Leila Basti

Effects of the Toxic Dinoflagellate Heterocapsa circularisquama on Larvae of the Pearl Oyster Pinctada Fucata Martensii (Dunker, 1873)

ABSTRACT The effects of the toxic dinoflagellate Heterocapsa circularisquama on the activity rate, development rate, prevalence of damage, and survival rate of trochophore and D-shaped larvae of the pearl oyster Pinctada fucata martensii were studied in relation to H. circularisquama cell densities and exposure duration. In addition, larvae were regularly processed via scanning electron microscopy to investigate morphological damage. The activity rate of both larval stages was significantly decreased after 3–6 h of exposure to H. circularisquama at densities ranging from 100 to 2 × 104 cells/mL. The prevalence of damage was significantly high after 3–6 h of exposure to H. circularisquama at densities of 100 to 2 × 104 cells/mL and 5 × 103 to 2 × 104 cells/mL for trochophores and D-shaped larvae, respectively. Cytoplasmic discharge, mass mucus production, irregular shape, delayed or inhibited mineralization of the shell, mantle protrusion, the appearance of abnormal masses in the velum, and the exfoliation of the larvae cilia coupled with epithelial desquamation were frequently observed. The activity rate of D-larvae transformed from trochophores exposed to H. circularisquama for 12–48 h at densities ranging from 10 to 2 × 104 cells/mL was significantly reduced. The survival of D-shaped larvae plummeted to less than 0.013 for densities ≥ 5 × 103 cells/mL. The results indicate that H. circularisquama blooms have detrimental impacts on bivalves at early life stages. Blooms of H. circularisquama occurring during the spawning periods will influence the natural recruitment in P. fucata martensii and will have profound impacts on its population biology. Therefore, shellfish farms should not be built in coastal areas where H. circularisquama occurs, or genitors should be relocated during potential blooming periods.

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.

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.

Physiological, Pathological, and Defense Alterations in Manila Clams (Short-Neck Clams), Ruditapes philippinarum, Induced by Heterocapsa circularisquama

ABSTRACT In a laboratory study, we investigated the clearance rates (CRs), respiration rates (RRs), total hemocyte count (THC), pathological alterations, and mucocyte densities in the gills of the short-neck clam Ruditapes philippinarum when exposed to the toxic dinoflagellate Heterocapsa circularisquama. Within 2 h of exposure to H. circularisquama at a concentration of 5–103 cells/mL, both the CRs and the RRs were significantly decreased by 43–52% compared with the control CRs and 43–93% compared with the control RRs, respectively (ANOVA, Newman-Keuls, P < 0.05). Clams exposed to H. circularisquama at 103 cells/mL for 96 h showed a set of defensive and degenerative pathologies that were absent in control clams. Within 3 h, the gills exhibited cilia matting, followed within 24 h by heavy hemocytic infiltration in the connective tissue of plicae, distortion of filaments, hyperplasia, and fusion of adjacent filaments with matted cilia. Within 48 h, the gill plicae became contracted and showed multifocal epithelial hyperplasia, increased filament fusion, exfoliation of the frontal cilia, epithelial desquamation, and abnormal epithelial masses. After 72 h, the gills presented extensive necrosis of epithelial and connective tissues, atrophy and fusion of the filaments, large masses of gill debris and mucus, and extensive exfoliation and loss of the frontal, laterofrontal, and lateral cilia. After 96 h, the gills of moribund clams exhibited more advanced stages of necrosis and degeneration. Quantitative analysis of the pathological alterations showed that both the prevalence and intensity of the defensive pathologies increased significantly, reaching their maximal values after 24 h of exposure, then decreasing. Meanwhile, the prevalence and intensity of the degenerative pathologies continued to increase throughout the experiments (ANOVA, Newman-Keuls/Fishers LSD, P < 0.05). In addition, significant decreases in the THC (t-test, P < 0.01) and the total, acid, and mixed mucocyte densities in the gills (ANOVA, Fishers LSD, P < 0.05) were observed after 48 h of exposure. The current study clearly showed physiological, pathological, and defense alterations induced by H. circularisquama in clams, highlighting the occurrence of cytotoxicity and tissue repair failure. Inhibition of feeding and respiration as well as extensive necrosis in the gills, coupled with depression of defense mechanisms resulting from depletion of hemocytes and mucocytes, ultimately resulted in the death of the clams.

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.

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.

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.

Haemolytic activity and reactive oxygen species production of four harmful algal bloom species

Abstract Based on haemolytic activity and reactive oxygen species (ROS) production of Chattonella marina, Chattonella antiqua, Heterocapsa circularisquama, Alexandrium tamiyavanichii and Karenia mikimotoi, the species were categorized into four types. (1) H. circularisquama: haemolytic activity was detected in both cell suspension and cell-free culture supernatant, but with greater activity in cell suspension than in the supernatant suggesting the presence of both cell surface and secreted haemolytic agents. (2) A. tamiyavanichii: equal haemolytic activities were detected in both the cell suspension and cell-free culture supernatant suggesting the presence of only secreted haemolytic agents. (3) K. mikimotoi: haemolytic activity was detected only in the cell suspension, indicating haemolytic agents occur only on the cell surface. (4) C. marina and C. antiqua: no significant haemolytic activity was detected in either cell suspension or cell-free culture supernatant, but high ROS were detected in the cell suspensions. Heterocapsa circularisquama and K. mikimotoi showed lethal effects on rotifers (Brachionus plicatilis), whereas A. tamiyavanichii, C. marina and C. antiqua had no effect. Our results suggest that H. circularisquama, K. mikimotoi and A. tamiyavanichii produce haemolytic agents with distinct characteristics, whereas C. marina and C. antiqua have an extremely potent ability to produce ROS.

Deleterious Effects of Harmful Dinoflagellates and Raphidophytes on Egg Viability and Spermatozoa Swimming Velocity in the Japanese Pearl Oyster Pinctada fucata martensii

ABSTRACT Several species of harmful algal blooms (HAB) exert inimical effects on bivalve molluscs, with mass mortalities and pathologies reported worldwide. Information on the effects of HAB on the reproduction of bivalve molluscs, however, remains very limited. The Japanese pearl oyster Pinctada fucata martensii is an important economic species cultured in Japan. In previous studies, harmful dinoflagellates and raphidophytes were shown to affect embryos and larvae of Japanese pearl oyster. In the present study, quantitative deleterious effects of Alexandrium catenella, Alexandrium affine, Heterosigma akashiwo, and Chattonella marina on egg viability and spermatozoa swimming velocity are reported. All four HAB species significantly reduced the swimming velocity of spermatozoa. Egg viability was affected only after exposure to A. catenella and A. affine. An additional cytotoxicity test using Vero cells showed that bioactive exudates are associated with the deleterious effects and these effects are more prominent during the death phase of the algal culture. The results of the present study warrant further research on the effects of HAB species on gamete quality of bivalves, further characterization of bioactive compounds secreted by Alexadnrium spp., and highlight the role of physical activation of the toxic mechanism of harmful raphidophytes suggested in a previous study and confirmed in the present study.

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.