth of ATHK, Chl a content, Fv/Fm and toxin production were not affected by MP shading. This is the first report on the effects of MP on the PST-producing microalgae, which will improve the understanding of the adverse impact of MP on the growth and toxin production of A. pacificum.