Toxicity and haemolytic activity of a newly described dinoflagellate, Heterocapsa bohainensis to the rotifer Brachionus plicatilis

Harmful Algae 84 (2019) 112–118

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Toxicity and haemolytic activity of a newly described dinoflagellate, Heterocapsa bohainensis to the rotifer Brachionus plicatilis

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Yiwen Zhanga, , Tongtong Fenga, Jing Qua, Na Sunb, Lifen Liua a b

School of Food and Environment, Dalian University of Technology, NO. 2 Dagong Road, New District of Liaodong Bay, Panjin City, Liaoning Province, 124221, China Guanghe Crab Industry Limited Company, Panjin 124200, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Dinoflagellate Heterocapsa bohaiensis Brachionus plicatilis Haemolytic activity Calcium homeostasis Membrane permeability

The algae Heterocapsa bohaiensis is a newly described species of dinoflagellate associated with Penaeus japonicus and larvae of Eriocheir sinensis in a coastal pond of Liaodong Bay China. The rotifer Brachionus plicatilis is used as live feed for aquaculture organisms including prawns and crabs larvae. To evaluate the potential toxicity of H. bohaiensis, the effects on Brachionus plicatilis and haemolytic activity were investigated in this study. The results showed that H. bohaiensis had significant toxic effect on B. plicatilis, and this effect was dependent on the cell concentration. Significant rotifer growth suppression was observed in the ruptured cells of H. bohaiensis with ultrasonic. Relatively similar rotifer mortalities were induced both in the light and in the dark. Interestingly, haemolysis to erythrocytes was also caused in a cell density-dependent and time-dependent manner, which meant the results of haemolytic activity were consistent with the toxicity. Therefore, haemolytic toxins were considered to be involved in the toxic mechanism of H. bohaiensis against rotifers. Then, the concentrations of calcium were measured in the mastax, stomach and ovary of B. plicatilis. Obviously increased fluorescence intensity was found in the stomach, which indicated the alteration of calcium homeostasis and membrane permeability after ingesting H. bohaiensis. These results implicated haemolytic activity as a causative factor linked to the toxicity of H. bohaiensis against B. plicatilis. The results contributed to research the production and control of H. bohaiensis toxins.

1. Introduction The frequency, magnitude and duration of harmful algae blooms (HABs) are increasing as a response to global climate change (Paerl and Huisman, 2008; Zou et al., 2010; Matsuyama, 2012; Li et al., 2016). Harmful algae blooms (HABs) frequently cause large-scale economic losses to aquaculture, fisheries and tourism; furthermore, they have increased concerns about environmental and human health risks (Hallegraeff, 1993; Kirkpatrick et al., 2004). The genus Heterocapsa is made up of bloom-forming dinoflagellates, including numerous toxic species. For example, Heterocapsa circularisquama has caused mass bivalve mortalities in the coastal embayment of western Japan since 1988 (Horiguchi, 1995; Matsuyama, 1996). This dinoflagellate shows high toxicity to shellfish, especially to bivalves such as pearl oysters (Pinctada fucata), short-necked clams (Tapes philippinarum), feral blue mussels (Mytilus galloprovincialis), and oysters (Crassostrea gigas), but not to fish and marine vertebrates (Yamamoto, 1990; Matsuyama, 1999). Blooms of algae Heterocapsa triquetra broke out in the fjord-like Mariehamn ferry harbor area (Aland, SW Finland) in 1996 (Lindholm and

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Nummelin, 1999). The algae Heterocapsa rotundata was reported as red tide algae in Manori Creek and Manim Bay, India (Shahi et al., 2015), Chesapeake Bay, USA (Millette et al., 2015) and the Baltic Sea, Germany (Jaschinski et al., 2015). Heterocapsa bohaiensis, which was successfully isolated and identified as a newly described dinoflagellate species, has caused mass mortality of Penaeus japonicas and Eriocheir sinensis in culture ponds along coastal areas of Liaoning China, since 2012 (Xiao et al., 2018). The algae H. bohaiensis can be distinguished from the other Heterocapsa spp. by a combination of cell size, morphology, cellular structure and body scale. Sequence analyses of both the ITS and LSU regions revealed the significant genetic divergence between H. bohaiensis and the other established species in this genus. The small (14.4 ± 1.6 μm in length) ellipsoid cells showed typical Heterocapsa thecal plate arrangement (Po, cp, 5′, 3a, 7′′, 6c, 5 s, 5′′′, 2′′′′). The episome was evidently bigger than the hyposome. And one to three spherical pyrenoids were located above the large elongated nucleus. The body scale was characterized by a triangle basal plate with one central upright and six peripheral spines (Xiao et al., 2018). The effects of temperature on the growth of H.

Corresponding author. E-mail address: [email protected] (Y. Zhang).

https://doi.org/10.1016/j.hal.2019.03.007 Received 21 July 2018; Received in revised form 18 March 2019; Accepted 18 March 2019 1568-9883/ © 2019 Elsevier B.V. All rights reserved.

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apparatus for 30 min at 20 °C. The apparatus worked under the conditions of 200 W power at a crushing time of 5 s and an interval time of 5 s. Microscopic observation confirmed that all cells were ruptured by the treatment. The cell-free culture supernatant and ruptured cell suspension were used immediately after their preparation for the rotifer toxicity test (Section 2.3) and haemolytic assay (Section 2.4).

bohaiensis were significant and the optimum growth temperature was 25℃˜30℃. The lethality of Ruditapes philippinarum feeded by H. bohaiensis was high (Liu and Liu, 2016). Due to its negative impacts on fisheries, it is a matter of urgency to study the ecological and physiological traits of this species. This study was to focus on the toxicity of H. bohaiensis and to determine the toxicological mechanism of action in aquaculture ponds. The rotifer Brachionus plicatilis is an important rotifer species that is used as live feed for aquaculture organisms including prawns and crab larvae. In addition to its nutritional value as a larval diet, B. plicatilis is widely used as an indicator in simple and sensitive bioassays to determine toxicity in estuarine water and seawater. It has been reported that H. circularisquama exhibited lethal effects on B. plicatilis in a cell density-dependent manner (Kim et al., 2000). Frequent contact of H. circularisquama cells with the cytoplasm around the oral plug of B. plicatilis and subsequent morphological changes of B. plicatilis were observed at high flagellate cell concentrations (Kim et al., 2000). The algae Karenia mikimotoi and Alexandrium species also have adverse effects on the rotifers (Wang et al., 2005; Zou et al., 2010). Toxic effects of H. bohaiensis on rotifers have not been reported. In addition, some previous studies suggested that haemolytic activity was involved in the toxicity of Heterocapsa spp. (Oda et al., 2001; Kim et al., 2002; Sato et al., 2002). For example, H. circularisquama cell suspension caused potent haemolysis toward rabbit erythrocytes in a cell density-dependent manner (Oda et al., 2001), and Kim et al. found that haemolytic activity and toxicity to shellfish were well correlated in several strains of H. circularisquama isolated from different localities in Japan (Kim et al., 2002). Regarding the compound responsible for the haemolytic activity of H. circularisquama, it was found that an ethanol extract prepared from the flagellate cells showed haemolytic activity, and this activity was light dependent (Sato et al., 2002). Furthermore, Matsuyama reported that an influx of Ca2+ was induced in trochophore larva of short-necked clams after exposure to H. circularisquama (Matsuyama, 2012). In this study, more detailed experiments were conducted and demonstrated that H. bohaiensis cells caused haemolysis of rabbit erythrocytes and showed toxic effects on the rotifer. At the same time, Ca2+ in both rabbit erythrocytes and rotifers were also detected.

2.3. Rotifer exposure experiment Before the experiment, rotifers were kept without food for 12 h and then concentrated using 96 μm mesh and resuspended in fresh seawater in 1000 mL beakers as stock for the experiments. All cultures were prepared using sterilized instruments. The test of rotifer exposure was conducted in 12-well plates (Becton-Dickinson) with the method described previously (Zou et al., 2010). Each well contained 10 individual rotifers in 4 mL of various concentrations of H. bohaiensis (1.0 × 105 cells mL−1; 1.0 × 104 cells mL−1; 1.0 × 103 cells mL−1; 1.0 × 102 cells mL−1) suspended in f/2 medium at 25 °C in either light (72 ± 5 μmol m-2 s−1) or dark for the indicated periods of time (0–72 h). A treatment with 10 individual rotifers was cultured in 4 mL of the f/2 medium alone as a negative control. Another treatment with 10 individual rotifers was cultured in 4 mL of Chlorella pyrenoides cell suspension (2.0 × 106 cells mL−1) as a positive control. The various concentrations of H. bohaiensis cell suspension, cell-free culture supernatant and ruptured cell suspension were also maintained in the same conditions. Then, the viable individuals, which were defined as those actively swimming in each well, were counted using a stereomicroscope. The number of rotifers was recorded after 12 h, 24 h, 48 h and 72 h. Three wells were used per treatment. 2.4. Measurement of haemolytic activity A haemolytic assay was conducted using rabbit erythrocytes as described previously (Oda et al.,2001). Rabbit erythrocytes were obtained from Baiji Kemao Company (Henan, China). Erythrocytes were washed three times with phosphate-buffered saline (PBS) and aliquoted into samples adjusted to a final concentration of 4% (v/v) in f/2 medium. Triplicate 50 μL aliquots of the intact cell suspension, cell-free culture, ruptured cell suspension or other samples in f/2 medium were added to round-bottom 96-well plates (Becton-Dickinson, Franklin Lake, NJ, USA). To the wells containing samples, 50 μL of a 4% (v/v) suspension of erythrocytes in f/2 medium were added, after incubation for 6 h at 25 °C under illumination from a fluorescent lamp (72 ± 5 μmol m−2 s-1) or in the dark, and the plates were centrifuged at 900×g for 10 min at 4 °C. Aliquots (50 μL) of supernatant were withdrawn from the wells and transferred to flat-bottom 96-well plates (Becton-Dickinson). The amount of hemoglobin released was determined by measuring absorbance at 560 nm using a microplate reader (Multiscan GO, Thermo Fisher Scientific Inc., MA, USA). Negative controls (zero haemolysis) and positive controls (100% haemolysis) were prepared using erythrocytes suspended in f/2 medium alone and in f/2 medium containing 1% v/v Triton X-100, respectively.

2. Materials and methods 2.1. Phytoplankton and rotifer cultures The strains of H. bohaiensis, Chlorella pyrenoidosa and B. plicatilis were provided by Panjin Guanghe Crab Industrial Co.,Ltd. in 2015. These phytoplankton species were maintained at 25 °C in 500 mL flasks containing 300 mL of a f/2 medium seawater medium at a salinity of 25 in a 12:12 h photoperiod using a cool-white fluorescent lamp (72 ± 5 μmol m−2 s-1) (Guillard and Ryther, 1962; Vasconcelos et al., 2002). The culture medium was autoclaved for 20 min at 121 °C. B. plicatilis was cultured in sterilized seawater and fed on C. pyrenoidosa. Cell densities were counted microscopically using a hemocytometer (Erma Inc., Tokyo, Japan). Microalgae in the late exponential growth phase were used in the toxicity experiments to B. plicatilis.

2.5. Calcium concentration assay in rotifers Living and robust adult rotifers were selected from rotifers precultured, and inoculated in H. bohaiensis cell suspension (100 mL, 1.017 × 105 cells mL−1) and sterilized seawater (100 mL) in 250 mL flasks. The initial rotifer density was 10 cell mL-1, and each experiment was conducted in triplicate. After incubation for 24 h, samples were filtered using a 300 mesh screen into a 10 mL centrifuge tube. The change in the intercellular concentrations of calcium ([Ca2+]in) was determined using Fluo-3/AM (Beyotime) following Garcia-Prieto et al. (Garciaprieto et al., 2013), with the following modifications: Rotifers were washed two times with phosphate-buffered saline (PBS 10×) (1.35 M NaCl, 47 mM KCl, 100 mM Na2HPO4, 20 mM NaH2PO4 and pH

2.2. Preparation of cell-free culture supernatant and ultra sonicruptured cell suspension of H. bohaiensis A cell-free culture supernatant of H. bohaiensis was prepared from a cell suspension in its late exponential growth phase (10–11 × 104 cell mL−1) through centrifugation at 5000×g for 10 min at 4 °C. A small amount of the filter residue after centrifugation was taken and observed under an optical microscope. It was found that the cells of the genus algae were intact and no cell lysis was observed. The ruptured cell suspension was prepared by performing an ultrasonic treatment in the cell suspension (10–11 × 104 cell mL−1) in a bath-type ultrasonic 113

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7.4), and then the precipitate was transferred to 1 mL centrifuge tubes. The investigated rotifers were loaded with 2 μM Fluo-3 AM (Beyotime Biotechnology, China) in the dark for 40 min at 37 °C (Meng et al., 2014). Then, the hatched samples were centrifuged and washed two times with PBS. The fluorescence intensity was detected using an inverted fluorescence microscope (Olympus IX 73, Japan). The [Ca2+] in value in the rotifers was calculated with Image-Pro Plus 6.0. The area and integrated optical density (IOD) value of the fluorescent part of the picture were measured (Li et al., 2016). The mean density contents were calculated with the following formula: Mean density = (IOD sum) / (area sum)

2.6. Calcium concentration assay in erythrocytes Erythrocytes were washed three times with PBS and aliquoted into samples adjusted to a final concentration of 4% (v/v) in f/2 medium. Triplicate 1 mL aliquots of the intact cell suspension (1.017 × 105 cells mL−1) and 1 mL of a 4% (v/v) suspension of erythrocytes were added to 5 mL centrifuge tubes, and then they were incubated lasted for 6 h at 25 °C, and sterilized f/2 medium was used as control. The change in the intercellular concentrations of calcium ([Ca2+]in) was determined using Fluo-3/AM (Beyotime) following Li et al. (Li et al., 2014), with the following modifications: Erythrocytes were washed three times with PBS, and then the precipitate was transferred to 5 mL centrifuge tubes. The precipitates were resuspended using Hanks` Balanced Salt Solution (with Ca2+ & Mg2+) (HBSS; 137.93 mM NaCl, 5.33 mM KCl, 4.17 mM NaHCO3, 1.26 mM CaCl2, 0.493 mM MgCl2, 0.407 mM MgSO4, 0.441 mM KH2PO4, 0.338 mM Na2HPO4, and 5.56 mM D-Glucose) and loaded with 2 μM Fluo-3 AM in the dark for 40 min at 37 °C; then, the tubes were centrifuged at 300×g for 5 min at 4 °C. After that, the fluorescently stained samples were pipetted into a standard flow tube and detected using flow cytometry (BD FACSCalibur, USA) equipped with a blue laser (excitation 488 nm). All particles analyzed by FCM were characterized according to their forward scatter (FSC), side scatter (SSC) and green fluorescence intensity (FL1; green emission filter band pass, 545/30 nm). A total of 10,000 events was collected per sample and the percentages of cells of each phenotype determined. Data were analyzed using Cellquest software and concentrations of erythrocytes were calculated (cells mL−1) from the number of events per unit time and the estimate of the FacsCalibur flow rate measured according to Marie et al. (Marie et al., 1999). It was possible to determine the intercellular concentrations of calcium by measuring the FL1 fluorescence intensity.

Fig. 1. Toxicity of H. bohaiensis to the rotifer. Rotifers in 12-well plates (10 rotifers well−1) were exposed to various concentrations of H. bohaiensis (—■—, 1 × 105 cells mL−1; —●—, 1 × 104 cells mL−1; —▲—, 1 × 103 cells mL−1; —▼—, 1 × 102 cells mL−1; —▶—, Chlorella pyrenoides; —◆—, f/2 medium) suspended in f/2 medium at 25 °C for the indicated period of time, and then the number of viable rotifers remaining were counted as described in the text. Each point represents the mean of triplicate measurements, and each bar represents the standard deviation.

Fig. 2. Densities of H. bohaiensis co-cultured with rotifers. Rotifers in 12-well plates (10 rotifers well−1) were exposed to various concentrations of H. bohaiensis (—■—, 1 × 105 cells mL−1; —●—, 1 × 104 cells mL−1; —▲—, 1 × 103 cells mL−1; —▼—, 1 × 102 cells mL−1) and no rotifer but seawater in 12-well(—□—, 1 × 105 cells mL-1 blank; —○—, 1 × 104 cells mL-1 blank; —△—, 1 × 103 cells mL-1 blank; —▽—, 1 × 102 cells mL-1 blank) suspended in f/2 medium at 25 °C for the indicated period of time. And then the number of H. bohaiensis cells remaining were counted as described in the text. Each point represents the mean of triplicate measurements, and each bar represents the standard deviation.

2.7. Statistical analysis The means and standard deviations from three replicates (n = 3) were calculated for each treatment. Data were analyzed by one-way analysis of variance (ANOVA) using OriginPro. 8.0, subsequent t-tests using SPSS 19.0 and the LC50 were calculated using Prism5. Sample differences were considered significant at P < 0.05.

algae densities (103 and 102 cells mL−1). The number of H. bohaiensis cells decreased during the exposure, indicating that these cells were preyed upon by the rotifers (Fig. 2). These results suggest that B. plicatilis remained alive by ingesting H. bohaiensis under conditions of sufficient algae density. In addition, H. bohaiensis appeared in the rotifers when rotifers were exposed to H. bohaiensis (Fig. 4B). Mortality data were subjected to probit analysis. LC50 were calculated using the method by Finney (Ronald and Rodney, 1987). The LC50 values of H. bohaiensis against B. plicatilis of 12 h, 24 h, 48 h and 72 h after the exposure to the cells of H. bohaiensis are shown in Table 1. The LC50 values was estimated to be approximately 1.38 × 104 cells mL−1 after 24 h and 4.16 × 103 cells mL-1 after 48 h (Table 1). Both in the light and in the dark were similarly (P > 0.05) lethal to

3. Results 3.1. Toxic effect of H. bohaiensis on rotifers and bioactivities of ruptured cells The algae H. bohaiensis had a significant lethal effect on the rotifer B. plicatilis, and all the rotifers eventually died after being immobilized during exposure to a high density of H. bohaiensis (105 cells mL−1) (Fig. 1). This effect was dependent on the cell concentration, and the mortality of the rotifer increased with higher concentrations of algae. For example, all the rotifers exposed to 105 cells mL−1 H. bohaiensis died after 24 h exposure. The rotifers were able to survive at lower 114

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shrinked, and the ovarian tissue shrinked to the middle of its body (Fig. 4). It was likely that the bioassay using the rotifer represented a simple and reproducible method for testing the potential toxicity of H. bohaiensis.

Table 1 LC50 of H. bohaiensis to rotifers. Time LC50

12h 1.357 × 10

24h 4

48h 3

4.161 × 10

3.295 × 10

72h 3

4.458 × 103

3.2. Haemolytic activity of H. bohaiensis

LC50 = lethal concentration that kills 50% of the exposed H. bohaiensis.

To examine the roles of haemolytic substances on the toxic effects of H. bohaiensis and their biological significance, a detailed haemolysis experiment was carried out in this study. Rabbit erythrocytes were highly sensitive to the haemolytic activity of H. bohaiensis. Since a previous study demonstrated that H. circularisquama causes the haemolysis most potently in rabbit erythrocytes among the erythrocytes from different species tested (Oda et al., 2001), a haemolytic assay was conducted using rabbit erythrocytes in this study. Haemolysis of erythrocytes was caused in a cell density-dependent manner, and the haemolysis of erythrocytes occurred with increasing algae concentrations. When the H. bohaiensis density was 104 cells mL−1, the haemolytic activity was close to 50%. Then, the haemolytic activity increased sharply. The haemolytic activity reached 100% at an algae density of only 105 cells mL−1. The cell suspension showed similar haemolytic activity both in the light and in the dark (P > 0.05), and thus its haemolytic activity was not affected by the light conditions, which was in accordance with results of the toxic effects on rotifers (Fig. 5).

rotifers, which meant that the lethal effect on the rotifers was not affected by the light conditions (Fig. 3C and D). ANOVA indicated that both in the cell-free cultured supernatant and seawater were also no significant differences (P > 0.05) among the rotifer numbers, which showed that the cell-free cultured supernatant was not toxic to B. plicatilis (Fig. 3A and B). Both in the live cell suspension and the ultrasonically ruptured cells of H. bohaiensis were significantly (P < 0.05) lethal to rotifers (Fig. 3A and B). These results confirmed that the toxins were present in algae cells or released from ruptured cells; however, no toxins were present in extracellular secretions. Thus, the toxicity of these algae to rotifers was mediated by intact cells rather than the filtrate. Slight morphological changes concomitant with the discharge of mucus-like substances were observed in B. plicatilis after exposure to a high concentration of H. bohaiensis (105 cells mL−1) before death. For instance, the interior of the rotifer body cavity shrinked, the stomach

Fig. 3. Toxicity of live cell suspension (—□—, —■—), cell-free cultured supernatant (—○—,—●—), ultrasonically ruptured cells (—△—, —▲—), and f/2 medium (—▽—,—▼—) of H. bohaiensis on the rotifer in the light (A)(□, ○, △, ▽) and in the dark (B)(■, ●, ▲, ▼). Toxicity of live cell suspension of H. bohaiensis on the rotifer in the light and in the dark (—□—, —■—) (C). Toxicity of cell-free cultured supernatant of H. bohaiensis on the rotifer in the light and in the dark (—□—, —■—) (D). Rotifers in 12-well plates (10 rotifers well−1) were exposed to various conditions of H. bohaiensis (final 10–11 × 104 cell mL−1) in f/2 medium at 25 °C, and then the number of viable rotifers remaining were counted as described in the text. Each point represents the mean of triplicate measurements, and each bar represents the standard deviation. 115

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Fig. 4. Photograph of a living B. plicatilis before (A) and after (B) exposure to H. bohaiensis. Slight morphological changes concomitant with the discharge of mucuslike substances was shown after 24 h exposure to H. bohaiensis at 105 cells ml−1. A high-resolution version of the image is available as eSlide: VM05644.

cell suspension was estimated to be equivalent to that in live cell suspension (P > 0.05). The haemolytic activity of the cell-free culture supernatant prepared from H. bohaiensis showed weak haemolytic activity (Fig. 6), which was consistent with the results of the rotifer exposure experiment. These results suggest that the haemolytic activity of H. bohaiensis on rabbit erythrocytes might be relevant to the potency of their rotifer toxicity.

3.3. Effect of H. bohaiensis on Ca erythrocytes

2+

concentration in rotifers and in

Compared with normal rotifers, the interior body cavity of the rotifer exposed by H. bohaiensis shrinked. For example, the stomach shrinked, and the ovarian tissue shrinked to the middle of its body. The light of the gap was showed in the micrograph (Fig. 4). Obviously increased fluorescence intensity was found in the stomach of exposed rotifers (Fig. 7). The mean density value of the control group was 219.69, and that of the test group was 228.66. The concentration of calcium in the erythrocytes was also tested. Fluorescence intensity of calcium in erythrocytes was tested using the visible-light fluorescent Ca2+ fluorescent indicator Fluo-3 AM. The Fluo-3 mean value of the control group was 40.26, and that of the test group was 35.91. The intercellular concentrations of calcium ([Ca2+] in) were significantly decreased (P < 0.05) (Figs. 8 and 9), which may be due to cell rupture caused by the haemolysis of erythrocytes. The permeability of the rotifer's stomach and erythrocyte membranes have changed, in the case of both the increase and decrease in concentrations of calcium ([Ca2+] in).

Fig. 5. Concentration dependence of H. bohaiensis induced haemolysis in rabbit erythrocytes. Rabbit erythrocytes were mixed with various concentrations of H. bohaiensis (incubation time, 6 h) and incubated at 25 °C in the light (—□—) and in the dark (—■—). Each point represents the mean of triplicate measurements, and each bar represents the standard deviation.

Fig. 6. Hemolytic activities of live cell suspension (—■—), cell-free cultured supernatant (—●—), and ultrasonically ruptured cells (—▲—) of Heterocapsa bohaiensis on rabbit erythrocytes. Each sample and all erythrocytes were incubated in 96 well-plates in f/2 medium at 25 °C for 6 h, and then the haemolysis was measured as described in the text. Each point represents the mean of triplicate measurements, and each bar represents standard deviation.

Haemolytic activity of H. bohaiensis was time-dependent, and haemolysis occurred more rapidly as time increased. When the incubation time was 4 h, the haemolytic activity of the live cell suspension was close to 100%. The extent of haemolysis caused by the ultrasonicated

Fig. 7. Inverted fluorescence microscope observation of B. plicatilis cocultured with H. bohaiensis(A) and seawater(B). The B. plicatilis were stained with Fluo-3 AM (final 2 μM) after coculture at 24 h. The scale bars indicate 50 μm. 116

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It has been reported that the symptoms observed in pearl oysters exposed to H. circularisquama were vigorous clapping, shrinkage of the mantle edges and gills, and subsequent cardiac arrest, all of which are affected by Ca2+ ion flux (Nagai et al., 1996). In fact, a dramatic influx of Ca2+ has been observed in trochophore larva of short-necked clams (Ruditapes philippinarum) after exposure to H. circularisquama (Matsuyama, 1999). Previous results suggest that the H. circularisquama toxin acts on erythrocyte membranes through an effect on ion channels, in a manner similar to that proposed for palytoxin. Further studies are required to clarify the relationship between the toxicity and haemolytic activity of H. bohaiensis. Elevated fluorescence intensity was found in the stomach of exposed rotifers. A previous study reported that oxidative stress can disrupt normal physiological pathways and induce cell death by affecting the calcium ion (Ca2+) signaling pathway (Chen et al., 2012). Another study found polybrominated diphenyl ethers (PBDEs) were toxic to rotifer B. plicatilis. The intercellular concentrations of calcium ([Ca2+] in) and the expression of calmodulin (CaM) mRNA were increased. This indicates the calcium ion (Ca2+) signaling channel is involved in PBDE stress (Zhang et al., 2016). In addition, fluorescence intensity of calcium in erythrocytes was tested using the visible-light fluorescent Ca2+ fluorescent indicator Fluo-3 AM. The intercellular concentrations of calcium ([Ca2+] in) were decreased. The biological functions of Ca2+ are extremely versatile, as it controls multiple processes, such as cellular metabolism, signal transduction, and gene expression (Zhang et al., 2011). The Ca2+ control mechanism in the cells is both sensitive to oxidative stress and able to modulate it (Ya et al., 2003; Kim et al., 2011). In both haemolysis assays and ichthyotoxicity assays, MTX required Ca2+ to exert its activity (Gusovsky et al., 1990). These results suggest that H. bohaiensis has a haemolytic toxin that may be a cause of rotifer mortality, but the mechanisms causing this toxicity are mostly still unclear. Previous studies have shown that the series of cellular events triggered by MTX were presumed to occur in the following sequence: increased Ca2+ entry in cells, activation of calmodulin, promotion of phospholipase A2 activity, and finally, destruction of the cell membrane resulting from the hydrolysis of membrane lipids (Igarashi et al., 1999). Several haemolytic compounds have been isolated from phytoplankton, and most of these compounds are fatty acids, such as polyunsaturated fatty acids (PUFAs) (Landsberg, 2002). Previous studies on the hemolytic properties of dinoflagellate cell suspensions suggested the presence of watersoluble high molecular weight haemolytic compounds that may be involved in the toxicity (Yamasaki et al., 2010). Further studies are required to purify and characterize the hemolytic toxin. In conclusion, H. bohaiensis cells showed toxic effects on rotifers, and the LC50 of H. bohaiensis against B. plicatilis has been estimated to be approximately 1.36 × 104 cells mL−1 after 24 h and 4.16 × 103 cells mL−1 after 48 h. Significant rotifer growth suppression was observed upon exposure to the live cell suspension and the ultrasonically ruptured cells of H. bohaiensis. The results of haemolytic activity assays were consistent with these observations. Moreover, H. bohaiensis cells caused haemolysis of rabbit erythrocytes in a cell density-dependent and time-dependent manner. The concentration of Ca2+ in the rotifer increased, indicating that the hemolytic toxin might be a cause of rotifer mortality.

Fig. 8. Representative original histograms of Ca2+-dependent Fluo-3 AM fluorescence from erythrocytes incubated for 6 h in seawater (A) or with H. bohaiensis (B).

Fig. 9. The effect of H. bohaiensis on calcium was determined using flow cytometric analysis with the fluorescent probe Fluo-3/AM. Each value represents the mean of triplicate measurements, and each bar represents the standard deviation.

4. Discussion The algae H. bohaiensis is a newly described species of dinoflagellate associated with Penaeus japonicus and larvae of Eriocheir sinensis in a coastal pond of Liaodong Bay China. The algae H. bohaiensis had a significant lethal effect on the rotifer B. plicatilis. Pearl oysters exposed to ＞106 cells mL−1 H. circularisquama in the laboratory immediately contracted their mantles markedly, closed their valves, became paralyzed, and eventually died (Nagai et al., 1996). The LC50 in this study showed a significant lethal effect of H. bohaiensis to B. plicatilis. Similar to the case for H. bohaiensis, the presence of toxins on the cell surface of H. circularisquama and the importance of intact cells for the lethal effect on the zooplankton rotifer were proposed (Kim et al., 2000). A previous study found that the haemolytic activities of H. circularisquama strains correlate to the potency of their shellfish toxicity (Kim et al., 2002).

Acknowledgement The project was supported by “the Fundamental Research Funds for the Central Universities”DUT17JC41. We are very grateful to the reviewers for their helpful comments, which improved considerably the early draft of this paper [CG]. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.hal.2019.03.007. 117

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