Growth, Toxin Content and Production of Dinophysis Norvegica in Cultured Strains Isolated from Funka Bay (Japan).

The successful cultivation of Dinophysis norvegica Claparède & Lachmann, 1859, isolated from Japanese coastal waters, is presented in this study, which also includes an examination of its toxin content and production for the first time. Maintaining the strains at a high abundance (>2000 cells per mL-1) for more than 20 months was achieved by feeding them with the ciliate Mesodinium rubrum Lohmann, 1908, along with the addition of the cryptophyte Teleaulax amphioxeia (W.Conrad) D.R.A.Hill, 1992. Toxin production was examined using seven established strains. At the end of the one-month incubation period, the total amounts of pectenotoxin-2 (PTX2) and dinophysistoxin-1 (DTX1) ranged between 132.0 and 375.0 ng per mL-1 (n = 7), and 0.7 and 3.6 ng per mL-1 (n = 3), respectively. Furthermore, only one strain was found to contain a trace level of okadaic acid (OA). Similarly, the cell quota of pectenotoxin-2 (PTX2) and dinophysistoxin-1 (DTX1) ranged from 60.6 to 152.4 pg per cell-1 (n = 7) and 0.5 to 1.2 pg per cell-1 (n = 3), respectively. The results of this study indicate that toxin production in this species is subject to variation depending on the strain. According to the growth experiment, D. norvegica exhibited a long lag phase, as suggested by the slow growth observed during the first 12 days. In the growth experiment, D. norvegica grew very slowly for the first 12 days, suggesting they had a long lag phase. However, after that, they grew exponentially, with a maximum growth rate of 0.56 divisions per day (during Days 24-27), reaching a maximum concentration of 3000 cells per mL-1 at the end of the incubation (Day 36). In the toxin production study, the concentration of DTX1 and PTX2 increased following their vegetative growth, but the toxin production still increased exponentially on Day 36 (1.3 ng per mL-1 and 154.7 ng per mL-1 of DTX1 and PTX2, respectively). The concentration of OA remained below detectable levels (≤0.010 ng per mL-1) during the 36-day incubation period, with the exception of Day 6. This study presents new information on the toxin production and content of D. norvegica, as well as insights into the maintenance and culturing of this species.

The impact of local and climate change drivers on the formation, dynamics, and potential recurrence of a massive fish-killing microalgal bloom in Patagonian fjord.

Harmful algal blooms (HABs) in southern Chile are a serious threat to public health, tourism, artisanal fisheries, and aquaculture in this region. Ichthyotoxic HAB species have recently become a major annual threat to the Chilean salmon farming industry, due to their severe economic impacts. In early austral autumn 2021, an intense bloom of the raphidophyte Heterosigma akashiwo was detected in Comau Fjord, Chilean Patagonia, resulting in a high mortality of farmed salmon (nearly 6000 tons of biomass) within 15 days. H. akashiwo cells were first detected at the head of the fjord on March 16, 2021 (up to 478 cells mL-1). On March 31, the cell density at the surface had reached a maximum of 2 × 105 cells mL-1, with intense brown spots visible on the water surface. Strong and persistent high-pressure anomalies over the southern tip of South America, consistent with the positive phase of the Southern Annular Mode (SAM), resulted in extremely dry conditions, high solar radiation, and strong southerly winds. A coupling of these features with the high water retention times inside the fjord can explain the spatial-temporal dynamics of this bloom event. Other factors, such as the internal local physical uplift process (favored by the north-to-south orientation of the fjord), salt-fingering events, and the uplift of subantarctic deep-water renewal, likely resulted in the injection of nutrients into the euphotic layer, which in turn could have promoted cell growth and thus high microalgal cell densities, such as reached by the bloom.

Hydrology, biogeochemistry and metabolism in a semi-arid mediterranean coastal wetland ecosystem.

A LOICZ Budget Model is applied to the Ichkeul Lake, a wetland ecosystem of the South Mediterranean-North African region, to evaluate its functioning in order to boost water management. The Ichkeul Lake water and nutrient budget, net ecosystem metabolism (NEM), nutrient availability, and their seasonal changes are estimated using field data. A considerable anthropogenic-driven amount of nitrogen is transferred into N2/N2O to the atmosphere during the dry season with predominance of denitrification-anammox processes. The primary production is impacted by forcing the ecosystem respiration to reduce the NEM so that the system is functioning as heterotrophic. Climate change and anthropogenic pressures are expected to exacerbate the current trends of water quality degradation, with possible negative impacts on Palearctic birds' population. Mitigation actions are possible, through the implementation of National Wetland Management Strategies that include nutrient load and water resources management.

Interactive effects of temperature and salinity on the growth and cytotoxicity of the fish-killing microalgal species Heterosigma akashiwo and Pseudochattonella verruculosa.

Fish-killing blooms of Heterosigma akashiwo and Pseudochattonella verruculosa have been devastating for the farmed salmon industry, but in Southern Chile the conditions that promote the growth and toxicity of these microalgae are poorly understood. This study examined the effects of different combinations of temperature (12, 15, 18 °C) and salinity (10, 20, 30 psu) on the growth of Chilean strains of these two species. The results showed that the optimal growth conditions for H. akashiwo and P. verruculosa differed, with a maximum rate of 0.99 day-1 obtained at 15 °C and a salinity of 20 psu for H. akashiwo, and a maximum rate of 1.06 day-1 obtained at 18 °C and a salinity of 30 psu for P. verruculosa. Cytotoxic assays (2 × 101 - 2 × 105 cell mL-1; cells, filtrates, and cell lysates) performed at salinities of 20 and 30 psu showed a 100% reduction in the viability of embryonic fish cells exposed to intact cells of H. akashiwo and a 39% reduction following exposure to culture filtrates of P. verruculosa. Differences in the fish-killing mechanisms (direct cell contact vs. extracellular substances) and physiological traits of H. akashiwo and P. verruculosa explain the recent occurrence of very large blooms under contrasting (cold-brackish vs. hot-salty) extreme climate conditions in Chile.

Development of an absolute quantification method for ribosomal RNA gene copy numbers per eukaryotic single cell by digital PCR.

Recent increase of Harmful Algal Blooms (HAB) causes world-wide ecological, economical, and health issues, and more attention is paid to frequent coastal monitoring for the early detection of HAB species to prevent or reduce such impacts. Use of molecular tools in addition to traditional microscopy-based observation has become one of the promising methodologies for coastal monitoring. However, as ribosomal RNA (rRNA) genes are commonly targeted in molecular studies, variability in the rRNA gene copy number within and between species must be considered to provide quantitative information in quantitative PCR (qPCR), digital PCR (dPCR), and metabarcoding analyses. Currently, this information is only available for a limited number of species. The present study utilized a dPCR technology to quantify copy numbers of rRNA genes per single cell in 16 phytoplankton species, the majority of which are toxin-producers, using a newly developed universal primer set accompanied by a labeled probe with a fluorophore and a double-quencher. In silico PCR using the newly developed primers allowed the detection of taxa from 8 supergroups, demonstrating universality and broad coverage of the primer set. Chelex buffer was found to be suitable for DNA extraction to obtain DNA fragments with suitable size to avoid underestimation of the copy numbers. The study successfully demonstrated the first comparison of absolute quantification of 18S rRNA copy numbers per cell from 16 phytoplankton species by the dPCR technology.

Sublethal and antioxidant effects of six ichthyotoxic algae on early-life stages of the Japanese pearl oyster.

Several species of harmful algae form blooms that are detrimental to aquatic organisms worldwide with severe economic loss to several industries. The cosmopolitan ichthyotoxic dinoflagellates and raphidophytes Karenia spp., Chattonella spp., Heterosigma spp., and Margalefidinium (Cochlodinium) polykrikoides are known to cause mass mortalities of fish and invertebrates, and the dinoflagellates Heterocapsa spp. are known to cause mass mortalities of shellfish, notably bivalve molluscs. The species K. mikimotoi, K. papilionacea, H. circularisquama, H. akashiwo, M. polykrikoides, and C. marina form recurrent harmful algal blooms (HAB) in coastal aquaculture areas of shellfish, coinciding with the reproduction seasons of natural and farmed bivalve molluscs. In the present study, their effects on eggs, fertilization, embryos, and three larval stages (D-shaped, umbo and pre-settling larvae) of a model bivalve species, the Japanese pearl oyster, Pinctada fucata martensii, are reported. The harmful algae had differential negative effects on each developmental stage, and had differential effects on larvae depending on their growth stage. Eggs were more affected by M. polykrikoides, K. mikimotoi and H. circularisquama than H. akashiwo and K. papilionacea. Fertilized eggs and developing embryos were more affected by M. polykrikoides and H. circularisquama than K. mikimotoi, K. papilionacea and H. akashiwo. Mortalities as well as abnormalities were not observed in any larval stage; however, motility of d-larvae and umbo larvae was more reduced by H. circularisquama and C. marina, than M. polykrikoides. In elder, 16 day-old larvae, all harmful algae induced a significant decrease in motility with the most severe effect observed during exposures to H. circularisquama, C. marina, H. akashiwo and M. polykrikoides. The superoxidase dismutase activity in larvae was not affected by exposure to any harmful alga; however, 6- and 16-day old larvae experienced a significant increase in GST activity following 48 h of exposures, with higher sensitivity of the elder larvae to C. marina, K. mikimotoi and M. polykrikoides. These results indicate that all tested harmful algae are differentially detrimental to the early-life development of the Japanese pearl oyster, with involvement of oxidative stress. Both M. polykrikoides and H. circularisquama were the most toxic followed by C. marina, K. mikimotoi, H. akashiwo and K. papilionacea. In addition, more developed larvae were most sensitive to these harmful algae in terms of motility-avoidance behavior and oxidative stress response, suggesting that ingestion of the harmful algae might enhance the toxicity of contact-dependent effects and dissolved extracellular compounds. The results also showed that superoxide anions were not associated with effects observed in larvae. Instead cellular detoxification was induced. The differential, stage-specific and growth-specific sublethal effects on bivalve development and recruitment also warrant further investigations of the oxidative stress and antioxidant enzyme activities in larvae of bivalves, to better address the toxicity mechanisms of ichthyotoxic HAB and their impacts on the reproduction, recruitment, and fitness of bivalve molluscs. Summary: The harmful algae Heterocapsa circularisquama, Chattonella marina, Hetrosigma akashiwo, Karenia mikimotoi, K. papilionacea, Margalefidinium (Cochlodinium) polykrikoides differentially affect early life stages of Japanese pearl oyster and activate detoxification enzymes in feeding larvae.

Thermal acclimation affects growth and lipophilic toxin production in a strain of cosmopolitan harmful alga Dinophysis acuminata.

Species of the harmful algal bloom (HAB) genera Dinophysis are causative of one of the most widespread and expanding HAB events associated with the human intoxication, diarrheic shellfish poisoning (DSP). The effects of warming temperature on the physiology and toxinology of these mixotrophic species remain intractable due to their low biomass in nature and difficulties in establishing and maintaining them in culture. Hence, the present study investigated the influence of warming temperature, encompassing present and predicted climate scenarios, on growth and toxin production in a strain of the most cosmopolitan DSP-causative species, Dinophysis acuminata. The strain was isolated from western Japan, acclimated, and cultured over extended time spans. The specific growth and toxin production rates were highest at 20-26 °C and 17-29 °C, respectively, and had significant linear relationships during exponential phase. The cellular toxin production of okadaic acid and pectenotoxin-2 were highest during early exponential growth phase at temperatures ≤17 °C but highest during late stationary phase at temperatures ≥20 °C. The cellular toxin production of Dinophysistoxin-1, however, increased from early exponential to late stationary growth phase independently from temperature. The net toxin productions were not affected by acclimation temperature but significantly affected by growth and were highest during early exponential growth phase. Warming water temperatures increase growth and promote toxin production of D. acuminata, potentially increasing incidence of diarrheic shellfish poisoning events and closures of shellfish production. It is likely that D. acuminata is more toxic at low cell densities during bloom initiation in winter, and at high cell densities during bloom termination in spring-autumn. The results of the present research are also of importance for the mass production of D. acuminata for subsequent studies of the toxicological and pharmacological bioactivities of DSTs and PTX2, and the fate of these toxins in the natural environment and the vectoring shellfish molluscs.

Intracellular haemolytic agents of Heterocapsa circularisquama exhibit toxic effects on H. circularisquama cells themselves and suppress both cell-mediated haemolytic activity and toxicity to rotifers (Brachionus plicatilis).

A harmful dinoflagellate, Heterocapsa circularisquama, is highly toxic to shellfish and the zooplankton rotifer Brachionus plicatilis. A previous study found that H. circularisquama has both light-dependent and -independent haemolytic agents, which might be responsible for its toxicity. Detailed analysis of the haemolytic activity of H. circularisquama suggested that light-independent haemolytic activity was mediated mainly through intact cells, whereas light-dependent haemolytic activity was mediated by intracellular agents which can be discharged from ruptured cells. Because H. circularisquama showed similar toxicity to rotifers regardless of the light conditions, and because ultrasonic ruptured H. circularisquama cells showed no significant toxicity to rotifers, it was suggested that live cell-mediated light-independent haemolytic activity is a major factor responsible for the observed toxicity to rotifers. Interestingly, the ultrasonic-ruptured cells of H. circularisquama suppressed their own lethal effect on the rotifers. Analysis of samples of the cell contents (supernatant) and cell fragments (precipitate) prepared from the ruptured H. circularisquama cells indicated that the cell contents contain inhibitors for the light-independent cell-mediated haemolytic activity, toxins affecting H. circularisquama cells themselves, as well as light-dependent haemolytic agents. Ethanol extract prepared from H. circularisquama, which is supposed to contain a porphyrin derivative that displays photosensitising haemolytic activity, showed potent toxicity to Chattonella marina, Chattonella antiqua, and Karenia mikimotoi, as well as to H. circularisquama at the concentration range at which no significant toxicity to rotifers was observed. Analysis on a column of Sephadex LH-20 revealed that light-dependent haemolytic activity and inhibitory activity on cell-mediated light-independent haemolytic activity existed in two separate fractions (f-2 and f-3), suggesting that both activities might be derived from common compounds. Our results suggest that the photosensitising haemolytic toxin discharged from ruptured H. circularisquama cells has a relatively broad spectrum of phytoplankton toxicity, and that physical collapse of H. circularisquama cells can lead not only to the disappearance of its own toxicity, but also to mitigation of the effects of other HABs.

Neuroenzymatic activity and physiological energetics in Manila clam, Ruditapes philippinarum, during short-term sublethal exposure to harmful alga, Heterocapsa circularisquama.

The harmful alga, Heterocapsa circularisquama, causes recurrent mortalities of bivalve molluscs in Japan, with demonstrated hemolysis and cytotoxicity in rabbit erythrocytes, HeLa cells, and bivalve tissues. Nonetheless, the effects of exposure to sublethal cell densities on the physiological energetics of bivalves have not been investigated, nor the potential involvement of neurotoxicity. In the present study, two sets of experiments were conducted with adult clams, Ruditapes philippinarum. In the first set, the clearance rate (CR), respiration rate (RR), absorption efficiency (AE), ingestion rate (IR), and absorption rate (AR) were examined in clams exposed to H. circularisquama to quantify the scope for growth (SFG) as an indicative of the bioenergetic status of clams (5, 50, 2.5×10(2), and 5×10(2)cellsml(-1); under 15°C and 20°C). In the second set, the activity of the biomarker of neurotoxic exposure, acetylcholinesterase (AChE), was monitored following 3, 6, 24, and 48h of exposure (5, 50, 5×10(2), and 10(3)cellsml(-1), at 20°C) in gills of R. philippinarum, and compared to that in Mediterranean mussels, Mytilus galloprovincialis; a species also affected by H. circualrisquama and in which AChE activity was more extensively studied. At 15°C, CR, IR, and AR were decreased for exposures to 50-5×10(2) cells ml(-1) resulting in a significant decrease in the absorbed energy (A), and a significant decrease in SFG at 5×10(2)cellsml(-1). At 20°C, AE was null for exposures to 2.5×10(2) and 5×10(2)cellsml(-1). RR was decreased at 2.5×10(2) and 5×10(2)cellsml(-1), CR, IR, and AR were decreased at 5-5×10(2)cellsml(-1), and the AE was null for 2.5×10(2)-5×10(2)cellsml(-1) resulting in a significant decrease in the respired energy (R), but mainly in (A) especially at 2.5×10(2) and 5×10(2)cellsml(-1) decreasing the SFG over the entire range of cell density with negative values for 2.5×10(2) and 5×10(2)cellsml(-1). The activity of AChE in both clams and mussels was significantly reduced following 3-48h of exposure to 5-5×10(2)cellsml(-1). Reduction in the energy available for clams to grow and reproduce was induced mainly via decreased energy acquisition, and delayed and/or reduced functions of the digestive organs. In addition, this is the first report of decreased neuroenzymatic activity in two bivalve molluscs induced by exposure to H. circularisquama potentially via either neurotoxic compounds affecting the activity of gill cilia and/or gill muscles, and/or potentially via either neurotoxic compounds affecting the activity of gill cilia and/or gill muscles, and/or non-cholinergic affects associated with other functions; both resulting in decreased SFG. The relationship between the decreased AChE and decreased SFG should be corroborated in future research.

Lethal effects of ichthyotoxic raphidophytes, Chattonella marina, C. antiqua, and Heterosigma akashiwo, on post-embryonic stages of the Japanese pearl oyster, Pinctada fucata martensii.

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5×102cellsml-1), C. antiqua (103cellsml-1), and H. akashiwo (5×103cellsml-1) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5×103cellsml-1 and C. marina at 8×103cellsml-1. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 105cellsml-1 more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8×103cellsml-1, 24h), C. marina (8×103cellsml-1, 24h), and H. akashiwo (104cellsml-1, 24h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (103cellsml-1, 24h),C. marina (8×103cellsml-1, 24h), and H. akashiwo (5×104cellsml-1, 24h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5×102-8×103cellsml-1, 48-86h) and C. antiqua (103-8×103cellsml-1, 72-86h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.

Influence of Temperature on Growth and Production of Pectenotoxin-2 by a Monoclonal Culture of Dinophysis caudata.

The effects of temperature on growth and production of Lipophilic Toxins (LT) by a monoclonal culture of Dinophysis caudata was studied. The cell density of D. caudata increased significantly with increasing temperature, and was the highest under 27, 30, and 32.5 °C. Temperature affected the average specific growth rate (µ) during the exponential growth phase (EG), which increased from 15 °C to 30 °C, and then decreased at 32.5 °C. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that this strain of D. caudata produced only pectenotoxin-2 (PTX-2) whose concentration increased significantly with incubation period, except at 32.5 °C. It was significantly different between temperatures ≤18 °C, ≥21 °C, and 32.5 °C. The cellular toxin production (CTP, pg·cell(-1)·day(-1)) showed variation with growth phase and temperature, except at 32.5 °C. The average net toxin production (Rtox) was not affected by temperature. During EG, the average specific toxin production rate (µtox) increased significantly with increase in temperature, reaching a peak of 0.66 ± 0.01 day(-1) at 30 °C, and then decreased. Over the entire growth span, µtox was significantly correlated to µ, and this correlation was most significant at 27 and 30 °C. During EG, µtox was affected by both temperature and growth. This study shows that temperature affects growth and toxin production of this strain of D. caudata during EG. In addition, a positive correlation was found between toxin production and growth.

Prevalence and intensity of pathologies induced by the toxic dinoflagellate, Heterocapsa circularisquama, in the Mediterranean mussel, Mytilus galloprovincialis.

The harmful dinoflagellate, Heterocapsa circularisquama, has been causing mass mortalities of bivalve molluscs in Japan, at relatively low cell densities. Although several studies have been conducted to determine the toxicity mechanisms, the specific cause of death is still unclear. In a previous study, in our laboratory, it was shown that H. circularisquama (10(3) cells ml(-1)) caused extensive cytotoxicity in the gills of short-neck clams, Ruditapes philippinarum. In the present study, Mediterranean mussels, Mytilus galloprovincialis, were exposed to H. circularisquama at four cell densities (5, 50, 500, 10(3) cells ml(-1)), three temperatures (15, 20, and 25°C), and three exposure durations (3, 24, and 48 h), and the pathologies in nine organs (gills, labial palps, mantle, hepatopancreas, stomach, intestines, exhalant siphon, adductor muscles, and foot) were assessed. Foot, adductor muscles, and exhalent siphons of mussels were not affected; however, 16 inflammatory (hemocytic infiltration and aggregation, diapedesis, hyperplasia, hypertrophy, edema, melanization, and firbrosis) and degenerative (thrombus, thrombosed edema, cilia matting and exfoliation, epithelial desquamation, atrophy, and necrosis) pathologies were identified in the gills, labial palps, mantle, hepatopancreas, stomach, and intestines. The total prevalence and total intensity of pathology in each individual mussel, and the prevalence and intensity of pathology in each organ increased significantly with increased cell density, exposure duration, and temperature. The prevalence of pathology was the highest in gills, followed by the prevalence in labial palps, mantle, stomach, and intestines. Pathology was least prevalent in the hepatopancreas. The intensity of pathology was the highest in the gills, followed by the labial palps and mantle, the stomach and intestines, and the hepatopancreas. This detailed quantitative histopathological study demonstrates that exposure to H. circularisquama induces a broad cytotoxic effect in six vital organs, even at low density (5 cells ml(-1)) and low temperature (15°C), but not in muscular organs. Combining cell density, time, and duration of exposure, the organ most affected by the harmful alga was the gill, followed by the labial palps and mantle, the stomach and intestines, and the hepatopancreas. The results of this pathological analysis show that exposure to H. ciruclarisquama severely affects the gills, the labial palps, and mantle thereby interfering with particle clearance and sorting, cleansing, and respiration, but also affects the stomach, intestines, and hepatopancreas, altering the digestive processes and possibly detoxification pathways, if mussels are able to detoxify the toxins of H. circularisquama. In the most severe cases, bivalves would most likely have died as a result of combined severe alterations of the vital functions, failure of tissue repair, and moderate to heavy hemorrhaging in both the external organs and the digestive organs concomitantly with light to moderate alterations in the detoxifying processes.