Toxic dinoflagellate Karenia mikimotoi induces apoptosis in Neuro-2a cells through an oxidative stress-mediated mitochondrial pathway.

The dinoflagellate Karenia mikimotoi is a toxic bloom-forming species that threatens aquaculture and public health worldwide. Previous studies showed that K. mikimotoi induces neurotoxicity; however, the underlying mechanism is poorly understood. In this study, three neural cell lines were used to investigate the potential neurotoxicity of K. mikimotoi. The tested cells were exposed to a ruptured cell solution (RCS) of K. mikimotoi at different concentrations (0.5 × 105, 1.0 × 105, 2.0 × 105, 4.0 × 105, and 6 × 105 cells mL-1) for 24 h, and the RCS decreased cell viabilities and promoted Neuro-2a (N2A) cell apoptosis in a dose-dependent manner. The underlying mechanism was further investigated in N2A cells. At the biochemical level, the RCS stimulated reactive oxygen species (ROS) and malondialdehyde (MDA) formation, decreased SOD activity, and reduced mitochondrial membrane potential (MMP). At the gene level, the moderate RCS treatment (2.0 × 105 cells mL-1) upregulated antioxidant response genes (e.g., nrf-2, HO-1, NQO-1, and cat) to alleviate RCS-induced oxidative stress, while the high RCS treatment (4.0 × 105 cells mL-1) downregulated these genes, thereby aggravating oxidative stress. Meanwhile, apoptosis-related genes (e.g., p53, caspase 3, and bax2) were significantly upregulated and the anti-apoptotic gene bcl2 was suppressed after RCS treatment. Western blotting results for Caspase 3, Bax2 and Bcl2 were consistent with the mRNA trends. These results revealed that K. mikimotoi RCS can induce neural cell apoptosis via the oxidative stress-mediated mitochondrial pathway, providing novel insights into the neurotoxicity of K. mikimotoi.

Whole-genome sequencing-based analysis of antimicrobial resistance, virulence factors, and genetic diversity in Yersinia isolated in Wenzhou, China 2020.

Yersinia spp. vary significantly in their ability to cause diseases that threaten public health. Their pathogenicity is frequently associated with increasing antimicrobial resistance (AMR) and various virulence factors. The aim of the study was to investigate the AMR genes, virulence factors, and genetic diversity of Yersinia strains isolated from meats and fish in Wenzhou in 2020 by using whole-genome sequencing (WGS). A total of 50 isolates were collected. The phylogenetic relationships among the Yersinia species were also analyzed using multilocus sequence typing (MLST), core genome multi-locus sequence typing (cgMLST), and single nucleotide polymorphism (SNP) analysis. According to the results, all the strains could be classified into five species, with most isolated from beef, followed by poultry, pork, and fish. AMR genes were identified in 23 strains. And the qnrD1 genes were all located in the Col3M plasmid. Virulence genes, such as yaxA, ystB, pla, and yplA, were also found in the 15 Y. enterocolitica strains. And this study also found the presence of icm/dot type IVB-related genes in one Yersinia massiliensis isolate. MLST analysis identified 43 sequence types (STs), 19 of which were newly detected in Yersinia. Moreover, cgMLST analysis revealed that no dense genotype clusters were formed (cgMLST 5341, 5344, 5346-5350, 5353-5390). Instead, the strains appeared to be dispersed over large distances, except when multiple isolates shared the same ST. Isolates Y4 and Y26 were closely related to strains originating from South Korea and Denmark. This study showed considerable diversity in Yersinia spp. isolated from local areas (Wenzhou City). The data generated in our study may enrich the molecular traceability database of Yersinia and provide a basis for the development of more effective antipathogen control strategies.

Transcriptome analysis of two bloom-forming Prorocentrum species reveals physiological changes related to light and temperature.

Temperature and light substantially influence red tide succession. However, it remains unclear whether the molecular mechanisms differ among species. In this study, we measured the variation in the physiological parameters of growth and pigments and transcriptional levels of two bloom-forming dinoflagellates, namely Prorocentrum micans and P. cordatum. This was undertaken in four treatments that represented two factorial temperature combinations (LT: 20 °C, HT: 28 °C) and light conditions (LL: 50 µmol photons m-2 s-1, HL: 400 µmol photons m-2 s-1) for 7-day batch culture. Growth under high temperature and high light (HTHL) was the fastest, while growth under high temperature and low light (HTLL) was the slowest. The pigments (chlorophyll a and carotenoids) decreased significantly in all high light (HL) treatments, but not in high temperature (HT) treatments. HL alleviated the low light-caused photolimitation and enhanced the growth of both species at low temperatures. However, HT inhibited the growth of both species by inducing oxidative stress under low light conditions. HL mitigated the HT-induced stress on growth in both species by upregulating photosynthesis, antioxidase activity, protein folding, and degradation. The cells of P. micans were more sensitive to HT and HL than those of P. cordatum. This study deepens our understanding of the species-specific mechanism of dinoflagellates at the transcriptomic level, adapting to the future ocean changes including higher solar radiation and higher temperatures in the upper mixed layer.

Dual characteristics of Bellamya aeruginosa encountering Microcystis aeruginosa: Algal control and toxin depuration.

The benthic gastropods Bellamya aeruginosa (B. aeruginosa) is ubiquitous in freshwater in China and neighboring countries with great edible value. It has been recognized as a potential manipulator to control harmful algal blooms due to its filtration on algal cells. In this study, the control effect of B. aeruginosa on toxic and non-toxic Microcystis aeruginosa (M. aeruginosa), and the accumulation and depuration of microcystins (MCs) in the snail were systematically explored. Results indicated that although toxic M. aeruginosa could protect itself via producing MCs, the introduction of B. aeruginosa could still effectively inhibit the algae with cell density below 1 × 106 cells/mL. Hepatopancreas was the primary target of MCs in all tissues of B. aeruginosa, presenting a maximum of 3089.60 ng/g DW when exposed to toxic M. aeruginosa of 1.0 × 107 cells/mL. The enrichment of MCs in other tissues following the order of digestive tract > gonad > mantle > muscle. Interestingly, snail could again excrete previously enriched MCs when transferred to non-toxic M. aeruginosa, giving rise to over 80% reduction of MCs in the body. After depuration, the estimated daily intake (EDI) of free MCs in intact individuals and the edible parts of B. aeruginosa were both lower than the tolerable daily intake (TDI). These results implicated that B. aeruginosa could control low density of M. aeruginosa in spring. Particularly, the snail could be perfectly safe to consume by purifying for a while after using as manipulator.

Cadmium chloride exposure impairs the growth and behavior of Drosophila via ferroptosis.

Cadmium (Cd) is a widely distributed toxic heavy metal that enters the environment via anthropogenic mobilization and accumulates in plants and animals, causing metabolic abnormalities even mortality. Although the toxic effects and stress damage of cadmium have been investigated extensively over the past few decades, research on its ability to trigger ferroptosis, growth retardation, and behavioral abnormalities is insufficient. As a result, the effects of CdCl2 exposure on growth and development, activity and sleep, and ferroptosis in this study were examined in fruit fly (Drosophila melanogaster). When exposed to 0.5 mM CdCl2, the entire growth period from larvae to adults was prolonged, and the rates of pupation and eclosion were decreased. Additionally, CdCl2 exposure resulted in a decrease in body weight and individual size of fruit fly and high lethality rate. Moreover, CdCl2 exposure altered fruit fly behavior, including decreased activity and increased sleep duration, particularly in females. Ferrostatin-1 (Fer-1) is a potent selective ferroptosis inhibitor that effectively slows lipid hydroperoxide accumulation to rescue body size reduction and restore activity and sleep in CdCl2-exposed female flies. CdCl2 exposure could induce ferroptosis in fruit fly mechanistically, as evidenced by inhibition of Nrf2 signaling pathway, accumulation of lipid peroxidation, impairment of GPX4 antioxidant system, and upregulation of iron metabolism. Our findings suggest that Cd exposure triggers ferroptosis, which leads to growth retardation and behavioral disorders in fruit fly.

Molecular Characterization and Phylogenetic Analysis of the 2019 Dengue Outbreak in Wenzhou, China.

In 2019, a dengue outbreak occurred with 290 confirmed cases in Wenzhou, a coastal city in southeast China. To identify the origin of the dengue virus (DENV) from this outbreak, viral RNA was extracted from four serum samples and sequenced for whole genome analysis. Then, phylogenetic analysis, gene mutation, secondary structure prediction, selection pressure analysis, and recombination analysis were performed. DENV strains Cam-03 and Cam-11 were isolated from patients traveling from Cambodia, while ZJWZ-18 and ZJWZ-62 strains were isolated from local patients without a record of traveling abroad. The whole genome sequence of all four strains was 10,735 nucleotides long. Phylogenetic tree analysis showed that the four strains belonged to genotype 1 of DENV-1, but the local Wenzhou strains and imported strains clustered in different branches. ZJWZ-18 and ZJWZ-62 were closely related to strain MF033254-Singapore-2016, and Cam-03 and Cam-11 were closely related to strain AB608788-China : Taiwan-1994. A comparison of the coding regions between the local strains and the DENV-1 standard strain (EU848545-Hawaii-1944) showed 82 amino acid mutations between the two strains. A total of 55 amino acid mutations were found between the coding regions of the local and imported strains. The overall secondary structure of the 3' UTR of the local strains had changed: apparent changes in the head and tail position were observed when compared to DENV-1 standard strain. Furthermore, selection pressure analysis and recombination detection using the 4 isolates and 41 reference strains showed two credible positive selection sites and eight credible recombination events, which warrant further studies. This study may enhance the understanding of viral replication, infection, evolution, virulence, and pathogenicity of DENV.

Antagonistic and synergistic effects of warming and microplastics on microalgae: Case study of the red tide species Prorocentrum donghaiense.

Bibliometric network analysis has revealed that the widespread distribution of microplastics (MPs) has detrimental effects on marine organisms; however, the combined effects of MPs and climate change (e.g., warming) is not well understood. In this study, Prorocentrum donghaiense, a typical red tide species in the East China Sea, was exposed to different MP concentrations (0, 1, 5, and 10 mg L-1) and temperatures (16, 22, and 28 °C) for 7 days to investigate the combined effects of MPs and simulated ocean warming by measuring different physiological parameters, such as cell growth, pigment contents (chlorophyll a and carotenoid), relative electron transfer rate (rETR), reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA), and adenosine triphosphate (ATP). The results demonstrated that MPs significantly decreased cell growth, pigment contents, and rETRmax, but increased the MDA, ROS, and SOD levels for all MP treatments at low temperature (16 °C). However, high temperatures (22 and 28 °C) increased the pigment contents and rETRmax, but decreased the SOD and MDA levels. Positive and negative effects of high temperatures (22 or 28 °C) were observed at low (1 and 5 mg L-1) and high MP (10 mg L-1) concentrations, respectively, indicating the antagonistic and synergistic effects of combined warming and MP pollution. These results imply that the effects of MPs on microalgae will likely not be substantial in future warming scenarios if MP concentrations are controlled at a certain level. These findings expand the current knowledge of microalgae in response to increasing MP pollution in future warming scenarios.

Environmentally relevant concentrations of triclosan exposure promote the horizontal transfer of antibiotic resistance genes mediated by Edwardsiella piscicida.

Aquaculture pathogen and antibiotic resistance genes (ARGs) co-occur in the aquatic environment. Accumulated evidence suggests that aquaculture pathogens can facilitate the horizontal transfer of plasmid-mediated ARGs. However, the role of Edwardsiella piscicida (E. piscicida) in ARG dissemination is still not fully understood. In addition, the potential impact of triclosan (TCS) on the spread of ARGs mediated by E. piscicida is still unknown, so a mating model system was established to investigate the transfer process of ARGs. The results showed that E. piscicida disseminated ARGs on RP4 by horizontal gene transfer (HGT). Furthermore, TCS exposure promoted this process. The conjugative transfer frequencies were enhanced approximately 1.2-1.4-fold by TCS at concentrations from 2 to 20 µg/L, when compared with the control. TCS promoted the HGT of ARGs by stimulating reactive oxygen species (ROS) production, increasing cell membrane permeability, and altering expressions of conjugative transfer-associated genes. Together, the results suggested that aquaculture pathogens spread ARGs and that the emerging contaminant TCS enhanced the transfer of ARGs between bacteria.

The responses of harmful dinoflagellate Karenia mikimotoi to simulated ocean acidification at the transcriptional level.

The HAB-forming, toxic dinoflagellate Karenia mikimotoi, previously found to benefit from ocean acidification (OA), was cultivated to investigate its transcriptional response to simulated OA for 30 generations. Batch cultures were grown under two CO2 concentrations, 450 (control) and 1100 (simulated OA) µatm, and physiological parameters [growth, pigments, catalase (CAT), glutathione reductase (GR), and superoxide dismutase (SOD) activity], as well as transcriptomes (obtained via RNA-seq), were compared. Chlorophyll a (Chl a) and carotenoid (Caro) contents, as well as CAT and GR activities, were significantly increased under OA conditions. Transcriptomic analysis revealed 2,490 differentially expressed unigenes in response to OA, which comprised 1.54% of all unigenes. A total of 1,121 unigenes were upregulated, and 1,369 unigenes were downregulated in OA compared to control conditions. The downregulated expression of bicarbonate transporter and carbonic anhydrase genes was a landmark of OA acclimation. Key genes involved in energy metabolism, e.g., photosynthesis, tricarboxylic acid cycle, oxidative phosphorylation, and nitrogen metabolism, were highly upregulated under OA, contributing to increases in the Chl a (55.05%) and Caro (28.37%). The enhanced antioxidant enzyme activities (i.e. CAT, GR) and upregulated genes (i.e. glutathione peroxidase, ascorbate peroxidase, heat shock protein, 20S proteasome, aldehyde dehydrogenase, and apolipoprotein) benefit cells against the potential lower pH stress condition under OA. In addition, the downregulation of four genes associated with motility suggested that the preserved energy could further boost growth. In conclusion, the present study suggests that K. mikimotoi exhibits efficient gene expression regulation for the utilization of energy and resistance to OA-induced stress. Taken together, K. mikimotoi appeared as a tolerant species in response to OA. Thus, more extensive algal blooms that threaten marine organisms are likely in the future. These findings expand current knowledge on the gene expression of HAB-forming species in response to future OA.

Microplastics impacts in seven flagellate microalgae: Role of size and cell wall.

The toxicity of microplastic particles (MPs) on aquatic environments has been widely reported; however, their effects on protists are still contradictory. For example, it is unclear if cell size and cell wall have a role in shaping the response of flagellates to MPs. In this study, seven marine flagellated microalgae (six Dinoflagellates and one Raphidophyceae) were incubated with 10 mg L-1 MPs (polystyrene plastic micro-spheres, 1 µm diameter) to address the above question by measuring different response variables, i.e., growth, optimal photochemical efficiency (Fv/Fm), chlorophyll-a (Chl-a) content, superoxide dismutase (SOD) activity, and cell morphology. The effect of MPs on growth and Fv/Fm showed species-specificity effects. Maximum and minimum MPs-induced inhibitions were detected in Karenia mikimotoi (76.43%) and Akashiwo sanguinea (10.16%), respectively, while the rest of the species showed intermediate responses. The presence of MPs was associated with an average reduction of Chl-a content in most cases and with a higher superoxide dismutase activity in all cases. Seven species were classified into two groups by the variation of Chl-a under MPs treatment. One group (Prorocentrum minimum and Karenia mikimotoi) showed increased Chl-a, while the other (P. donghaiense, P. micans, Alexandrium tamarense, Akashiwo sanguinea, Heterosigma akashiwo) showed decreased Chl-a content. The MPs-induced growth inhibition was negatively correlated with cell size in the latter group. SEM images further indicated that MPs-induced malformation in the smaller cells (e.g., P. donghaiense and K. mikimotoi) was more severe than the bigger cells (e.g., A. sanguinea and P. micans), probably due to a relatively higher ratio of the cell surface to cell volume in the former. These results implicate that the effect of MPs on marine flagellated microalgae was related to the cell size among most species but not cell wall. Thus plastic pollution may have size-dependent effects on phytoplankton in future scenarios.

Toxic effects of the dinoflagellate Karenia mikimotoi on zebrafish (Danio rerio) larval behavior.

Karenia mikimotoi is a toxic dinoflagellate that forms harmful blooms in coastal waters, threatening aquaculture worldwide. However, we do not know whether K. mikimotoi has a neurotoxic effect on aquatic animal behavior. Thus, this study investigated potential K. mikimotoi neurotoxicity in zebrafish larvae. Cells of K. mikimotoi were collected at the mid-exponential phase from a batch culture to prepare ruptured cell solutions (RCS). At 6 h post-fertilization (hpf), zebrafish embryos were exposed to different RCS concentrations (0, 102, 103, 104, and 2.5 × 104 cells mL-1). After 120 hpf, treated larvae were collected to analyze locomotor behavior; activities of acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT); and expression of genes related to neurodevelopment. We found that RCS did not affect survival rate, but significantly decreased larval locomotion, as well as their AChE, SOD, and CAT activity. Additionally, the examination of the day-night behavioral experiment revealed RCS decreased locomotion only at night. Zebrafish larvae were also significantly hypoactive in response to light and sound stimulations. Of the neurodevelopment genes, three (th, neurog1, and neurod1) were downregulated, while two (bdnf and manf) were upregulated. Our study suggests that K. mikimotoi neurotoxicity occurs through causing oxidative damage, as well as disorders in the cholinergic system and nervous system development. The results provide new insight that K. mikimotoi in low abundance did not cause significant lethal effect but still exhibited significant neurotoxicity on aquatic animals.

Ghrelin modulates dopaminergic neuron formation and attention deficit hyperactivity disorder-like behaviors: From animals to human models.

Attention deficit hyperactivity disorder (ADHD) is one of the most prevalent psychiatric disorders in children. The orexigenic hormone ghrelin is important in neuroprotection and neurodevelopment, which may play an important role in psychopathogenesis of ADHD. This study aimed to systematically investigate the genomic and pharmacological manipulations of ghrelin functioning in ADHD-like symptoms in zebrafish models and validated the effects of ghrelin polymorphisms in human subjects with ADHD. We firstly generated ghrelinΔ/Δ zebrafish mutant, which displayed hyperactive, attention deficit-like and impulsive-like behaviors, as well as endophenotypes, mimicking human ADHD. GhrelinΔ/Δ zebrafish exhibited downregulated expression levels of wnt1, wnt3a, wnt5a that are critical for dopaminergic neuron development to possibly regulate their number and spatial organization. Pharmacological blockade of wnt signaling with XAV939 induced a reduced moving activity and less dopaminergic neurons; whereas, wnt agonist SB415286 rescued hyperactivity and dopaminergic neuron loss in ghrelinΔ/Δ zebrafish. In addition, we further identified and validated a SNP, rs696217, on orexigenic hormone preproghrelin/ghrelin (T408T, Met72Met) to be associated with a higher risk of ADHD in a case-controlled association study with 248 subjects with ADHD and 208 subjects of healthy controls. Together, our results reveal a novel endogenous role for orexigenic hormone ghrelin in ADHD, which provides insights into genetic regulation and drug screens for the identification of novel treatments of ADHD.

Transcriptome sequencing of a toxic dinoflagellate, Karenia mikimotoi subjected to stress from solar ultraviolet radiation.

Solar ultraviolet radiation (UVR) is a stress factor in aquatic environments and may act directly or indirectly on orgnisms in the upper layers of the water column. However, UVR effects are usually species-specific and difficult to extrapolate. Here we use the HAB-forming, toxic dinoflagellate Karenia mikimotoi (which was found to be relatively resistant in previous studies) to investigate its transcriptional responses to a one-week UVR exposure. For this, batch cultures of K. mikimotoi were grown with and without UVR, and their transcriptomes (generated via RNAseq technology) were compared. RNA-seq generated 45.31 million reads, which were further assembled to 202600 unigenes (>300bp). Among these, ca. 61% were annotated with NCBI, NR, GO, KOG, PFAM, Swiss-Prot, and KEGG database. Transcriptomic analysis revealed 722 differentially expressed unigenes (DEGs, defined as being within a |log2 fold change| ≥ 2 and padj < 0.05) responding to solar UVR, which were only 0.36% of all unigenes. 716 unigenes were down-regulated, and only 6 unigenes were up-regulated in the UVR compared to non-UVR treatment. KEGG pathway further analysis revealed DEGs were involved in the different pathway; genes involved in the ribosome, endocytosis and steroid biosynthesis pathways were highly down-regulated, but this was not the case for those involved in the energy metabolisms (including photosynthesis, oxidative phosphorylation) which may contribute to the sustainable growth observed in UVR treatment. The up-regulated expression of both zinc-finger proteins (ZFPs) and ribosomal protein L11 (RPL11) may be one of the acclimated mechanisms against UVR. In addition, this work identified down-regulated genes involved in fatty acid degradation and the hydrophobic branched chain amino acids (e.g., Valine, leucine, and isoleucine), which act as structural components of cell membranes modulating lipid homeostasis or turnover. In conclusion, the present study suggests that the toxic dinoflagellate K. mikimotoi has limited transcriptomic regulation but confirms that it appears as a tolerant species in response to solar UVR. These findings expand current knowledge of gene expression in HAB-forming species in response to natural environment factors such as solar radiation.

Effects of ocean acidification and phosphate limitation on physiology and toxicity of the dinoflagellate Karenia mikimotoi.

This work demonstrated a 10-day batch culture experiment to test the physiology and toxicity of harmful dinoflagellate Karenia mikimotoi in response to ocean acidification (OA) under two different phosphate concentrations. Cells were previously acclimated in OA (pH = 7.8 and CO2 = 1100 µatm) condition for about three months before testing the responses of K. mikimotoi cells to a two-factorial combinations experimentation. This work measured the variation in physiological parameters (growth, rETR) and toxicity (hemolytic activity and its toxicity to zebrafish embryos) in four treatments, representing two factorial combinations of CO2 (450 and 1100 µatm) and phosphate concentration (37.75 and 4.67 umol l-1). Results: OA stimulated the faster growth, and the highest rETRmax in high phosphate (HP) treatment, low phosphate (LP) and a combination of high CO2 and low phosphate (HC*LP) inhibited the growth and Ek in comparison to low CO2*high phosphate (LCHP) treatment. The embryotoxicity of K. mikimotoi cells enhanced in all high CO2 (HC) conditions irrespective of phosphate concentration, but the EC50 of hemolytic activity increased in all high CO2 (HC) and low phosphate (LP) treatments in comparison of LCHP. Ocean acidification (high CO2 and lower pH) was probably the main factor that affected the rETRmax, hemolytic activity and embryotoxicity, but low phosphate was the main factor that affected the growth, α, and Ek. There were significant interactive effects of OA and low phosphate (LP) on growth, rETRmax, and hemolytic activity, but there were no significant effects on α, Ek, and embryotoxicity. If these results are extrapolated to the aquatic environment, it can be hypothesized that the K. mikimotoi cells were impacted significantly by future changing ocean (e.g., ocean acidification and nutrient stoichiometry).

Developmental neurotoxicity of reserpine exposure in zebrafish larvae (Danio rerio).

Reserpine is widely used for treatment of hypertension and schizophrenia. As a specific inhibitor of monoamine transporters, reserpine is known to deplete monoamine neurotransmitters and cause decreased movement symptoms. However, how zebrafish larvae respond to reserpine treatment is not well studied. Here we show that swimming distance and average velocity are significantly reduced after reserpine exposure under various stimulatory conditions. Using liquid chromatograph-mass spectrometer analysis, decreased levels of monoamines (e.g. dopamine, noradrenaline, and serotonin) were detected in reserpine-treated larvae. Moreover, reserpine treatment significantly reduced the number of dopaminergic neurons, which was identified with th (Tyrosine Hydroxylase) in situ hybridization in the preoptic area. Interestingly, dopaminergic neuron development-associated genes, such as otpa, otpb, wnt1, wnt3, wnt5 and manf, were downregulated in reserpine treated larvae. Our data indicates that 2 mg/L reserpine exposure induces dopaminergic neuron damage in the brain, demonstrating a chemical induced depression-like model in zebrafish larvae for future drug development.

Effects of ocean acidification and solar ultraviolet radiation on physiology and toxicity of dinoflagellate Karenia mikimotoi.

A batch culture experiment was conducted to study the interactive effects of ocean acidification (OA) and solar ultraviolet radiation (UVR, 280-400 nm) on the harmful dinoflagellate Karenia mikimotoi. Cells were incubated in 7-days trials under four treatments. Physiological (growth, pigments, UVabc) and toxicity (hemolytic activity and its toxicity to zebrafish embryos) response variables were measured in four treatments, representing two factorial combinations of CO2 (400 and 1000 µatm) and solar irradiance (with or without UVR). Toxic species K. mikimotoi showed sustained growth in all treatments, and there was not statistically significant difference among four treatments. Cell pigment content decreased, but UVabc and hemolytic activity increased in all HC treatments and PAB conditions. The toxicity to zebrafish embryos of K. mikimotoi was not significantly different among four treatments. All HC and UVR conditions and the combinations of HC*UVR (HC-PAB) positively affected the UVabc, hemolytic activity in comparison to the LC*P (LC-P) treatment, and negatively affected the pigments. Ocean acidification (OA) was probably the main factor that affected the chlorophyll-a (Chl-a) and UVabc, but UVR was the main factor that affected the carotenoid (Caro) and hemolytic activity. There were no significant interactive effects of OA*UVR on growth, toxicity to zebrafish embryos. If these results are extrapolated to the natural environment, it can be hypothesized that this strain (DP-C32) of K. mikimotoi cells have the efficient mechanisms to endure the combination of ocean acidification and solar UVR. It is assumed that this toxic strain could form harmful bloom and enlarge the threatening to coastal communities, marine animals, even human health under future conditions.

Interactive effect of nitrogen source and high CO2 concentration on the growth of the dinoflagellate Alexandrium tamarense and its toxicity to zebrafish (Danio rerio) embryos.

The effects and interactive effects of different nitrogen (N) sources (ammonium, nitrate, and urea) and carbon dioxide (CO2) concentrations were investigated on Alexandrium tamarense, a harmful marine dinoflagellate, by measuring its growth (µ), extracellular carbonic anhydrase (CA), and its toxicity to zebrafish (Danio rerio) embryo. The µ and CA were influenced more strongly by CO2 concentrations rather than by N sources; significant effects of CO2 on µ and CA were observed under low CO2 concentration (LC) conditions compared to high CO2 concentration (HC) conditions. The ammonium and nitrate media under LC conditions had the maximum µ and CA, which was inhibited under HC conditions. The embryotoxic effects were influenced more strongly by the N sources than by CO2 concentrations, thus excluding the lower deformation in urea under HC conditions. Moreover, the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST), and catalase (CAT) were detected in normal (untreated) zebrafish embryos, and among them, the level of SOD was the highest. In summary, this study provides a clear insight for understanding the effects and interactive effects of N sources and CO2 concentrations on the growth and toxicity of harmful dinoflagellates.

The dinoflagellate Akashiwo sanguinea will benefit from future climate change: The interactive effects of ocean acidification, warming and high irradiance on photophysiology and hemolytic activity.

Due to global climate change, marine phytoplankton will likely experience low pH (ocean acidification), high temperatures and high irradiance in the future. Here, this work report the results of a batch culture experiment conducted to study the interactive effects of elevated CO2, increased temperature and high irradiance on the harmful dinoflagellate Akashiwo sanguinea, isolated at Dongtou Island, Eastern China Sea. The A. sanguinea cells were acclimated in high CO2 condition for about three months before testing the responses of cells to a full factorial matrix experimentation during a 7-day period. This study measured the variation in physiological parameters and hemolytic activity in 8 treatments, representing full factorial combinations of 2 levels each of exposure to CO2 (400 and 1000µatm), temperature (20 and 28°C) and irradiance (50 and 200µmol photons m-2s-1). Sustained growth of A. sanguinea occurred in all treatments, but high CO2 (HC) stimulated faster growth than low CO2 (LC). The pigments (chlorophyll a and carotenoid) decreased in all HC treatments. The quantum yield (Fv/Fm) declined slightly in all high-temperature (HT) treatments. High irradiance (HL) induced the accumulation of ultraviolet-absorbing compounds (UVabc) irrespective of temperature and CO2. The hemolytic activity in the LC treatments, however, declined when exposed to HT and HL, but HC alleviated the adverse effects of HT and HL on hemolytic activity. All HC and HL conditions and the combinations of high temperature*high light (HTHL) and high CO2*high temperature*high light (HCHTHL) positively affected the growth in comparison to the low CO2*low temperature*low light (LCLTLL) treatment. High temperature (HT), high light (HL) and a combination of HT*HL, however, negatively impacted hemolytic activity. CO2 was the main factor that affected the growth and hemolytic activity. There were no significant interactive effects of CO2*temperature*irradiance on growth, pigment, Fv/Fm or hemolytic activity, but there were effects on Pm, α, and Ek. If these results are extrapolated to the natural environment, it can be hypothesized that A. sanguinea cells will benefit from the combination of ocean acidification, warming, and high irradiance that are likely to occur under future climate change. It is assumed that faster growth and higher hemolytic activity and UVabc of this species will occur under future conditions compared with those the current CO2 (400µatm) and temperature (20°C) conditions.

Light histories influence the impacts of solar ultraviolet radiation on photosynthesis and growth in a marine diatom, Skeletonema costatum.

Phytoplanktonic species acclimated to high light are known to show less photoinhibition. However, little has been documented on how cells grown under indoor conditions for decades without exposure to UV radiation (UVR, 280-400 nm) would respond differently to solar UVR compared to those in situ grown under natural solar radiation. Here, we have shown the comparative photosynthetic and growth responses to solar UVR in an indoor- (IS) and a naturally grown (WS) Skeletonema costatum type. In short-term experiment (<1 day), Phi(PSII) and photosynthetic carbon fixation rate were more inhibited by UVR in the IS than in the WS cells. The rate of UVR-induced damages of PSII was faster and their repair was significantly slower in IS than in WS. Even under changing solar radiation simulated for vertical mixing, solar UVR-induced higher inhibition of photosynthetic rate in IS than in WS cells. During long-term (10 days) exposures to solar radiation, the specific growth rate was much lower in IS than WS at the beginning, then increased 3 days later to reach an equivalent level as that of WS. UVR-induced inhibition of photosynthetic carbon fixation in the IS was identical with that of WS at the end of the long-term exposure. The photosynthetic acclimation was not accompanied with increased contents of UV-absorbing compounds, indicating that repair processes for UVR-induced damages must have been accelerated or upgraded.

Effects of solar ultraviolet radiation on photosynthesis of the marine red tide alga Heterosigma akashiwo (Raphidophyceae).

In order to assess the short- and long-term impacts of UV radiation (UVR, 280-400nm) on the red tide alga, Heterosigma akashiwo, we exposed the cells to three different solar radiation treatments (PAB: 280-700nm, PA: 320-700nm, P: 400-700nm) under both solar and artificial radiation. A significant decrease in the effective quantum yield (Y) during high irradiance periods (i.e., local noon) was observed, but the cells partially recovered during the evening hours. Exposure to high irradiances for 15, 30, and 60min under a solar simulator followed by the recovery (8h) under dark, 9 and 100micromolphotonsm(-2)s(-1) of PAR, highlighted the importance of the irradiance level during the recovery period. Regardless the radiation treatments, the highest recovery (both in rate and total Y) was found at a PAR irradiance of 9micromolphotonsm(-2)s(-1), while the lowest was observed at 100micromolphotonsm(-2)s(-1). In all experiments, PAR was responsible for most of the observed inhibition; nevertheless, the cells exposed only to PAR had the highest recovery in any condition, as compared to the other radiation treatments. In long-term experiments (10 days) using semi-continuous cultures, there was a significant increase of UV-absorbing compounds (UV(abc)) per cell from 1.2 to >4x10(-6)microgUV(abc)cell(-1) during the first 3-5 days of exposure to solar radiation. The highest concentration of UV(abc) was found in samples exposed in the PAB as compared to PA and P treatments. Growth rates (mu) mimic the behavior of UV-absorbing compounds, and during the first 5 days mu increased from <0.2 to ca. 0.8, and stayed relatively constant at this value during the rest of the experiment. The inhibition of the Y decreased with increasing acclimation of cells. All our data indicates that H. akashiwo is a sensitive species, but was able acclimate relatively fast (3-5 days) synthesizing UV-absorbing compounds and thus reducing any impact either on photosystem II or on growth.