Regulation of photosynthetic and hemolytic activity of Phaeocystis globosa under different light spectra.

Phaeocystis globosa frequently proliferates in eutrophic waters and forms ichthyotoxic algal blooms that cause massive fish mortalities in marine ecosystems. One of the ichthyotoxic metabolites was identified as the glycolipid-like hemolytic toxin, reported to be initiated under light conditions. However, the association between hemolytic activity (HA) and photosynthesis of P. globosa remained unclear. Light spectra (blue, red, green, and white) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) were selected as the stressors to stimulate the hemolytic response of P. globosa in relation to the light and dark photosynthesis reaction. Hemolytic activity in P. globosa was sensitive to the light spectrum as it decreased from 93% to nearly undetectable (1.6%) within 10 min of transfer from red (630 nm) to green light (520 nm). This indicates that the vertical transformation of P. globosa from deep to surface waters (dominated by green light and all light spectra, respectively) may drive the hemolytic response in coastal waters. However, regulation of photosynthetic electron transfer in the light reaction of P. globosa was excluded by the evidence of inconsistent response of HA to photosynthetic activity. The biosynthesis of HA may interfere with the pathway of photopigments diadinoxanthin or fucoxanthin, and the metabolism of three- and five-carbon sugars (GAP and Ru5P, respectively), which ultimately lead to changes in the alga's hemolytic carbohydrate metabolism.

Hemolytic Activity in Relation to the Photosynthetic System in Chattonella marina and Chattonella ovata.

Chattonella species, C. marina and C. ovata, are harmful raphidophycean flagellates known to have hemolytic effects on many marine organisms and resulting in massive ecological damage worldwide. However, knowledge of the toxigenic mechanism of these ichthyotoxic flagellates is still limited. Light was reported to be responsible for the hemolytic activity (HA) of Chattonella species. Therefore, the response of photoprotective, photosynthetic accessory pigments, the photosystem II (PSII) electron transport chain, as well as HA were investigated in non-axenic C. marina and C. ovata cultures under variable environmental conditions (light, iron and addition of photosynthetic inhibitors). HA and hydrogen peroxide (H2O2) were quantified using erythrocytes and pHPA assay. Results confirmed that% HA of Chattonella was initiated by light, but was not always elicited during cell division. Exponential growth of C. marina and C. ovata under the light over 100 µmol m-2 s-1 or iron-sufficient conditions elicited high hemolytic activity. Inhibitors of PSII reduced the HA of C. marina, but had no effect on C. ovata. The toxicological response indicated that HA in Chattonella was not associated with the photoprotective system, i.e., xanthophyll cycle and regulation of reactive oxygen species, nor the PSII electron transport chain, but most likely occurred during energy transport through the light-harvesting antenna pigments. A positive, highly significant relationship between HA and chlorophyll (chl) biosynthesis pigments, especially chl c2 and chl a, in both species, indicated that hemolytic toxin may be generated during electron/energy transfer through the chl c2 biosynthesis pathway.

Phosphorus-Induced Lipid Class Alteration Revealed by Lipidomic and Transcriptomic Profiling in Oleaginous Microalga Nannochloropsis sp. PJ12.

Phytoplankton are primary producers in the marine ecosystem, where phosphorus is often a limiting factor of their growth. Hence, they have evolved strategies to recycle phosphorus by replacing membrane phospholipids with phosphorus-free lipids. However, mechanisms for replacement of lipid classes remain poorly understood. To improve our understanding, we performed the lipidomic and transcriptomic profiling analyses of an oleaginous marine microalga Nannochloropsis sp. PJ12 in response to phosphorus depletion (PD) and replenishing. In this study, by using (liquid chromatography couple with tandem mass spectrometry) LC-MS/MS-based lipidomic analysis, we show that membrane phospholipid levels are significantly reduced upon PD, while phosphorus-free betaine lipid levels are increased. However, levels of phosphorus-free photosynthetic galactolipid and sulfolipid are not increased upon PD, consistent with the reduced photosynthetic activity. RNA-seq-based transcriptomic analysis indicates that enzymes involved in phospholipid recycling and phosphorus-free lipid synthesis are upregulated, supporting the lipidomic analysis. Furthermore, enzymes involved in FASII (type II fatty acid synthesis) elongation cycle upon PD are transcriptionally downregulated. EPA (eicosapentaenoic acid) level decrease upon PD is revealed by both GC-MS (gas chromatography coupled with mass spectrometry) and LC-MS/MS-based lipidomic analyses. PD-induced alteration is reversed after phosphorus replenishing. Taken together, our results suggest that the alteration of lipid classes upon environmental change of phosphorus is a result of remodeling rather than de novo synthesis in Nannochloropsis sp. PJ12.

Characterization and comparison of toxin-producing isolates of Dinophysis acuminata from New England and Canada.

Following the identification of the first toxic isolate of Dinophysis acuminata from the northwestern Atlantic, we conducted detailed investigations into the morphology, phylogeny, physiology, and toxigenicity of three isolates from three sites within the northeastern U.S./Canada region: Eel Pond and Martha's Vineyard, Massachusetts, and the Bay of Fundy. Another isolate, collected from the Gulf of Mexico, was grown under the same light, temperature, and prey conditions for comparison. Despite observed phenotypic heterogeneity, morphometrics and molecular evidence classified the three northwestern Atlantic isolates as D. acuminata Claparède & Lachmann, whereas the isolate from the Gulf of Mexico was morphologically identified as D. cf. ovum. Physiological and toxin analyses supported these classifications, with the three northwestern Atlantic isolates being more similar to each other with respect to growth rate, toxin profile, and diarrhetic shellfish poisoning (DSP) toxin content (okadaic acid + dinophysistoxin 1/cell) than they were to the isolate from the Gulf of Mexico, which had toxin profiles similar to those published for D. cf. ovum F. Schütt. The DSP toxin content, 0.01-1.8 pg okadaic acid (OA) + dinophysistoxin (DTX1) per cell, of the three northwestern Atlantic isolates was low relative to other D. acuminata strains from elsewhere in the world, consistent with the relative scarcity of shellfish harvesting closures due to DSP toxins in the northeastern U.S. and Canada. If this pattern is repeated with the analyses of more geographically and temporally dispersed isolates from the region, it would appear that the risk of significant DSP toxin outbreaks in the northwestern Atlantic is low to moderate. Finally, the morphological, physiological, and toxicological variability within D. acuminata may reflect spatial (and/or temporal) population structure, and suggests that sub-specific resolution may be helpful in characterizing bloom dynamics and predicting toxicity.

Genomic reconstruction to improve bioethanol and ergosterol production of industrial yeast Saccharomyces cerevisiae.

Baker's yeast (Saccharomyces cerevisiae) is the common yeast used in the fields of bread making, brewing, and bioethanol production. Growth rate, stress tolerance, ethanol titer, and byproducts yields are some of the most important agronomic traits of S. cerevisiae for industrial applications. Here, we developed a novel method of constructing S. cerevisiae strains for co-producing bioethanol and ergosterol. The genome of an industrial S. cerevisiae strain, ZTW1, was first reconstructed through treatment with an antimitotic drug followed by sporulation and hybridization. A total of 140 mutants were selected for ethanol fermentation testing, and a significant positive correlation between ergosterol content and ethanol production was observed. The highest performing mutant, ZG27, produced 7.9 % more ethanol and 43.2 % more ergosterol than ZTW1 at the end of fermentation. Chromosomal karyotyping and proteome analysis of ZG27 and ZTW1 suggested that this breeding strategy caused large-scale genome structural variations and global gene expression diversities in the mutants. Genetic manipulation further demonstrated that the altered expression activity of some genes (such as ERG1, ERG9, and ERG11) involved in ergosterol synthesis partly explained the trait improvement in ZG27.

Using solid phase adsorption toxin tracking and extended local similarity analysis to monitor lipophilic shellfish toxins in a mussel culture ranch in the Yangtze River Estuary.

Harmful algal blooms (HABs) and their associated phycotoxins are increasing globally, posing great threats to local coastal ecosystems and human health. Nutrients have been carried by the freshwater Yangtze River and have entered the estuary, which was reported to be a biodiversity-rich but HAB-frequent region. Here, in situ solid phase adsorption toxin tracking (SPATT) was used to monitor lipophilic shellfish toxins (LSTs) in seawaters, and extended local similarity analysis (eLSA) was conducted to trace the temporal and special regions of those LSTs in a one-year trail in a mussel culture ranch in the Yangtze River Estuary. Nine analogs of LSTs, including okadaic acid (OA), dinophysistoxin-1 (DTX1), yessotoxin (YTX), homoyessotoxin (homoYTX), 45-OH-homoYTX, pectenotoxin-2 (PTX2), 7-epi-PTX2 seco acid (7-epi-PTX2sa), gymnodimine (GYM) and azaspiracids-3 (AZA3), were detected in seawater (SPATT) or rope farmed mussels. The concentrations of OA + DTX1 and homoYTX in mussels were positively correlated with those in SPATT samplers (Pearson test, p < 0.05), indicating that SPATT (with resin HP20) would be a good monitoring tool and potential indicator for OA + DTX1 and homoYTX in mussel Mytilus coruscus. The eLSA results indicated that late summer and early autumn were the most phycotoxin-contaminated seasons in the Yangtze River Estuary. OA + DTX1, homoYTX, PTX2 and GYM were most likely driven by the local growing HAB species in spring and summer, while Yangtze River diluted water may impact the accumulation of HAB species, causing potential phycotoxin contamination in the Yangtze River Estuary in autumn and winter. Together, the results showed that the mussel harvesting season, late summer and early autumn, would be the season with the greatest phycotoxin risk and would be the most contaminated by local growing toxic algae. Routine monitoring sites should be set up close to the local seawaters.

Prokaryotic and eukaryotic microbial communities associated with coral species have high host specificity in the South China Sea.

Reef-building corals are well known for their obligate association with Symbiodiniaceae, and an array of other microbes, including bacteria, fungi, and symbiotic algae (i.e., total microbiome), which together form the coral holobiont. The total microbiome plays an intricate part in maintaining the homeostasis of the coral holobiont and is closely associated with host health. However, the composition of the coral associated microbiome and interaction between its different members remains elusive because few analyses have bridged taxonomically disparate groups. This research gaps have prevented a holistic understanding of the total microbiome. Thus, to simultaneously characterize the bacterial, fungal and symbiotic algal communities associated with different coral species, and explore the relationship between these symbionts and coral health, healthy and bleached tissues from four coral species, Acropora muricata, Galaxea fascicularis, Platygyra daedalea, and Pavona explanulata, were collected from the Xisha Islands of the South China Sea. Using high throughput sequencing, a high degree of host-specificity was observed among bacterial, fungal, and algal groups across coral species. There were no obvious changes in the microbial community structure of apparently healthy and bleached corals, but host bleaching allowed colonization of the holobionts by diverse opportunistic microbes, resulting in a significant elevation in the α-diversity of microbial communities. In addition, co-occurrence analysis of the coral microbiota also identified more complex microbial interactions in bleached corals than in healthy ones. In summary, this study characterized the structure of coral-associated microbiomes across four coral species, and systematically studied microbiome differences between healthy and bleached corals. The findings improve our understanding of the heterogeneity of symbiotic microorganisms and the impact of coral's physiological status on its associated microbial communities composition.

Indole-3-acetic acid as a cross-talking molecule in algal-bacterial interactions and a potential driving force in algal bloom formation.

Most signaling molecules are involved in inter-or intra-species communication, and signaling involving cross-kingdom cell-to-cell communication is limited. Howerver, algae and bacteria exchange nutrients and information in a range of interactions in marine environments. Multiple signaling molecules exist between algae and bacteria, including quorum-sensing molecules, nitric oxide, and volatile organic compounds. Recently, indole-3-acetic acid (IAA), an auxin hormone that is a well-studied signaling molecule in terrestrial ecosystems, was found to act as a cue in cross-kingdom communication between algae and bacteria in aquatic environments. To increase understanding of the roles of IAA in the phycosphere, the latest evidence regarding the ecological functions of IAA in cross-kingdom communication between algae and bacteria has been compiled in this review. The pathways of IAA biosynthesis, effects of IAA on algal growth & reproduction, and potential mechanisms at phenotypic and molecular levels are summarized. It is proposed that IAA is an important molecule regulating algal-bacterial interactions and acts as an invisible driving force in the formation of algal blooms.

Bacterial Communities Vary from Different Scleractinian Coral Species and between Bleached and Non-Bleached Corals.

Bleaching is one of the most relevant factors implicated in the integrity of coral reef ecosystems, with the increasing frequency and intensity of damaging events representing a serious threat to reef biodiversity. Here, we analyzed changes in coral-associated bacteria from three types of non-bleached and bleached scleractinian corals (Acropora digitifera, Galaxea fascicularis, and Porites pukoensis) in Hainan Luhuitou peninsula coastal areas. The community structure of symbiotic bacteria differed significantly among the three apparently healthy corals. The bleached corals had higher bacterial alpha diversity and some specific bacteria genera, including Ruegeria, Methyloceanibacter, Filomicrobium, Halioglobus, Rubripirellula, Rhodopirellula, Silicimonas, Blastopirellula, Sva0996 marine group, Woeseia, and unclassified_c_Gammaproteobacteria, were consistently increased in bleached groups. Network analysis revealed significantly different degrees of modularity between bleached and non-bleached groups at the bacterial genus level, and a higher proportion of links was dominated by positive co-occurrences. Functional prediction analysis illustrated that coral-associated bacteria remained relatively consistent in the bleached and non-bleached groups. Structure equation modeling revealed that the bacterial community diversity and function were directly influenced by host and environment factors. These findings suggested that coral-associated bacterial responses to bleaching occur in a host-dependent manner, informing novel strategies for restoring coral and aiding adaption to bleaching stress. IMPORTANCE Accumulating evidence indicates that coral-associated bacteria play an important role in the health of holobionts. However, the variability of the symbiotic bacterial community structure among coral species with different coral health statuses remains largely unknown. Here, we investigated three apparent non-bleached (healthy) and bleached coral species (sampled in situ), involving related symbiotic bacterial profiles, including composition, alpha diversity, network relationship, and potential function. Structural equation modeling analysis was used to analyze the relationship between coral status and abiotic and biotic factors. The bacterial community structure of different groups was shown to exhibit host-specific traits. Both host and environmental impacts had primary effects on coral-associated microbial communities. Future studies are needed to identify the mechanisms that mediate divergent microbial consortia.

Integrated metagenomic and metaproteomic analyses reveal bacterial micro-ecological mechanisms in coral bleaching.

IMPORTANCE: Coral reefs worldwide are facing rapid decline due to coral bleaching. However, knowledge of the physiological characteristics and molecular mechanisms of coral symbionts respond to stress is scarce. Here, metagenomic and metaproteomic approaches were utilized to shed light on the changes in the composition and functions of coral symbiotic bacteria during coral bleaching. The results demonstrated that coral bleaching significantly affected the composition of symbionts, with bacterial communities dominating in bleached corals. Through differential analyses of gene and protein expression, it becomes evident that symbionts experience functional disturbances in response to heat stress. These disturbances result in abnormal energy metabolism, which could potentially compromise the health and resilience of the symbionts. Furthermore, our findings highlighted the highly diverse microbial communities of coral symbionts, with beneficial bacteria providing critical services to corals in stress responses and pathogenic bacteria driving coral bleaching. This study provides comprehensive insights into the complex response mechanisms of coral symbionts under heat stress from the micro-ecological perspective and offers fundamental data for future monitoring of coral health.

A rapid fluorescence approach on differentiation of typical dinoflagellate of East China Sea.

Detecting the marine phytoplankton by the means of absorption or fluorescence spectra were successfully deployed in the past decades, however, the differentiation are mainly limited in levels of class, such as bacillariophytas, dinophytas, raphidophytes, chlorophytes, cyanobacteria, etc. which are characterized by their specific composition of photosynthetic pigments. To further differentiate the typical dinoflagellate Prorocentrum donghaiense, Amphidinium carterae, Scrippsiella trochoidea, Karenia mikimotoi out of the common diatom Skeletonema costatum and haptonema Phaeocystis globosa at East China Sea, a rapid 3D-fluorescence method equipped with CHEMTAX model were conducted. Initial fluorescence excitation spectra of each algal species (under variable environmental conditions) were captured by 3D-fluorometer first. Then fingerprints of each algae were characterized by ten-point discrete excitation spectrum with the excitation wavelengths of 405, 420, 435, 470, 490, 505, 535, 555, 570 and 590 nm, which closely reflecting the difference of photosynthetic pigments. By equipping with CHEMTAX model, the standard spectra and norm spectra were constructed for FS-CHEMTAX (Fluorescence spectra-CHEMTAX) model to further identify the algal species and estimate the cell density. The developed method performed a better way of identifying the toxic species Amphidinium carterae, Phaeocystis globosa, and Karenia mikimotoi out of the non-toxic ones, with the identification accuracy rates of 83.3%, 90% and 100%, in monocultures, and 77.8%, 90% and 100%, in the bi-mixed cultures, respectively. Meanwhile, the detection limits for the three toxic species were found as low as 250, 1,400 and 120 cells/mL. The concentrations estimated are in good agreement with the microscopic cell counts for all the algae groups (correlation coefficients (R2) exceed 0.8). The relative error of predict concentration was lowest for small cells, i.e., Phaeocystis globosa (10.0%) and Amphidinium carterae (21.1%), but the highest for big cells, i.e. Karenia mikimotoi (41.8%) when the target algae become the dominant species. The overall concentration detection error was no more than one order of magnitude, indicating that this method could provide an important technical support for monitoring the related harmful algal blooms.

Progress on the investigation and monitoring of marine phycotoxins in China.

Marine phycotoxins associated with paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), ciguatera fish poisoning (CFP), tetrodotoxin (TTX), palytoxin (PLTX) and neurotoxin ß-N-methylamino-L-alanine (BMAA) have been investigated and routinely monitored along the coast of China. The mouse bioassay for monitoring of marine toxins has been progressively replaced by the enzyme-linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which led to the discovery of many new hydrophilic and lipophilic marine toxins. PSP toxins have been detected in the whole of coastal waters of China, where they are the most serious marine toxins. PSP events in the Northern Yellow Sea, the Bohai Sea and the East China Sea are a cause of severe public health concern. Okadaic acid (OA) and dinophysistoxin-1 (DTX1), which are major toxin components associated with DSP, were mainly found in coastal waters of Zhejiang and Fujian provinces, and other lipophilic toxins, such as pectenotoxins, yessotoxins, azaspiracids, cyclic imines, and dinophysistoxin-2(DTX2) were detected in bivalves, seawater, sediment, as well as phytoplankton. CFP events mainly occurred in the South China Sea, while TTX events mainly occurred in Jiangsu, Zhejiang and Fujian provinces. Microalgae that produce PLTX and BMAA were found in the phytoplankton community along the coastal waters of China.

CoT-XNet: contextual transformer with Xception network for diabetic retinopathy grading.

Objective.Diabetic retinopathy (DR) grading is primarily performed by assessing fundus images. Many types of lesions, such as microaneurysms, hemorrhages, and soft exudates, are available simultaneously in a single image. However, their sizes may be small, making it difficult to differentiate adjacent DR grades even using deep convolutional neural networks (CNNs). Recently, a vision transformer has shown comparable or even superior performance to CNNs, and it also learns different visual representations from CNNs. Inspired by this finding, we propose a two-path contextual transformer with Xception network (CoT-XNet) to improve the accuracy of DR grading.Approach.The representations learned by CoT through one path and those by the Xception network through another path are concatenated before the fully connected layer. Meanwhile, the dedicated pre-processing, data resampling, and test time augmentation strategies are implemented. The performance of CoT-XNet is evaluated in the publicly available datasets of DDR, APTOS2019, and EyePACS, which include over 50 000 images. Ablation experiments and comprehensive comparisons with various state-of-the-art (SOTA) models have also been performed.Main results.Our proposed CoT-XNet shows better performance than available SOTA models, and the accuracy and Kappa are 83.10% and 0.8496, 84.18% and 0.9000 and 84.10% and 0.7684 respectively, in the three datasets (listed above). Class activation maps of CoT and Xception networks are different and complementary in most images.Significance.By concatenating the different visual representations learned by CoT and Xception networks, CoT-XNet can accurately grade DR from fundus images and present good generalizability. CoT-XNet will promote the application of artificial intelligence-based systems in the DR screening of large-scale populations.

Sulfamethoxazole induced systematic and tissue-specific antioxidant defense in marine mussels (Mytilus galloprovincialis): Implication of antibiotic's ecotoxicity.

Sulfamethoxazole (SMX), recognized as emerging pollutant, has been frequently detected in aquatic environment. However, effects induced by SMX and the underneath mechanism on non-target aquatic organisms, marine mussels (Mytilus galloprovincialis), are still largely unknown. In present study, marine mussels were exposed to SMX (nominal concentrations 0.5, 50 and 500 µg/L) for 6 days, followed by 6 days depuration and responses of antioxidant defenses, e.g. superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST), etc., at transcriptional, translational and functional levels were evaluated in two vital tissues, gills and digestive glands. Results showed SMX can be accumulated in mussels while the bio-accumulative ability was low under the experimental condition. A systemic but not completely synchronous antioxidant defense at different levels upon SMX exposure. The transcriptional alteration was more sensitive and had the potential to be used as early warning of SMX induced ecotoxicity. Complementary function of antioxidant enzymes with specific alteration of metabolism related gene (gst) suggested that further researches should focused on SMX metabolism and SMX induced effects simultaneously. Significant tissue-specific antioxidant responses were discovered and gills showed earlier and quicker reacting ability than digestive glands, which was closely related to the functional diversity and different thresholds of xenobiotics allowance.

Effect of allelochemicals on photosynthetic and antioxidant defense system of Ulva prolifera.

Ulva prolifera is a macroalgae that forms massive blooms, negatively impacting natural communities, aquaculture operations and recreation. The effects of the natural products, eugenol, ß-myrcene, citral and nonanoic acid on the growth rate, antioxidative defense system and photosynthesis of Ulva prolifera were investigated as a possible control strategy for this harmful taxon. Negative effects on growth were observed with all four chemicals, due to the excessive production of reactive oxygen species and oxidative damage to the thalli. However, the response of U. prolifera under the four chemicals stress was different at the cellular level. ß-myrcene, the most effective compound in terms of growth inhibition, induced oxidative stress as shown by the damage of total antioxidant capacity (T-AOC) and the downregulation of the glutathione-ascorbate (GSH-ASA) cycle which inhibited the antioxidative system. This chemical also inhibited photosynthesis and photoprotection mechanisms in U. prolifera, resulting in growth limitation. In contrast, U. prolifera was less affected by the second tested chemical, eugenol, and showed no significant change on photosynthetic efficiency in the presence of the chemical. The inhibition effects of the third and fourth tested chemicals, nonanoic acid and citralon, on growth and on the antioxidant defense system in U. prolifera were inferior. These results provide a potential avenue for controlling green tides in the future.

Cell cycle regulation of the mixotrophic dinoflagellate Dinophysis acuminata: Growth, photosynthetic efficiency and toxin production.

The mixotrophic dinoflagellate Dinophysis acuminata is a widely distributed diarrhetic shellfish poisoning (DSP) producer. Toxin variability of Dinophysis spp. has been well studied, but little is known of the manner in which toxin production is regulated throughout the cell cycle in these species, in part due to their mixotrophic characteristics. Therefore, an experiment was conducted to investigate cell cycle regulation of growth, photosynthetic efficiency, and toxin production in D. acuminata. First, a three-step synchronization approach, termed "starvation-feeding-dark", was used to achieve a high degree of synchrony of Dinophysis cells by starving the cells for 2 weeks, feeding them once, and then placing them in darkness for 58 h. The synchronized cells started DNA synthesis (S phase) 10 h after being released into the light, initiated G2 growth stage eight hours later, and completed mitosis (M phase) 2 h before lights were turned on. The toxin content of three dominant toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-2 (PTX2), followed a common pattern of increasing in G1 phase, decreasing on entry into the S phase, then increasing again in S phase and decreasing in M phase during the diel cell cycle. Specific toxin production rates were positive throughout the G1 and S phases, but negative during the transition from G1 to S phase and late in M phase, the latter reflecting cell division. All toxins were initially induced by the light and positively correlated with the percentage of cells in S phase, indicating that biosynthesis of Dinophysis toxins might be under circadian regulation and be most active during DNA synthesis.

Prey Lysate Enhances Growth and Toxin Production in an Isolate of Dinophysis acuminata.

The physiological and toxicological characteristics of Dinophysis acuminata have been increasingly studied in an attempt to better understand and predict diarrhetic shellfish poisoning (DSP) events worldwide. Recent work has identified prey quantity, organic nitrogen, and ammonium as likely contributors to increased Dinophysis growth rates and/or toxicity. Further research is now needed to better understand the interplay between these factors, for example, how inorganic and organic compounds interact with prey and a variety of Dinophysis species and/or strains. In this study, the exudate of ciliate prey and cryptophytes were investigated for an ability to support D. acuminata growth and toxin production in the presence and absence of prey, i.e., during mixotrophic and phototrophic growth respectively. A series of culturing experiments demonstrated that the addition of ciliate lysate led to faster dinoflagellate growth rates (0.25 ± 0.002/d) in predator-prey co-incubations than in treatments containing (1) similar levels of prey but without lysate (0.21 ± 0.003/d), (2) ciliate lysate but no live prey (0.12 ± 0.004/d), or (3) monocultures of D. acuminata without ciliate lysate or live prey (0.01 ± 0.007/d). The addition of ciliate lysate to co-incubations also resulted in maximum toxin quotas and extracellular concentrations of okadaic acid (OA, 0.11 ± 0.01 pg/cell; 1.37 ± 0.10 ng/mL) and dinophysistoxin-1 (DTX1, 0.20 ± 0.02 pg/cell; 1.27 ± 0.10 ng/mL), and significantly greater total DSP toxin concentrations (intracellular + extracellular). Pectenotoxin-2 values, intracellular or extracellular, did not show a clear trend across the treatments. The addition of cryptophyte lysate or whole cells, however, did not support dinoflagellate cell division. Together these data demonstrate that while certain growth was observed when only lysate was added, the benefits to Dinophysis were maximized when ciliate lysate was added with the ciliate inoculum (i.e., during mixotrophic growth). Extrapolating to the field, these culturing studies suggest that the presence of ciliate exudate during co-occurring dinoflagellate-ciliate blooms may indirectly and directly exacerbate D. acuminata abundance and toxigenicity. More research is required, however, to understand what direct or indirect mechanisms control the predator-prey dynamic and what component(s) of ciliate lysate are being utilized by the dinoflagellate or other organisms (e.g., ciliate or bacteria) in the culture if predictive capabilities are to be developed and management strategies created.

Analysis of multiple pesticide residues in polyphenol-rich agricultural products by UPLC-MS/MS using a modified QuEChERS extraction and dilution method.

An efficient method for the rapid extraction and clean-up of 20 pesticide residues from polyphenol-rich agricultural samples (tea, apple, broccoli, and shallot) for analysis by UPLC-MS/MS was developed. Following extraction, PVPP was used to precipitate polyphenols, supernatant was diluted to minimize matrix effects. Homogenized samples were vortexed in acetonitrile prior to cleanup with a combination of PVPP (150 mg), PSA (50 mg) and GCB (10 mg). Supernatant (1 mL) was filtered and diluted 10-fold before analysis. In 4 agricultural products that usually produce high interference, the matrix effects were overcome for all pesticides expect in green and oolong tea for acephate, omethoate, dinotefuran and nitenpyram. Mean recoveries ranged from 73% to 106%, and RSD ≦ 13%. Limits of quantification ranged from 0.01 to 0.02 mg kg-1. PVPP as one of excellent QuEChERS material combined with dilution was verified as a promising method for multiple pesticide residues analysis in complex matrices.

Impact of Dinophysis acuminata Feeding Mesodinium rubrum on Nutrient Dynamics and Bacterial Composition in a Microcosm.

The development of Dinophysis populations, producers of diarrhetic shellfish toxins, has been attributed to both abiotic (e.g., water column stratification) and biotic (prey availability) factors. An important process to consider is mixotrophy of the Dinophysis species, which is an intensive feeding of the Mesodinium species for nutrients and a benefit from kleptochloroplasts. During the feeding process, the nutritional status in the environment changes due to the preference of Mesodinium and/or Dinophysis for different nutrients, prey cell debris generated by sloppy feeding, and their degradation by micro-organisms changes. However, there is little knowledge about the role of the bacterial community during the co-occurrence of Mesodinium and Dinophysis and how they directly or indirectly interact with the mixotrophs. In this study, laboratory experiments were performed to characterize the environmental changes including those of the prey present, the bacterial communities, and the ambient dissolved nutrients during the co-occurrence of Mesodinium rubrum and Dinophysis acuminata. The results showed that, during the incubation of the ciliate prey Mesodinium with its predator Dinophysis, available dissolved nitrogen significantly shifted from nitrate to ammonium especially when the population of M. rubrum decayed. Growth phases of Dinophysis and Mesodinium greatly affected the structure and composition of the bacterial community. These changes could be mainly explained by both the changes of the nutrient status and the activity of Dinophysis cells. Dinophysis feeding activity also accelerated the decline of M. rubrum and contamination of cultures with okadaic acid, dinophysistoxin-1, and pectenotoxin-2, but their influence on the prokaryotic communities was limited to the rare taxa (<0.1%) fraction. This suggests that the interaction between D. acuminata and bacteria is species-specific and takes place intracellularly or in the phycosphere. Moreover, a majority of the dominant bacterial taxa in our cultures may also exhibit a metabolic flexibility and, thus, be unaffected taxonomically by changes within the Mesodinium-Dinophysis culture system.

Effect of ciliate strain, size, and nutritional content on the growth and toxicity of mixotrophic Dinophysis acuminata.

Previous studies indicate differences in bloom magnitude and toxicity between regional populations, and more recently, between geographical isolates of Dinophysis acuminata; however, the factors driving differences in toxicity/toxigenicity between regions/strains have not yet been fully elucidated. Here, the roles of prey strains (i.e., geographical isolates) and their associated attributes (i.e., biovolume and nutritional content) were investigated in the context of growth and production of toxins as a possible explanation for regional variation in toxicity of D. acuminata. The mixotrophic dinoflagellate, D. acuminata, isolated from NE North America (MA, U.S.) was offered a matrix of prey lines in a full factorial design, 1 × 2 × 3; one dinoflagellate strain was fed one of two ciliates, Mesodinium rubrum, isolated from coastal regions of Japan or Spain, which were grown on one of three cryptophytes (Teleaulax/Geminigera clade) isolated from Japan, Spain, or the northeastern USA. Additionally, predator: prey ratios were manipulated to explore effects of the prey's total biovolume on Dinophysis growth or toxin production. These studies revealed that the biovolume and nutritional status of the two ciliates, and less so the cryptophytes, impacted the growth, ingestion rate, and maximum biomass of D. acuminata. The predator's consumption of the larger, more nutritious prey resulted in an elevated growth rate, greater biomass, and increased toxin quotas and total toxin per mL of culture. Grazing on the smaller, less nutritious prey, led to fewer cells in the culture but relatively more toxin exuded from the cells on per cell basis. Once the predator: prey ratios were altered so that an equal biovolume of each ciliate was delivered, the effect of ciliate size was lost, suggesting the predator can compensate for reduced nutrition in the smaller prey item by increasing grazing. While significant ciliate-induced effects were observed on growth and toxin metrics, no major shifts in toxin profile or intracellular toxin quotas were observed that could explain the large regional variations observed between geographical populations of this species.