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.

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.

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.

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.

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.

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.

Photosynthesis acclimation under severely fluctuating light conditions allows faster growth of diatoms compared with dinoflagellates.

BACKGROUND: Diatoms contribute 20% of the global primary production and are adaptable in dynamic environments. Diatoms always bloom earlier in the annual phytoplankton succession instead of dinoflagellates. However, how diatoms acclimate to a dynamic environment, especially under changing light conditions, remains unclear. RESULTS: We compared the growth and photosynthesis under fluctuating light conditions of red tide diatom Skeletonema costatum, red tide dinoflagellate Amphidinium carterae, Prorocentrum donghaiense, Karenia mikimotoi, model diatom Phaeodactylum tricornutum, Thalassiosira pseudonana and model dinoflagellate Dinophycae Symbiodinium. Diatoms grew faster and maintained a consistently higher level of photosynthesis. Diatoms were sensitive to the specific inhibitor of Proton Gradient Regulation 5 (PGR5) depending photosynthetic electron flow, which is a crucial mechanism to protect their photosynthetic apparatus under fluctuating light. In contrast, the dinoflagellates were not sensitive to this inhibitor. Therefore, we investigate how PGR5 functions under light fluctuations in the model diatom P. tricornutum by knocking down and overexpressing PGR5. Overexpression of PGR5 reduced the photosystem I acceptor side limitation (Y (NA)) and increased growth rate under severely fluctuating light in contrast to the knockdown of PGR5. CONCLUSION: Diatoms acclimatize to fluctuating light conditions better than dinoflagellates. PGR5 in diatoms can regulate their photosynthetic electron flow and accelerate their growth under severe light fluctuation, supporting fast biomass accumulation under dynamic environments in pioneer blooms.

Micro-size plankton abundance and assemblages in the western North Pacific Subtropical Gyre under microscopic observation.

While primary productivity in the oligotrophic North Pacific Subtropical Gyre (NPSG) is changing, the micro-size plankton community has not been evaluated in the last 4 decades, prompting a re-evaluation. We collected samples over three years (2016-2018) from depths of 10 to 200 m (n = 127), and the micro-size plankton were identified and counted to understand the heterogeneity of micro-size plankton community structure. The assemblages were consistent to the those of 4 decades ago. Dinophyceae (dinoflagellates) were the most numerically abundant, followed by Cryptophyceae and Bacillariophyceae (diatoms). The other micro-size plankton classes (Cyanophyceae, Haptophyceae, Dictyochophyceae, Euglenophyceae, and Prasinophyceae) were not always detected, whereas only Trichodesmium spp. was counted in the Cyanophyceae. Other unidentified autotrophic and heterotrophic flagellates were also significantly present, and their numeric abundance was higher than or at the same level as was that of the Dinophyceae. In the Dinophyceae, Gymnodiniaceae and Peridiniales were abundant. The chlorophyll a concentration and these class-level assemblages suggested micro-size plankton is not a major primary producer in this area. We applied generalized additive models (GAMs) and principal coordination analyses (PCoAs) to evaluate the habitats of every plankton group and the heterogeneity of the assemblages. The GAMs suggested that every classified plankton abundance showed a similar response to salinity, and we observed differences in habitats in terms of temperature and nitrate concentrations. Based on the PCoAs, we observed unique communities at the 200 m depth layer compared with those at the other sampling layers. The site scores of PCoAs indicated that the micro-size plankton assemblages are most heterogeneous at the 10 m depth layer. At such depth, diazotrophic Cyanophyceae (Trichodesmium spp.) are abundant, particularly in less-saline water. Therefore, nitrogen fixation may contribute to the heterogeneity in the abundance and assemblages in the western NPSG.

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.

Dynamic bacterial community response to Akashiwo sanguinea (Dinophyceae) bloom in indoor marine microcosms.

We investigated the dynamics of the bacterial composition and metabolic function within Akashiwo sanguinea bloom using a 100-L indoor microcosm and metagenomic next-generation sequencing. We found that the bacterial community was classified into three groups at 54% similarity. Group I was associated with "during the A. sanguinea bloom stage" and mainly consisted of Alphaproteobacteria, Flavobacteriia and Gammaproteobacteria. Meanwhile, groups II and III were associated with the "late bloom/decline stage to post-bloom stage" with decreased Flavobacteriia and Gammaproteobacteria in these stages. Upon the termination of the A. sanguinea bloom, the concentrations of inorganic nutrients (particularly PO43-, NH4+ and dissolved organic carbon) increased rapidly and then decreased. From the network analysis, we found that the A. sanguinea node is associated with certain bacteria. After the bloom, the specific increases in NH4+ and PO43- nodes are associated with other bacterial taxa. The changes in the functional groups of the bacterial community from chemoheterotrophy to nitrogen association metabolisms were consistent with the environmental impacts during and after A. sanguinea bloom. Consequently, certain bacterial communities and the environments dynamically changed during and after harmful algal blooms and a rapid turnover within the bacterial community and their function can respond to ecological interactions.

Molecular Identification and Toxin Analysis of Alexandrium spp. in the Beibu Gulf: First Report of Toxic A. tamiyavanichii in Chinese Coastal Waters.

The frequency of harmful algal blooms (HABs) has increased in China in recent years. Information about harmful dinoflagellates and paralytic shellfish toxins (PSTs) is still limited in China, especially in the Beibu Gulf, where PSTs in shellfish have exceeded food safety guidelines on multiple occasions. To explore the nature of the threat from PSTs in the region, eight Alexandrium strains were isolated from waters of the Beibu Gulf and examined using phylogenetic analyses of large subunit (LSU) rDNA, small subunit (SSU) rDNA, and internal transcribed spacer (ITS) sequences. Their toxin composition profiles were also determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). All eight strains clustered in the phylogenetic tree with A. pseudogonyaulax, A. affine, and A. tamiyavanichii from other locations, forming three well-resolved groups. The intraspecific genetic distances of the three Alexandrium species were significantly smaller than interspecific genetic distances for Alexandrium species. Beibu Gulf isolates were therefore classified as A. pseudogonyaulax, A. affine, and A. tamiyavanichii. No PSTs were identified in A. pseudogonyaulax, but low levels of gonyautoxins (GTXs) 1 to 5, and saxitoxin (STX) were detected in A. tamiyavanichii (a total of 4.60 fmol/cell). The extremely low level of toxicity is inconsistent with PST detection above regulatory levels on multiple occasions within the Beibu Gulf, suggesting that higher toxicity strains may occur in those waters, but were unsampled. Other explanations including biotransformation of PSTs in shellfish and the presence of other PST-producing algae are also suggested. Understanding the toxicity and phylogeny of Alexandrium species provides foundational data for the protection of public health in the Beibu Gulf region and the mitigation of HAB events.

Combined characterization of a new member of Marivita cryptomonadis strain LZ-15-2 isolated from cultivable phycosphere microbiota of highly toxic HAB dinoflagellate Alexandrium catenella LZT09.

During our conveying the microbial structures of phycosphere microbiota (PM) derived from diverse marine harmful algal bloom (HAB) dinoflagellates, a new rod-sharped, white-colored cultivable bacterial strain, designated as LZ-15-2, was isolated from the PM of highly toxic Alexandrium catenella LZT09. Phylogenetic analysis of 16S rRNA gene sequence indicated that strain LZ-15-2 belonged to the genus Marivita within the family Rhodobacteraceae, and demonstrated the highest gene similarity of 99.2% to M. cryptomonadis CL-SK44T, and less than 98.65% with other type strains of Marivita. Phylogenomic calculations on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the new isolate and M. cryptomonadis CL-SK44T were 99.86% and 99.88%, respectively. Genomic comparison of strain LZ-15-2 with available genomes of Marivita species further verified its taxonomic position within the genus of Marivita. Moreover, comparative genomics analysis showed a proximal similarity of strain LZ-15-2 with M. cryptomonadis CL-SK44T, and it also revealed an open pan-genome status based on constructed gene accumulation curves among Marivita members with 9,361 and 1,712 genes for the pan- and core-genome analysis, respectively. Based on combined polyphasic taxonomic characteristics, strain LZ-15-2 represents a new member of M. cryptomonadis, and proposed as a potential candidate for further exploration of the detailed mechanisms governing the dynamic cross-kingdom algae-bacteria interactions (ABI) between PM and their algal host LZT09.

Bacteria enhance the production of extracellular polymeric substances by the green dinoflagellate Lepidodinium chlorophorum.

High biomasses of the marine dinoflagellate Lepidodinium chlorophorum cause green seawater discolorations along Southern Brittany (NE Atlantic, France). The viscosity associated to these phenomena has been related to problems in oyster cultivation. The harmful effect of L. chlorophorum might originate from the secretion of Extracellular Polymeric Substances (EPS). To understand whether the EPS are produced by L. chlorophorum or its associated bacteria, or if they are a product of their interaction, batch cultures were performed under non-axenic and pseudo-axenic conditions for three strains. Maximum dinoflagellate cell abundances were observed in pseudo-axenic cultures. The non-sinking fraction of polymers (Soluble Extracellular Polymers, SEP), mainly composed of proteins and the exopolysaccharide sulphated galactan, slightly increased in pseudo-axenic cultures. The amount of Transparent Exopolymer Particles (TEP) per cell increased under non-axenic conditions. Despite the high concentrations of Particulate Organic Carbon (POC) measured, viscosity did not vary. These results suggest that the L. chlorophorum-bacteria interaction could have a detrimental consequence on the dinoflagellate, translating in a negative effect on L. chlorophorum growth, as well as EPS overproduction by the dinoflagellate, at concentrations that should not affect seawater viscosity.

Lobster Supply Chains Are Not at Risk from Paralytic Shellfish Toxin Accumulation during Wet Storage.

Lobster species can accumulate paralytic shellfish toxins (PST) in their hepatopancreas following the consumption of toxic prey. The Southern Rock Lobster (SRL), Jasus edwardsii, industry in Tasmania, Australia, and New Zealand, collectively valued at AUD 365 M, actively manages PST risk based on toxin monitoring of lobsters in coastal waters. The SRL supply chain predominantly provides live lobsters, which includes wet holding in fishing vessels, sea-cages, or processing facilities for periods of up to several months. Survival, quality, and safety of this largely exported high-value product is a major consideration for the industry. In a controlled experiment, SRL were exposed to highly toxic cultures of Alexandrium catenella at field relevant concentrations (2 × 105 cells L-1) in an experimental aquaculture facility over a period of 21 days. While significant PST accumulation in the lobster hepatopancreas has been reported in parallel experiments feeding lobsters with toxic mussels, no PST toxin accumulated in this experiment from exposure to toxic algal cells, and no negative impact on lobster health was observed as assessed via a wide range of behavioural, immunological, and physiological measures. We conclude that there is no risk of PST accumulation, nor risk to survival or quality at the point of consumption through exposure to toxic algal cells.

Establish axenic cultures of armored and unarmored marine dinoflagellate species using density separation, antibacterial treatments and stepwise dilution selection.

Academic research on dinoflagellate, the primary causative agent of harmful algal blooms (HABs), is often hindered by the coexistence with bacteria in laboratory cultures. The development of axenic dinoflagellate cultures is challenging and no universally accepted method suit for different algal species. In this study, we demonstrated a promising approach combined density gradient centrifugation, antibiotic treatment, and serial dilution to generate axenic cultures of Karenia mikimotoi (KMHK). Density gradient centrifugation and antibiotic treatments reduced the bacterial population from 5.79 ± 0.22 log10 CFU/mL to 1.13 ± 0.07 log10 CFU/mL. The treated KMHK cells were rendered axenic through serial dilution, and algal cells in different dilutions with the absence of unculturable bacteria were isolated. Axenicity was verified through bacterial (16S) and fungal internal transcribed spacer (ITS) sequencing and DAPI epifluorescence microscopy. Axenic KMHK culture regrew from 1000 to 9408 cells/mL in 7 days, comparable with a normal culture. The established methodology was validated with other dinoflagellate, Alexandrium tamarense (AT6) and successfully obtained the axenic culture. The axenic status of both cultures was maintained more than 30 generations without antibiotics. This efficient, straightforward and inexpensive approach suits for both armored and unarmored dinoflagellate species.