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Control of ichthyotoxic Cochlodinium polykrikoides using the mixotrophic dinoflagellate Alexandrium pohangense: A potential effective sustainable method
Harmful Algae 63 (2017) 109–118
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Control of ichthyotoxic Cochlodinium polykrikoides using the mixotrophic dinoflagellate Alexandrium pohangense: A potential effective sustainable method An Suk Lima,b , Hae Jin Jeongb,c,* , Ji Hye Kima , Sung Yeon Leea a Brain Korea 21 Plus Program, School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 08826, Republic of Korea b School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 08826, Republic of Korea c Advanced Institutes of Convergence Technology, Suwon, Gyeonggi-do, 16229, Republic of Korea
A R T I C L E I N F O
Article history: Received 14 September 2016 Received in revised form 3 February 2017 Accepted 3 February 2017 Available online 17 February 2017 Keywords: Biological control Harmful algal bloom Olive flounder Red tide
A B S T R A C T
Red tides dominated by Cochlodinium polykrikoides often lead to great economic losses and some methods of controlling these red tides have been developed. However, due to possible adverse effects and the short persistence of their control actions, safer and more effective sustainable methods should be developed. The non-toxic dinoflagellate Alexandrium pohangense is known to grow well mixotrophically feeding on C. polykrikoides, and populations are also maintained by photosynthesis. Thus, compared with other methods, the use of mass-cultured A. pohangense is safer and the effects can be maintained in the long term. To develop an effective method, the concentrations of A. pohangense cells and culture filtrate resulting in the death of C. polykrikoides cells were determined by adding the cells or filtrates to cultured and natural populations of C. polykrikoides. Cultures containing 800 A. pohangense cells ml1 eliminated almost all cultured C. polykrikoides cells at a concentration of 1000 cells ml1 within 24 h. Furthermore, the addition of A. pohangense cultures at a concentration of 800 cells ml1 to C. polykrikoides populations from a red-tide patch resulted in the death of most C. polykrikoides cells (99.8%) within 24 h. This addition of A. pohangense cells also lowered the abundances of total phototrophic dinoflagellates excluding C. polykrikoides, but did not lower the abundance of total diatoms. Filtrate from 800 cells ml1 A. pohangense cultures reduced the population of cultured C. polykrikoides by 80% within 48 h. This suggests that A. pohangense cells eliminate C. polykrikoides by feeding and releasing extracellular compounds. Over time, A. pohangense concentrations gradually increased when incubated with C. polykrikoides. Thus, an increase in the concentration of A. pohangense by feeding may lead to A. pohangense cells eliminating more C. polykrikoides cells in larger volumes. Based on the results of this study, a 1 m3 stock culture of A. pohangense at 4000 cells ml1 is calculated to remove all C. polykrikoides cells in ca. 200 m3 within 6 days. Furthermore, maintenance of A. pohangense populations through photosynthesis prepared A. pohangense to eliminate C. polykrikoides cells in future red-tide patches. Moreover, incubation of A. pohangense at 2000 cells ml1 with juvenile olive flounder Paralichthys olivaceus for 3 days did not result in the death of fish. Therefore, the method developed in this study is a safe and effective way of controlling C. polykrikoides populations and can be easily applied to aqua-tanks on land. © 2017 Elsevier B.V. All rights reserved.
1. Introduction The mixotrophic dinoflagellate Cochlodinium polykrikoides often forms red-tide patches in the waters of many countries and has caused great losses in aquaculture industries in Korea, Japan,
* Corresponding author at: School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 08826, Republic of Korea. E-mail address: [email protected] (H.J. Jeong). http://dx.doi.org/10.1016/j.hal.2017.02.001 1568-9883/© 2017 Elsevier B.V. All rights reserved.
Canada, and other countries (Onoue et al., 1985; Qi et al., 1993; Kim et al., 1999, 2000; Zingone and Enevoldsen, 2000; Whyte et al., 2001; Gobler et al., 2008; Imai and Kimura, 2008; Kudela et al., 2008; Mulholland et al., 2009; Park et al., 2013; Jeong et al., 2015). C. polykrikoides red tides have often resulted in the large-scale mortality of fish and/or shellfish in both coastal aqua-cages and aqua-tanks on land (Whyte et al., 2001; Fukuyo et al., 2002; Park et al., 2013; Lee et al., 2014). C. polykrikoides is known to kill finfish and shellfish in these restricted facilities by producing reactive
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oxygen species after clogging their gills (Kim et al., 1999, 2000; Tang and Gobler, 2009a,b; Dorantes-Aranda et al., 2010; Rountos et al., 2014; Griffith and Gobler, 2016). Furthermore, the accumulation of Cochlodinium cells on the bottom of land aquatanks depletes dissolved oxygen (Ryu et al., 1998; He, 2015). Thus, to prevent the death of finfish and/or shellfish in land-based aquaculture farms, it is important to remove C. polykrikoides cells in the influent seawater of land-based flow-through tanks. Global land-based aquaculture has been extended for several decades (Bostock et al., 2010). Land-based aquaculture farms use seawater, which is pumped into the aqua-tanks (Bostock et al., 2010). When seawater is taken from nearby water columns, diverse microorganisms including C. polykrikoides enter the tanks. To minimize loss due to Cochlodinium red-tide patches in aqua-tanks, several protocols have been suggested to prevent the death of finfish and/or shellfish (NFRDI, 2013; Lee et al., 2014); (1) pumping deep water in which Cochlodinium concentrations are usually low and then filtering the water before use; (2) refraining from pumping and supplying water to aqua-tanks and instead supplying liquefied and solidified oxygen into the tanks to increase the concentration of dissolved oxygen; and (3) withholding feed from fish to minimize their oxygen consumption. Despite such efforts, economic losses due to C. polykrikoides in land-based aquaculture have been steadily incurred. Thus, to directly remove C. polykrikoides from influent seawaters, several methods such as chemical treatment, UV sterilization, and screen filtrations have been developed (Kang et al., 1998; Ryu et al., 1998; Kim, 2006). However, these physico-chemical methods may have adverse effects and thus biological methods may be safer in land-based aquaculture farms. Mass cultured protist grazers have been suggested as an effective biological method of controlling red tides because some protist grazers sometimes have considerable grazing impact on populations of red tide species (Stoecker et al., 2002; Jeong et al., 2003, 2008; Tillmann, 2004; Kamiyama et al., 2005; Kim, 2006). For example, the large naked ciliates Strombidinopsis spp. are known to effectively eliminate Cochlodinium cells and are able to divide twice per day after feeding on them (Jeong et al., 2008). Thus, this has been suggested as an effective method to control Cochlodinium populations in restricted waters. However, maintaining seed populations of these ciliates during nonCochlodinium red-tide periods is not easy. Furthermore, the ciliates cannot easily be reused to eliminate Cochlodinium cells when subsequent red-tide patches occur 1–2 weeks of the ciliates eliminating all Cochlodinium cells in the first red-tide patches,
because ciliates starve to death within a few days without prey. Thus, a method in which grazers can be maintained or remain available during non-red tide events is needed. Mixotrophic protists, which are able to feed on Cochlodinium and maintain their populations photosynthetically, may be ideal grazers. The recently described mixotrophic dinoflagellate Alexandrium pohangense is known to grow well together with C. polykrikoides and can lyse C. polykrikoides cells (Lim et al., 2015a,b). Moreover, this species does not carry saxitoxin (STX)-related genes and it proved not to be toxic towards Artemia (Lim et al., 2015b; Kim et al., 2016). Thus, A. pohangense may be an ideal grazer for the removal of C. polykrikoides cells in aqua-tanks. To develop an effective method of controlling C. polykrikoides populations in aqua-tanks using mass-cultured A. pohangense, the concentrations of A. pohangense and culture filtrates able to kill all C. polykrikoides cells were determined. A. pohangense cells or filtrates were added to C. polykrikoides cultures and field seawater samples from the waters off Tongyoung, Korea in 2015. Simultaneously, the abundances of protists in the collected waters with and without cultured A. pohangense cells or culture filtrates were determined. Furthermore, to investigate possible adverse effects, survival of the juvenile olive flounder Paralichthys olivaceus was determined after the juveniles were incubated with dense A. pohangense cells for 3 days. Finally, based on the results of the incubation experiments, the culture volume of dense A. pohangense cells over time following the addition of C. polykrikoides cells, and the volume of C. polykrikoides waters treatable with these cultures were calculated. This study provides a basis for the development of an effective biological method of controlling C. polykrikoides in land-based aqua-tanks. 2. Materials and methods 2.1. Preparation of experimental organisms Alexandrium pohangense (GenBank Accession no. = LN811348) was originally isolated from plankton samples collected off the coast of Pohang, a city in southeastern South Korea, in September 2014. At that time, the water temperature and salinity were 23.3 C and 31.1, respectively (Lim et al., 2015b). A. pohangense was grown mixotrophically with Cochlodinium polykrikoides (prey concentrations = 500–1000 cells ml1) at 20 C under a 14:10 h light-dark (LD) cycle at 100 mE m2 s1 provided by cool-white fluorescent lights (Lim et al., 2015a).
Table 1 Design of experiments investigating the effects of Alexandrium pohangense cells (culture containing cells) and cell-free culture filtrates on cultured (A) and natural (B) populations of Cochlodinium polykrikoides, and juveniles of the flounder Paralichthys olivaceus (C). The numbers in the potential predator and prey columns represent the initial densities. A. Effects of A. pohangense cells and culture filtrates on cultured populations of C. polykrikoides Expt.
Species or filtrate
Density (cells ml1)
Species
Density
1 2
Alexandrium pohangense Filtrate of A. pohangense
0, 100, 200, 400, 800 Filtrates from a culture with a cell concentration of 0, 100, 200, 400, 800
Cochlodinium polykrikoides Cochlodinium polykrikoides
1000 1000
B. Effects of A. pohangense cells and culture filtrates on the natural populations of Cochlodinium (see text) Expt.
Species or filtrate
Density
Species
Density (cells ml1)
3 4
Alexandrium pohangense Filtrate of A. pohangense
800 Filtrates from a culture with a cell concentration of 800
Natural population of Cohlodinium Natural population of Cohlodinium
1500 1500
C. Effects of A. pohangense cells and culture filtrates on juveniles of Paralichthys olivaceus Expt.
Species or conditions
Density
No. of fish used
Length of fish (means SD) (cm)
5 5 5
Alexandrium pohangense Filtrate of A. pohangense Control
2000 2000 0
15 15 15
7.5 1.7 8.1 1.8 9.0 1.6
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C. polykrikoides was originally isolated from water samples collected off the coast of Tongyoung, Korea, in August 2002, when the water temperature and salinity were 22.4 C and 27.0, respectively (Jeong et al., 2004). C. polykrikoides was grown in f/ 2 seawater medium (Guillard and Ryther, 1962) at 20 C under the light conditions described above.
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2.2. Effects of different concentrations of A. pohangense cells and culture filtrate on populations of cultured C. polykrikoides Experiment 1 was designed to investigate changes in the abundance of cultured C. polykrikoides following the addition of A.
Fig. 1. Changes in the abundance (cells ml1) of Cochlodinium polykrikoides and Alexandrium pohangense cells as a function of elapsed incubation time. The abundance of C. polykrikoides in the control bottle (i.e., without added A. pohangense cells; closed circles, dot line) and those with A. pohangense cells at concentrations (closed squares, solid line) of 100 (100C; A), 200 (200C; C), 400 (400C; E), and 800 cells ml1 (800C; G). Simultaneously, the abundance of A. pohangense in the control (closed circles, dot line) and at C. polykrikoides concentrations (closed squares, solid line) of 100 (100C; B), 200 (200C; D), 400 (400C; F), and 800 cells ml1 (800C; H) was assessed. Symbols represent treatment means SE. The curve was fitted by an applied Eq. (1) using all treatments in the experiment. (A) C. polykrikoides abundance (CPA) = 1000 [1 1.37x/(x + 27.0)], r2 = 0.953; (C) CPA = 1000 [1 1.06x/(x + 5.04)], r2 = 0.987; (E) CPA = 1000 [1 1.22x/(x + 12.7)], r2 = 0.957; (G) CPA = 1000 [1 1.10x/(x + 3.71)], r2 = 0.907.
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pohangense cells, and experiment 2 involved the addition of filtrates from A. pohangense cultures (Table 1). A. pohangense was cultured mixotrophically for 2 weeks, and then photosynthetically for another 2 weeks in enriched f/2 seawater medium. Three 1-ml aliquots taken from the cultures were examined using a compound microscope to determine the concentrations of A. pohangense. Cultures of A. pohangense were filtered through 5-mm membrane filters (Millipore Ltd., Cork, Republic of Ireland) to obtain filtrates. The target concentrations of A. pohangense cells and equivalent culture filtrates (i.e., 100, 200, 400, and 800 cells ml1) were determined in 270-ml polycarbonate (PC) bottles using an autopipette to deliver predetermined volumes of known A. pohangense concentrations and freshly filtered seawater into the bottles. Furthermore, 10 ml of f/2 medium was added to each bottle. In experiment 1, triplicate 270-ml PC experimental bottles (containing mixtures of A. pohangense and Cochlodinium), triplicate Cochlodinium control bottles (Cochlodinium culture only), and triplicate A. pohangense control bottles (A. pohangense culture only) were established. Similarly, in experiment 2, triplicate 270-ml PC bottles (containing mixtures of A. pohangense cell-free culture media and Cochlodinium culture), triplicate Cochlodinium control bottles (Cochlodinium culture only), and triplicate A. pohangense control bottles (A. pohangense culture only) were established. The bottles were incubated for 72 h at 20 C under 100 mE m2 s1 and a 14:10 h LD cycle. After 2, 6, 12, 24, 48, and 72 h of
incubation, 5-ml aliquots from each bottle were subsampled and fixed with 5% Lugol’s solution. The concentrations of C. polykrikoides and A. pohangense were determined by enumerating all or >200 cells in triplicate 1-ml Sedgwick-Rafter chambers (SRC). 2.3. Effects of A. pohangense cells and culture filtrate on natural populations of C. polykrikoides During August 2015, when C. polykrikoides red tide occurred (water temperature 24.3 C, salinity 32.5), a plankton net sample was collected from the surface of coastal water near Tongyoung, South Korea and placed into a 20-l bottle. Three 1-ml aliquots from the bottle were examined under a compound microscope to determine the concentration of C. polykrikoides. Experiment 3 was designed to investigate how the addition of A. pohangense cells affects the abundance of C. polykrikoides originated from a bloom sample, under laboratory conditions, whereas experiment 4 included the addition of filtrates from A. pohangense cultures (Table 1). In experiment 3, triplicate 270-ml PC experimental bottles (containing mixtures of A. pohangense and natural water containing C. polykrikoides) and triplicate control bottles (natural water only) were established. Similarly, in experiment 4, triplicate 270-ml PC bottles (containing mixtures of A. pohangense cell-free culture media and natural water containing C. polykrikoides) and triplicate control bottles (natural water only) were established.
Fig. 2. Change in the abundance (cells ml1) of Cochlodinium polykrikoides incubated with culture filtrate from Alexandrium pohangense over time. The abundance of C. polykrikoides in the control (i.e., without added A. pohangense filtrates; closed circles, dot line) and those with added A. pohangense filtrates (closed squares, solid line) of 100 (100F; A), 200 (200F; B), 400 (400F; C), and 800 cells ml1 (800F; D). Symbols represent treatment means SE. The curve was fitted by an applied Eq. (1) using all treatments in the experiment. (B) C. polykrikoides abundance (CPA) = 1000 [1 0.88x/(x + 23.5)], r2 = 0.643; (C) CPA = 1000 [1 1.02x/(x + 24.6)], r2 = 0.896; (D) CPA = 1000 [1 1.27x/ (x + 25.8)], r2 = 0.903.
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The bottles were incubated for 24 h at 25 C under an illumination intensity of 100 mE m2 s1 with cool-white fluorescent light and a 14:10-h LD cycle. After 2, 6, 12, and 24 h incubation, a 10-ml aliquot was removed from each bottle and fixed with 5% Lugol’s solution. All or >200 protist cells in triplicate 1-ml Sedgwick-Rafter chambers in each bottle were counted.
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where C0 is the initial concentration of A. pohangense or C. polykrikoides, and Ct is the final concentration after time t. 2.5. Acute ichthyotoxic test of A. pohangense
ð2Þ
Experiment 5 was designed to investigate the effects of A. pohangense cells and culture filtrates on the survival of juvenile fish (Table 1). The flounder Paralichthys olivaceus occupies the highest percentage (51%) of aqua-cultured fish in Korea (KOSTAT, 2013) and it is one of the main affected species during C. polykrikoides red tides. Thus, juvenile flounders were used for this experiment. Approximately 200 P. olivaceus juveniles (ca. 8-cm long) were obtained from an adjacent fish farm. They were transferred into a 200-l tank with a continuous water supply, and then subjected to a 1–2-day period of adaptation before use. During this acclimation, the average temperature and salinity were 18.4 C and 32, respectively. A. pohangense cells or equivalent culture filtrate (final concentration = ca. 2000 cells ml1) were added to 8–l plastic boxes containing 4 l seawater. Air was continuously supplied. Five
Fig. 3. Change in the abundance (cells ml1) of Cochlodinium polykrikoides and Alexandrium pohangense in a natural red-tide water over time. The water was collected from the coast of Tongyoung, Korea. (A) The abundances of C. polykrikoides in the control (i.e., without added A. pohangense cells and filtrates; closed squares, dot line) and those with A. pohangense cells at 800 cells ml1 (closed circles, thick solid line) and equivalent A. pohangense culture filtrate (open circles, thin solid line). (B) The abundance of A. pohangense (closed circles, thick solid line) in mixtures of C. polykrikoides and A. pohangense. Symbols represent treatment means SE.
Fig. 4. Change in the abundance (cells ml1) of total diatoms (Dia) and phototrophic dinoflagellates excluding Cochlodinium polykrikoides (PDEC) in the natural red-tide water as a function of elapsed incubation times. The water was collected from the coast of Tongyoung, Korea. (A) The abundance of Dia in the control (closed squares, dot line) and those with Alexandrium pohangense cells at 800 cells ml1 (closed circles, thick solid line) and equivalent A. pohangense culture filtrate (open circles, thin solid line). (B) The abundance of PDEC in the control (closed squares, dot line) and with A. pohangense cells at 800 cells ml1 (closed circles, thick solid line) and equivalent A. pohangense culture filtrate (open circles, thin solid line). Symbols represent treatment means SE.
2.4. Data analysis Data on the abundance of C. polykrikoides (ACp) were fitted to the following equation: ACp (cells ml1) = Amax [1 (a x/(b + x))]
(1) 1
where x is the A. pohangense concentration (cells ml ) or equivalent culture filtrate. The value of Amax was 1000 for all experiments. Data were iteratively fitted to the model using DeltaGraph1 (SPSS Inc., Chicago, USA). The specific growth rates of A. pohangense and C. polykrikoides in experimental and control bottles were calculated as follows:
m¼
lnðCt =C0 Þ t
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juveniles were introduced into each of triplicate experimental boxes (containing fish and A. pohangense cells or filtrates). In addition, 5 juveniles were added to triplicate control boxes (fish only). The conditions of the fish were examined every 12 h for 72 h. Furthermore, the water temperature, salinity, pH, and dissolved oxygen in the containers were measured every day using a YSI Professional Plus instrument (YSI Inc., Yellow Springs, OH, USA). 3. Results 3.1. Effects of A. pohangense cell concentration and culture filtrate on the survival of Cochlodinium At a concentration of 100 A. pohangense cells ml1, the abundance of C. polykrikoides in the experimental bottle slowly decreased with increasing incubation time and reduced <10 cells ml1 at 72 h, while that in the control bottles gradually increased over the 72-h incubation period (Fig. 1A). The abundance of C. polykrikoides in the experimental bottle at 6, 12, 24, 48, and 72 h was significantly lower than that at 0 h (p < 0.05, one-tailed t-test), although the abundance of C. polykrikoides at 2 h was not significantly lower than that at 0 h (p > 0.1, one-tailed t-test). Simultaneously, the abundance of A. pohangense in the
experimental bottle gradually increased from 100 to 350 cells ml1, while that in the control bottle was maintained (Fig. 1B). At a concentration of 200 A. pohangense cells ml1, the abundance of C. polykrikoides in the experimental bottle rapidly decreased at <2 h and reached 10 cells ml1 at 48 h, while that in the control bottles gradually increased over the 72-h incubation period (Fig. 1C). The abundance of C. polykrikoides in the experimental bottle at 2, 6, 12, 24, 48, and 72 h was significantly lower than that at 0 h (p < 0.05 or 0.001, one-tailed t-test). Simultaneously, the abundance of A. pohangense in the experimental bottle gradually increased from 200 to 570 cells ml1, while that in the control bottle was maintained (Fig. 1D). At a concentration of 400 A. pohangense cells ml1, the abundance of C. polykrikoides in the experimental bottle rapidly decreased at <2 h and reached 10 cells ml1 at 48 h, while that in the control bottles gradually increased over the 72-h incubation period (Fig. 1E). The abundance of C. polykrikoides in the experimental bottle at 2, 6, 12, 24, 48, and 72 h was significantly lower than that at 0 h (p < 0.05 or 0.001, one-tailed t-test). Simultaneously, the abundance of A. pohangense cells in the experimental bottle gradually increased from 390 to 940 cells ml1, while that in the control bottle was maintained (Fig. 1F). At a concentration of 800 A. pohangense cells ml1, the abundance of C. polykrikoides in the experimental bottle rapidly decreased at <2 h and reached zero at 48 h, while that in the control bottles gradually increased over the 72-h incubation period (Fig. 1G). The abundance of C. polykrikoides in the experimental bottle at 2, 6, 12, 24, 48, and 72 h was significantly lower than that at 0 h (p < 0.05 or 0.001, one-tailed t-test). Simultaneously, the abundance of A. pohangense in the experimental bottle gradually increased from 790 to 1430 cells ml1, while that in the control bottle was maintained (Fig. 1H). Over time, the abundance of C. polykrikoides in filtrates from the cultures containing A. pohangense at concentrations of 200, 400, and 800 cells ml1 gradually decreased over the 72-h incubation period, whereas that in the control gradually increased (Fig. 2). However, the abundance of C. polykrikoides in the presence of 100 cells ml1 of A. pohangense filtrates was maintained for the 72-h incubation (Fig. 2). 3.2. Effects of A. pohangense cells and filtrate on the survival of Cochlodinium from natural populations
Fig. 5. Change in the abundance (cells ml1) of heterotrophic dinoflagellates (HD) and ciliates (CIL) in the natural red-tide water over time. The water was collected from the coast of Tongyoung, Korea. (A) The abundance of HD in the control (closed squares, dot line) and with Alexandrium pohangense at 800 cells ml1 (closed circles, thick solid line) and equivalent A. pohangense culture filtrate (open circles, thin solid line). (B) The abundance of CIL in the control (closed squares, dot line) and with A. pohangense at 800 cells ml1 (closed circles, thick solid line) and equivalent A. pohangense culture filtrate (open circles, thin solid line). Symbols represent treatment means SE.
During the C. polykrikoides red tides in Tongyoung, the phototrophic dinoflagellates Karenia mikimotoi, Prorocentrum donghaiense, Prorocentrum triestinum, Scrippsiella trochoidea, Polykrikos hartmanii, Gymnodinium spp., and heterotrophic dinoflagellate Gyrodinium spp., Protoperidinium spp., Pfiesteria-like dinoflagellates, and diatom Skeletonema costatum, Pseudonitzschia spp., Thalassiosira spp., Chaetoceros spp., and the naked ciliates (<50 mm) co-occurred. Over time, the abundance of C. polykrikoides in the control bottles slowly increased, while that in the presence of 800 cells ml1 of A. pohangense or equivalent filtrates rapidly decreased after 2 h, but slowly decreased thereafter (Fig. 3A). C. polykrikoides cells incubated with A. pohangense cells were completely removed within 24 h, while those with the A. pohangense filtrates were maintained after 6 h. The abundance of C. polykrikoides incubated with A. pohangense cells at 2, 6, 12, and 24 h was significantly lower than that at 0 h (p < 0.005, one-tailed t-test). Furthermore, the abundance of C. polykrikoides incubated with the A. pohangense filtrates at 2, 6, 12, and 24 h was significantly lower than that at 0 h (p < 0.005, one-tailed t-test). However, the abundance of A. pohangense in the experiment gradually increased over the 24-h incubation period (Fig. 3B).
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Among the other protists co-occurring with C. polykrikoides, the abundance of total diatoms in the control and experimental bottles followed a similar pattern. The abundance of total diatoms slightly decreased from ca. 70 to ca. 40 cells ml1 at 6 h, but increased to 170–290 cells ml1 in both the control and experimental bottles (Fig. 4A). However, the abundance of total phototrophic dinoflagellates excluding C. polykrikoides, in the control bottles increased, while those containing A. pohangense cells and equivalent filtrates slowly decreased until 6 h and were maintained or increased thereafter (Fig. 4B). The abundance of heterotrophic dinoflagellates in the control bottles slowly increased, while the abundance in bottles containing A. pohangense cells or equivalent filtrates decreased until 6 h but slightly increased thereafter (Fig. 5A). The abundance of total ciliates in both the control and experimental bottles decreased at 6 h and was almost zero at 24 h (Fig. 5B). 3.3. Ichthyotoxicity test During the experiments, the water temperature, salinity, pH, and dissolved oxygen in the experimental plastic boxes were 17– 20 C, 31–33, 7.3–8.1, and 5.9–7.9 mg l1, respectively. Over elapsed time, P. olivaceus juveniles in the control and experimental boxes (A. pohangense cells and equivalent filtrates) survived during the 72-h incubation period. During the experiment, no abnormal behaviors, such as disorientated swimming and/or turning upside down, were observed in the fish.
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4. Discussion The results of the present study clearly show that both cells and culture filtrates of A. pohangense are effective at controlling both cultured and natural populations of C. polykrikoides. The minimum A. pohangense cell concentrations causing 100% mortality of C. polykrikoides at 24 h and 48 h were 800 cells ml1 and 200 cells ml1, respectively. Thus, depending on the intensity of a C. polykrikoides bloom, A. pohangense cell concentrations between 200 and 800 cells ml1 may be used to mitigate the bloom. However, further field experiments are still required because these results were obtained from laboratory experiments. The minimum A. pohangense cell concentration producing culture filtrates in which all culture C. polykrikoides cells were dead within 72 h was 800 cells ml1. Thus, the culture filtrates of A. pohangense are less effective materials than A. pohangense cells. Within 24 h of 800 A. pohangense cells ml1 being introduced to natural populations of C. polykrikoides at 1500 cells ml1, all C. polykrikoides cells had been removed. A. pohangense cells can remove both cultured and natural populations of C. polykrikoides at similar rates. In land-based aquaculture, seawater is pumped up and stored in flow-through tanks for recirculation. Once seawater is pumped into a large seawater reservoir tank, the waters distribute into each fish-rearing tank. Thus, if dense C. polykrikoides cells in the tank are observed, the C. polykrikoides cells may be removed by the introduction of mass cultured A. pohangense (final concentration 200 cells ml1).
Fig. 6. Schematic diagrams showing differences between heterotrophic ciliates (A1–A3) and mixtrophic dinoflagellate Alexandrium pohangense (B1–B3) as grazers removing Cochlodinium polykrikoides (Cp) when mass-cultured grazers are introduced to waters containing Cp red-tide patches in aqua-tanks on land. (A1) Introduced ciliate grazers remove Cp cells in the first arrived red-tide patch. (A2) The ciliates starve to death after eliminating all Cp cells if no prey is supplied. (A3) New ciliate populations should be introduced to remove Cp cells in newly arrived Cp red-tide patch. (B1) Introduced Alexandrium removes Cp cells in the first arrived Cp red-tide patch. (B2) The Alexandrium cells are maintained for a long time after the elimination of all Cp cells. (A3) The maintained Alexandrium populations can remove Cp cells in a newly arrived Cp red tide patch.
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A. pohangense can grow fast on C. polykrikoides prey (maximum mixotrophic growth rate = 0.5 d1), but they can also grow slowly photosynthetically or be maintained without the addition of prey (phototrophic growth rate = 0.1 d1 at 20 C, 100 mE m2 s1; Lim et al., 2015a). Thus, it is much easier to increase or maintain A. pohangense populations than those of heterotrophic protist grazers, to which optimal prey should be constantly supplied. Some heterotrophic protist grazers have been shown to be effective grazers on C. polykrikoides, and have thus been suggested as biological control agents. Jeong et al. (2008) suggested that large-scale culture of the large naked ciliates Strombidinopsis jeokjo could be used to control C. polykrikoides red tides. This ciliate can be isolated from natural seawaters, and grows rapidly upon ingestion of C. polykrikoides (growth and ingestion rates = 0.72 d1 and 72 cells grazer1 d1). However, S. jeokjo is not easy to maintain because it needs to be fed with optimal prey every day. This heterotrophic ciliate divides more than once a day, but it starves to death within 2 days without prey. In natural environments, S. jeokjo feeds on C. polykrikoides cells until complete removal and grazes other organisms. Thus, S. jeokjo can grow continuously. However, in land-based aqua-tanks, S. jeokjo may starve to death within 2–3 days of the complete removal of C. polykrikoides. Thus, new populations of cultured S. jeokjo would need to be introduced when C. polykrikoides blooms again. However, A. pohangense is able to survive photosynthetically once C. polykrikoides has been eliminated. Thus, it is not necessary to introduce new populations of A. pohangense to remove newly arrived C. polykrikoides red tide patches (Fig. 6). A. pohangense may have an advantage over S. jeokjo in the maintenance of stock cultures for controlling C. polykrikoides in land-based aqua-tanks. Furthermore, using A. pohangense cultures to control C. polykrikoides red tides may be more cost-efficient than using S. jeokjo cultures because an additional system for S. jeokjo would be required to maintain. This method of controlling C. polykrikoides red-tide patches in land-based aqua-tanks may be cost-effective because populations of A. pohangense increase when feeding on C. polykrikoides. These larger populations can remove more C. polykrikoides cells. An A. pohangense culture with a concentration of 200 cells ml1 is able to remove all C. polykrikoides cells within 2 days in aqua-tanks when the initial concentration of C. polykrikoides is 1000 cells ml1 as in experiment 1. Based on the results of the present study (i.e., the growth rates of A. pohangense when feeding on red-tide blooming C. polykrikoides = 0.41 d1), the population of the A. pohangense culture is calculated to increase by 60% every day. Thus, theoretically the 1 m3 A. pohangense cultures of 4000 cells ml1 increase to 2.27, 5.15, and 11.7 m3 at the same concentration after 2, 4, and 6 days, respectively (Fig. 7A). In turn, these populations can remove all C. polykrikoides cells within 45, 103, and 234 m3 mixed waters, respectively (final A. pohangense and C. polykrikoides cell concentrations = 200 and ca. 1000 cells ml1, respectively, in the mixed waters of A. pohangense culture and C. polykrikoides water, Fig. 7B). The cost for the production of 1 m3 of A. pohangense stock culture is approximately 250 US dollars in Korea when calculated by summing all costs such as purchase of f/2 medium for cultivating C. polykrikoides as prey, labor, and electricity charge. The introduced 1 m3 of A. pohangense stock culture can remove C. polykrikoides cells in 20 m3 waters within 2 days in an aqua-tank. Thus, this method is cost-effective and could be used to remove C. polykrikoides cells in land-based aqua-tanks. The patterns in temporal variations in the abundances of total diatoms and ciliates in the experimental and control bottles after being with both A. pohangense cells and filtrates were similar, while those of the other phototrophic dinoflagellates and heterotrophic dinoflagellates were different. However, the abundance of heterotrophic dinoflagellates incubated with A.
Fig. 7. Calculated volumes of Alexandrium pohangense stock cultures and treatable waters containing Cochlodinium polykrikoides red-tide patches. (A) Volume (m3) of the Alexandrium stock cultures containing 4000 A. pohangense cells ml1 as a function of elapsed time following the addition of a 1 m3 Alexandrium stock culture to a population of Cochlodinium (1000 cells ml1), calculated with the assumption that the growth rate of A. pohangense feeding on C. polykrikoides is 0.41 day1 (see text). (B) Calculated volumes (m3) of treatable waters containing C. polykrikoides using A. pohangense culture (final concentration = 200 cells ml1), which can be obtained by a 1/20 dilution of the Alexandrium stock culture.
pohangense cells or equivalent filtrates almost maintained. Thus, introduced A. pohangense cells and filtrates do not cause marked adverse effects on total diatoms, heterotrophic dinoflagellates, and ciliates, but may cause some adverse effects on other phototrophic dinoflagellates. However, the dominant species in the other phototrophic dinoflagellates was Karenia mikimotoi, which is harmful. Thus, this method may have the advantage of removing other harmful species, although this still needs to be investigated. In the last few decades, several physical and chemical methods of controlling C. polykrikoides red tides, such as clay and dredged sediment disposal, and spraying with sodium hypochlorite (NaOCl), copper sulfate (CuSO4), and thiazolidinedione derivative (TD49), have been developed and some of these have been used in Korean waters (Haas, 1999; Jeong et al., 2002; Sengco and Anderson, 2004; Kim, 2010; Song et al., 2010; Baek et al., 2014; Ebenzer et al., 2014). However, these methods are not speciestargeted because they can kill diverse marine organisms and some of them produce unpleasant odors (Shumway et al., 2003; Kim, 2010; Jung et al., 2015). Thus, the use of biological mitigation methods can be a safer and more sustainable technique.
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5. Conclusions The present study demonstrated that: (1) A. pohangense can control both cultured and natural population of C. polykrikoides in the laboratory condition. Addition of A. pohangense cultures at a concentration of 800 cells ml1 killed most C. polykrikoides cells in both cultured and natural populations within 24 h. (2) Culture filtrates of 800 A. pohangense cells ml1 reduced 80% of cultured C. polykrikoides populations within 48 h. Thus, A. pohangense cells remove C. polykrikoides cells by feeding and/or releasing extracellular compounds. However, A. pohangense cells or filtrates did not affect the abundance of total diatoms and heterotrophic dinoflagellates in the natural waters. (3) Moreover, none of the olive flounder juveniles incubated with 2000 cells ml1 of A. pohangense for 3 days died. Based on the results of the present study, a 1 m3 stock culture of A. pohangense with 4000 cells ml1 is calculated to remove all C. polykrikoides cells in ca. 200 m3 mixed waters in 6 days. Therefore, this method of controlling C. polykrikoides populations may result as a safe and effective method that can be easily applied to land-based aqua-tanks. Acknowledgements This paper was supported by the Useful Dinoflagellate Program of Korea Institute of Marine Science and Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries (MOF) and Management of marine organisms causing ecological disturbance and harmful effect Program of KIMST and the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2015M1A5A1041806) award to HJJ.[SS] References Baek, S.H., Shin, K., Son, M., Bae, S.W., Cho, H., Na, D.H., Kim, Y.O., Kim, S.W., 2014. Algicidal effects of yellow clay and the thiazolidinedione derivative TD49 on the fish-killing dinoflagellate Cochlodinium polykrikoides in microcosm experiments. J. Appl. Phycol. 26, 2367–2378. Bostock, J., McAndrew, B., Rishards, R., Jauncey, K., Telfer, T., Lorenzen, K., Little, D., Ross, L., Handisyde, N., Gatward, I., Corner, R., 2010. Aquaculture: global status and trends. Philos. Trans. R. Soc. B 365, 2897–2912. Dorantes-Aranda, J.J., García-de la Parra, L.M., Alonso-Rodríguez, R., Morquecho, L., Voltolina, D., 2010. Toxic effect of the harmful dinoflagellate Cochlodinium polykrikoides on the spotted rose snapper Lutjanus guttatus. Environ. Toxicol. 25 (4), 319–326. Ebenzer, V., Lim, W.A., Ki, J., 2014. Effects of the algicides CuSO4 and NaOCl on various physiological parameters in the harmful dinoflagellate Cochlodinium polykrikoides. J. Appl. Phycol. 26, 2357–2365. Fukuyo, Y., Imai, I., Kodama, M., Tamai, K., 2002. Red tide and other harmful algal blooms in Japan. In: Tayler, F.R.J., Trainer, V.L. (Eds.), Harmful Algal Blooms in the PICES Region of the North Pacific. PICES Sci. Rep. No. 23 pp. 152. Gobler, C.J., Berry, D.L., Anderson, O.R., Burson, A., Koch, F., Rodger, B.S., Moore, L.K., Goleski, J.A., Allam, B., Bowser, P., Tang, Y., Nuzzi, R., 2008. Characterization, dynamics, and ecological impacts of harmful Cochlodinium polykrikoides blooms on eastern Long Island, NY, USA. Harmful Algae 7, 293–307. Griffith, A.W., Gobler, C.J., 2016. Temperature controls the toxicity of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides. Mar. Ecol. Prog. Ser. 545, 63–76. Guillard, R.R.L., Ryther, J.H., 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Grun. Can. J. Microbiol. 8, 229– 239. Haas, C.N., 1999. Disinfection, In: Letterman, R.D. (Ed.), Water Quality and Treatment a Handbook of Community Water Supplies. 5th edition McGraw-Hill, New York, pp. 14.1–14.6 American water works association. He, P., 2015. Harmful algal blooms. In: Sahoo, D., Seckbach, J. (Eds.), The Algae World. Springer, Netherlands, pp. 339–356. Imai, I., Kimura, S., 2008. Resistance of the fish-killing dinoflagellate Cochlodinium polykrikoides against algicidal bacteria isolated from the coastal Sea of Japan. Harmful Algae 7 (3), 360–367. Jeong, H.J., Kim, H.R., Kim, G.Y., Park, K.H., Kim, S.T., Yoo, Y.D., Song, J.Y., Kim, J.S., Seong, K.A., Yih, Y.H., Pae, S.J., Lee, C.H., Huh, M.D., Lee, S.H., 2002. NaOCl produced by electrolysis of natural seawater as a potential method to control marine red-tide dinoflagellate. Phycologia 41 (6), 643–656. Jeong, H.J., Kim, J.S., Yoo, Y.D., Kim, S.T., Kim, T.H., Park, M.G., Lee, C.H., Seong, K.A., Kang, N.S., Shim, J.H., 2003. Feeding by the heterotrophic dinoflagellate Oxyrrhis marina on the red-tide raphidophyte Heterosigma akashiwo: a potential
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Control of ichthyotoxic Cochlodinium polykrikoides using the mixotrophic dinoflagellate Alexandrium pohangense: A potential effective sustainable method An Suk Lima,b , Hae Jin Jeongb,c,* , Ji Hye Kima , Sung Yeon Leea a Brain Korea 21 Plus Program, School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 08826, Republic of Korea b School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 08826, Republic of Korea c Advanced Institutes of Convergence Technology, Suwon, Gyeonggi-do, 16229, Republic of Korea
A R T I C L E I N F O
Article history: Received 14 September 2016 Received in revised form 3 February 2017 Accepted 3 February 2017 Available online 17 February 2017 Keywords: Biological control Harmful algal bloom Olive flounder Red tide
A B S T R A C T
Red tides dominated by Cochlodinium polykrikoides often lead to great economic losses and some methods of controlling these red tides have been developed. However, due to possible adverse effects and the short persistence of their control actions, safer and more effective sustainable methods should be developed. The non-toxic dinoflagellate Alexandrium pohangense is known to grow well mixotrophically feeding on C. polykrikoides, and populations are also maintained by photosynthesis. Thus, compared with other methods, the use of mass-cultured A. pohangense is safer and the effects can be maintained in the long term. To develop an effective method, the concentrations of A. pohangense cells and culture filtrate resulting in the death of C. polykrikoides cells were determined by adding the cells or filtrates to cultured and natural populations of C. polykrikoides. Cultures containing 800 A. pohangense cells ml1 eliminated almost all cultured C. polykrikoides cells at a concentration of 1000 cells ml1 within 24 h. Furthermore, the addition of A. pohangense cultures at a concentration of 800 cells ml1 to C. polykrikoides populations from a red-tide patch resulted in the death of most C. polykrikoides cells (99.8%) within 24 h. This addition of A. pohangense cells also lowered the abundances of total phototrophic dinoflagellates excluding C. polykrikoides, but did not lower the abundance of total diatoms. Filtrate from 800 cells ml1 A. pohangense cultures reduced the population of cultured C. polykrikoides by 80% within 48 h. This suggests that A. pohangense cells eliminate C. polykrikoides by feeding and releasing extracellular compounds. Over time, A. pohangense concentrations gradually increased when incubated with C. polykrikoides. Thus, an increase in the concentration of A. pohangense by feeding may lead to A. pohangense cells eliminating more C. polykrikoides cells in larger volumes. Based on the results of this study, a 1 m3 stock culture of A. pohangense at 4000 cells ml1 is calculated to remove all C. polykrikoides cells in ca. 200 m3 within 6 days. Furthermore, maintenance of A. pohangense populations through photosynthesis prepared A. pohangense to eliminate C. polykrikoides cells in future red-tide patches. Moreover, incubation of A. pohangense at 2000 cells ml1 with juvenile olive flounder Paralichthys olivaceus for 3 days did not result in the death of fish. Therefore, the method developed in this study is a safe and effective way of controlling C. polykrikoides populations and can be easily applied to aqua-tanks on land. © 2017 Elsevier B.V. All rights reserved.
1. Introduction The mixotrophic dinoflagellate Cochlodinium polykrikoides often forms red-tide patches in the waters of many countries and has caused great losses in aquaculture industries in Korea, Japan,
* Corresponding author at: School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 08826, Republic of Korea. E-mail address: [email protected] (H.J. Jeong). http://dx.doi.org/10.1016/j.hal.2017.02.001 1568-9883/© 2017 Elsevier B.V. All rights reserved.
Canada, and other countries (Onoue et al., 1985; Qi et al., 1993; Kim et al., 1999, 2000; Zingone and Enevoldsen, 2000; Whyte et al., 2001; Gobler et al., 2008; Imai and Kimura, 2008; Kudela et al., 2008; Mulholland et al., 2009; Park et al., 2013; Jeong et al., 2015). C. polykrikoides red tides have often resulted in the large-scale mortality of fish and/or shellfish in both coastal aqua-cages and aqua-tanks on land (Whyte et al., 2001; Fukuyo et al., 2002; Park et al., 2013; Lee et al., 2014). C. polykrikoides is known to kill finfish and shellfish in these restricted facilities by producing reactive
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oxygen species after clogging their gills (Kim et al., 1999, 2000; Tang and Gobler, 2009a,b; Dorantes-Aranda et al., 2010; Rountos et al., 2014; Griffith and Gobler, 2016). Furthermore, the accumulation of Cochlodinium cells on the bottom of land aquatanks depletes dissolved oxygen (Ryu et al., 1998; He, 2015). Thus, to prevent the death of finfish and/or shellfish in land-based aquaculture farms, it is important to remove C. polykrikoides cells in the influent seawater of land-based flow-through tanks. Global land-based aquaculture has been extended for several decades (Bostock et al., 2010). Land-based aquaculture farms use seawater, which is pumped into the aqua-tanks (Bostock et al., 2010). When seawater is taken from nearby water columns, diverse microorganisms including C. polykrikoides enter the tanks. To minimize loss due to Cochlodinium red-tide patches in aqua-tanks, several protocols have been suggested to prevent the death of finfish and/or shellfish (NFRDI, 2013; Lee et al., 2014); (1) pumping deep water in which Cochlodinium concentrations are usually low and then filtering the water before use; (2) refraining from pumping and supplying water to aqua-tanks and instead supplying liquefied and solidified oxygen into the tanks to increase the concentration of dissolved oxygen; and (3) withholding feed from fish to minimize their oxygen consumption. Despite such efforts, economic losses due to C. polykrikoides in land-based aquaculture have been steadily incurred. Thus, to directly remove C. polykrikoides from influent seawaters, several methods such as chemical treatment, UV sterilization, and screen filtrations have been developed (Kang et al., 1998; Ryu et al., 1998; Kim, 2006). However, these physico-chemical methods may have adverse effects and thus biological methods may be safer in land-based aquaculture farms. Mass cultured protist grazers have been suggested as an effective biological method of controlling red tides because some protist grazers sometimes have considerable grazing impact on populations of red tide species (Stoecker et al., 2002; Jeong et al., 2003, 2008; Tillmann, 2004; Kamiyama et al., 2005; Kim, 2006). For example, the large naked ciliates Strombidinopsis spp. are known to effectively eliminate Cochlodinium cells and are able to divide twice per day after feeding on them (Jeong et al., 2008). Thus, this has been suggested as an effective method to control Cochlodinium populations in restricted waters. However, maintaining seed populations of these ciliates during nonCochlodinium red-tide periods is not easy. Furthermore, the ciliates cannot easily be reused to eliminate Cochlodinium cells when subsequent red-tide patches occur 1–2 weeks of the ciliates eliminating all Cochlodinium cells in the first red-tide patches,
because ciliates starve to death within a few days without prey. Thus, a method in which grazers can be maintained or remain available during non-red tide events is needed. Mixotrophic protists, which are able to feed on Cochlodinium and maintain their populations photosynthetically, may be ideal grazers. The recently described mixotrophic dinoflagellate Alexandrium pohangense is known to grow well together with C. polykrikoides and can lyse C. polykrikoides cells (Lim et al., 2015a,b). Moreover, this species does not carry saxitoxin (STX)-related genes and it proved not to be toxic towards Artemia (Lim et al., 2015b; Kim et al., 2016). Thus, A. pohangense may be an ideal grazer for the removal of C. polykrikoides cells in aqua-tanks. To develop an effective method of controlling C. polykrikoides populations in aqua-tanks using mass-cultured A. pohangense, the concentrations of A. pohangense and culture filtrates able to kill all C. polykrikoides cells were determined. A. pohangense cells or filtrates were added to C. polykrikoides cultures and field seawater samples from the waters off Tongyoung, Korea in 2015. Simultaneously, the abundances of protists in the collected waters with and without cultured A. pohangense cells or culture filtrates were determined. Furthermore, to investigate possible adverse effects, survival of the juvenile olive flounder Paralichthys olivaceus was determined after the juveniles were incubated with dense A. pohangense cells for 3 days. Finally, based on the results of the incubation experiments, the culture volume of dense A. pohangense cells over time following the addition of C. polykrikoides cells, and the volume of C. polykrikoides waters treatable with these cultures were calculated. This study provides a basis for the development of an effective biological method of controlling C. polykrikoides in land-based aqua-tanks. 2. Materials and methods 2.1. Preparation of experimental organisms Alexandrium pohangense (GenBank Accession no. = LN811348) was originally isolated from plankton samples collected off the coast of Pohang, a city in southeastern South Korea, in September 2014. At that time, the water temperature and salinity were 23.3 C and 31.1, respectively (Lim et al., 2015b). A. pohangense was grown mixotrophically with Cochlodinium polykrikoides (prey concentrations = 500–1000 cells ml1) at 20 C under a 14:10 h light-dark (LD) cycle at 100 mE m2 s1 provided by cool-white fluorescent lights (Lim et al., 2015a).
Table 1 Design of experiments investigating the effects of Alexandrium pohangense cells (culture containing cells) and cell-free culture filtrates on cultured (A) and natural (B) populations of Cochlodinium polykrikoides, and juveniles of the flounder Paralichthys olivaceus (C). The numbers in the potential predator and prey columns represent the initial densities. A. Effects of A. pohangense cells and culture filtrates on cultured populations of C. polykrikoides Expt.
Species or filtrate
Density (cells ml1)
Species
Density
1 2
Alexandrium pohangense Filtrate of A. pohangense
0, 100, 200, 400, 800 Filtrates from a culture with a cell concentration of 0, 100, 200, 400, 800
Cochlodinium polykrikoides Cochlodinium polykrikoides
1000 1000
B. Effects of A. pohangense cells and culture filtrates on the natural populations of Cochlodinium (see text) Expt.
Species or filtrate
Density
Species
Density (cells ml1)
3 4
Alexandrium pohangense Filtrate of A. pohangense
800 Filtrates from a culture with a cell concentration of 800
Natural population of Cohlodinium Natural population of Cohlodinium
1500 1500
C. Effects of A. pohangense cells and culture filtrates on juveniles of Paralichthys olivaceus Expt.
Species or conditions
Density
No. of fish used
Length of fish (means SD) (cm)
5 5 5
Alexandrium pohangense Filtrate of A. pohangense Control
2000 2000 0
15 15 15
7.5 1.7 8.1 1.8 9.0 1.6
A.S. Lim et al. / Harmful Algae 63 (2017) 109–118
C. polykrikoides was originally isolated from water samples collected off the coast of Tongyoung, Korea, in August 2002, when the water temperature and salinity were 22.4 C and 27.0, respectively (Jeong et al., 2004). C. polykrikoides was grown in f/ 2 seawater medium (Guillard and Ryther, 1962) at 20 C under the light conditions described above.
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2.2. Effects of different concentrations of A. pohangense cells and culture filtrate on populations of cultured C. polykrikoides Experiment 1 was designed to investigate changes in the abundance of cultured C. polykrikoides following the addition of A.
Fig. 1. Changes in the abundance (cells ml1) of Cochlodinium polykrikoides and Alexandrium pohangense cells as a function of elapsed incubation time. The abundance of C. polykrikoides in the control bottle (i.e., without added A. pohangense cells; closed circles, dot line) and those with A. pohangense cells at concentrations (closed squares, solid line) of 100 (100C; A), 200 (200C; C), 400 (400C; E), and 800 cells ml1 (800C; G). Simultaneously, the abundance of A. pohangense in the control (closed circles, dot line) and at C. polykrikoides concentrations (closed squares, solid line) of 100 (100C; B), 200 (200C; D), 400 (400C; F), and 800 cells ml1 (800C; H) was assessed. Symbols represent treatment means SE. The curve was fitted by an applied Eq. (1) using all treatments in the experiment. (A) C. polykrikoides abundance (CPA) = 1000 [1 1.37x/(x + 27.0)], r2 = 0.953; (C) CPA = 1000 [1 1.06x/(x + 5.04)], r2 = 0.987; (E) CPA = 1000 [1 1.22x/(x + 12.7)], r2 = 0.957; (G) CPA = 1000 [1 1.10x/(x + 3.71)], r2 = 0.907.
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pohangense cells, and experiment 2 involved the addition of filtrates from A. pohangense cultures (Table 1). A. pohangense was cultured mixotrophically for 2 weeks, and then photosynthetically for another 2 weeks in enriched f/2 seawater medium. Three 1-ml aliquots taken from the cultures were examined using a compound microscope to determine the concentrations of A. pohangense. Cultures of A. pohangense were filtered through 5-mm membrane filters (Millipore Ltd., Cork, Republic of Ireland) to obtain filtrates. The target concentrations of A. pohangense cells and equivalent culture filtrates (i.e., 100, 200, 400, and 800 cells ml1) were determined in 270-ml polycarbonate (PC) bottles using an autopipette to deliver predetermined volumes of known A. pohangense concentrations and freshly filtered seawater into the bottles. Furthermore, 10 ml of f/2 medium was added to each bottle. In experiment 1, triplicate 270-ml PC experimental bottles (containing mixtures of A. pohangense and Cochlodinium), triplicate Cochlodinium control bottles (Cochlodinium culture only), and triplicate A. pohangense control bottles (A. pohangense culture only) were established. Similarly, in experiment 2, triplicate 270-ml PC bottles (containing mixtures of A. pohangense cell-free culture media and Cochlodinium culture), triplicate Cochlodinium control bottles (Cochlodinium culture only), and triplicate A. pohangense control bottles (A. pohangense culture only) were established. The bottles were incubated for 72 h at 20 C under 100 mE m2 s1 and a 14:10 h LD cycle. After 2, 6, 12, 24, 48, and 72 h of
incubation, 5-ml aliquots from each bottle were subsampled and fixed with 5% Lugol’s solution. The concentrations of C. polykrikoides and A. pohangense were determined by enumerating all or >200 cells in triplicate 1-ml Sedgwick-Rafter chambers (SRC). 2.3. Effects of A. pohangense cells and culture filtrate on natural populations of C. polykrikoides During August 2015, when C. polykrikoides red tide occurred (water temperature 24.3 C, salinity 32.5), a plankton net sample was collected from the surface of coastal water near Tongyoung, South Korea and placed into a 20-l bottle. Three 1-ml aliquots from the bottle were examined under a compound microscope to determine the concentration of C. polykrikoides. Experiment 3 was designed to investigate how the addition of A. pohangense cells affects the abundance of C. polykrikoides originated from a bloom sample, under laboratory conditions, whereas experiment 4 included the addition of filtrates from A. pohangense cultures (Table 1). In experiment 3, triplicate 270-ml PC experimental bottles (containing mixtures of A. pohangense and natural water containing C. polykrikoides) and triplicate control bottles (natural water only) were established. Similarly, in experiment 4, triplicate 270-ml PC bottles (containing mixtures of A. pohangense cell-free culture media and natural water containing C. polykrikoides) and triplicate control bottles (natural water only) were established.
Fig. 2. Change in the abundance (cells ml1) of Cochlodinium polykrikoides incubated with culture filtrate from Alexandrium pohangense over time. The abundance of C. polykrikoides in the control (i.e., without added A. pohangense filtrates; closed circles, dot line) and those with added A. pohangense filtrates (closed squares, solid line) of 100 (100F; A), 200 (200F; B), 400 (400F; C), and 800 cells ml1 (800F; D). Symbols represent treatment means SE. The curve was fitted by an applied Eq. (1) using all treatments in the experiment. (B) C. polykrikoides abundance (CPA) = 1000 [1 0.88x/(x + 23.5)], r2 = 0.643; (C) CPA = 1000 [1 1.02x/(x + 24.6)], r2 = 0.896; (D) CPA = 1000 [1 1.27x/ (x + 25.8)], r2 = 0.903.
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The bottles were incubated for 24 h at 25 C under an illumination intensity of 100 mE m2 s1 with cool-white fluorescent light and a 14:10-h LD cycle. After 2, 6, 12, and 24 h incubation, a 10-ml aliquot was removed from each bottle and fixed with 5% Lugol’s solution. All or >200 protist cells in triplicate 1-ml Sedgwick-Rafter chambers in each bottle were counted.
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where C0 is the initial concentration of A. pohangense or C. polykrikoides, and Ct is the final concentration after time t. 2.5. Acute ichthyotoxic test of A. pohangense
ð2Þ
Experiment 5 was designed to investigate the effects of A. pohangense cells and culture filtrates on the survival of juvenile fish (Table 1). The flounder Paralichthys olivaceus occupies the highest percentage (51%) of aqua-cultured fish in Korea (KOSTAT, 2013) and it is one of the main affected species during C. polykrikoides red tides. Thus, juvenile flounders were used for this experiment. Approximately 200 P. olivaceus juveniles (ca. 8-cm long) were obtained from an adjacent fish farm. They were transferred into a 200-l tank with a continuous water supply, and then subjected to a 1–2-day period of adaptation before use. During this acclimation, the average temperature and salinity were 18.4 C and 32, respectively. A. pohangense cells or equivalent culture filtrate (final concentration = ca. 2000 cells ml1) were added to 8–l plastic boxes containing 4 l seawater. Air was continuously supplied. Five
Fig. 3. Change in the abundance (cells ml1) of Cochlodinium polykrikoides and Alexandrium pohangense in a natural red-tide water over time. The water was collected from the coast of Tongyoung, Korea. (A) The abundances of C. polykrikoides in the control (i.e., without added A. pohangense cells and filtrates; closed squares, dot line) and those with A. pohangense cells at 800 cells ml1 (closed circles, thick solid line) and equivalent A. pohangense culture filtrate (open circles, thin solid line). (B) The abundance of A. pohangense (closed circles, thick solid line) in mixtures of C. polykrikoides and A. pohangense. Symbols represent treatment means SE.
Fig. 4. Change in the abundance (cells ml1) of total diatoms (Dia) and phototrophic dinoflagellates excluding Cochlodinium polykrikoides (PDEC) in the natural red-tide water as a function of elapsed incubation times. The water was collected from the coast of Tongyoung, Korea. (A) The abundance of Dia in the control (closed squares, dot line) and those with Alexandrium pohangense cells at 800 cells ml1 (closed circles, thick solid line) and equivalent A. pohangense culture filtrate (open circles, thin solid line). (B) The abundance of PDEC in the control (closed squares, dot line) and with A. pohangense cells at 800 cells ml1 (closed circles, thick solid line) and equivalent A. pohangense culture filtrate (open circles, thin solid line). Symbols represent treatment means SE.
2.4. Data analysis Data on the abundance of C. polykrikoides (ACp) were fitted to the following equation: ACp (cells ml1) = Amax [1 (a x/(b + x))]
(1) 1
where x is the A. pohangense concentration (cells ml ) or equivalent culture filtrate. The value of Amax was 1000 for all experiments. Data were iteratively fitted to the model using DeltaGraph1 (SPSS Inc., Chicago, USA). The specific growth rates of A. pohangense and C. polykrikoides in experimental and control bottles were calculated as follows:
m¼
lnðCt =C0 Þ t
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juveniles were introduced into each of triplicate experimental boxes (containing fish and A. pohangense cells or filtrates). In addition, 5 juveniles were added to triplicate control boxes (fish only). The conditions of the fish were examined every 12 h for 72 h. Furthermore, the water temperature, salinity, pH, and dissolved oxygen in the containers were measured every day using a YSI Professional Plus instrument (YSI Inc., Yellow Springs, OH, USA). 3. Results 3.1. Effects of A. pohangense cell concentration and culture filtrate on the survival of Cochlodinium At a concentration of 100 A. pohangense cells ml1, the abundance of C. polykrikoides in the experimental bottle slowly decreased with increasing incubation time and reduced <10 cells ml1 at 72 h, while that in the control bottles gradually increased over the 72-h incubation period (Fig. 1A). The abundance of C. polykrikoides in the experimental bottle at 6, 12, 24, 48, and 72 h was significantly lower than that at 0 h (p < 0.05, one-tailed t-test), although the abundance of C. polykrikoides at 2 h was not significantly lower than that at 0 h (p > 0.1, one-tailed t-test). Simultaneously, the abundance of A. pohangense in the
experimental bottle gradually increased from 100 to 350 cells ml1, while that in the control bottle was maintained (Fig. 1B). At a concentration of 200 A. pohangense cells ml1, the abundance of C. polykrikoides in the experimental bottle rapidly decreased at <2 h and reached 10 cells ml1 at 48 h, while that in the control bottles gradually increased over the 72-h incubation period (Fig. 1C). The abundance of C. polykrikoides in the experimental bottle at 2, 6, 12, 24, 48, and 72 h was significantly lower than that at 0 h (p < 0.05 or 0.001, one-tailed t-test). Simultaneously, the abundance of A. pohangense in the experimental bottle gradually increased from 200 to 570 cells ml1, while that in the control bottle was maintained (Fig. 1D). At a concentration of 400 A. pohangense cells ml1, the abundance of C. polykrikoides in the experimental bottle rapidly decreased at <2 h and reached 10 cells ml1 at 48 h, while that in the control bottles gradually increased over the 72-h incubation period (Fig. 1E). The abundance of C. polykrikoides in the experimental bottle at 2, 6, 12, 24, 48, and 72 h was significantly lower than that at 0 h (p < 0.05 or 0.001, one-tailed t-test). Simultaneously, the abundance of A. pohangense cells in the experimental bottle gradually increased from 390 to 940 cells ml1, while that in the control bottle was maintained (Fig. 1F). At a concentration of 800 A. pohangense cells ml1, the abundance of C. polykrikoides in the experimental bottle rapidly decreased at <2 h and reached zero at 48 h, while that in the control bottles gradually increased over the 72-h incubation period (Fig. 1G). The abundance of C. polykrikoides in the experimental bottle at 2, 6, 12, 24, 48, and 72 h was significantly lower than that at 0 h (p < 0.05 or 0.001, one-tailed t-test). Simultaneously, the abundance of A. pohangense in the experimental bottle gradually increased from 790 to 1430 cells ml1, while that in the control bottle was maintained (Fig. 1H). Over time, the abundance of C. polykrikoides in filtrates from the cultures containing A. pohangense at concentrations of 200, 400, and 800 cells ml1 gradually decreased over the 72-h incubation period, whereas that in the control gradually increased (Fig. 2). However, the abundance of C. polykrikoides in the presence of 100 cells ml1 of A. pohangense filtrates was maintained for the 72-h incubation (Fig. 2). 3.2. Effects of A. pohangense cells and filtrate on the survival of Cochlodinium from natural populations
Fig. 5. Change in the abundance (cells ml1) of heterotrophic dinoflagellates (HD) and ciliates (CIL) in the natural red-tide water over time. The water was collected from the coast of Tongyoung, Korea. (A) The abundance of HD in the control (closed squares, dot line) and with Alexandrium pohangense at 800 cells ml1 (closed circles, thick solid line) and equivalent A. pohangense culture filtrate (open circles, thin solid line). (B) The abundance of CIL in the control (closed squares, dot line) and with A. pohangense at 800 cells ml1 (closed circles, thick solid line) and equivalent A. pohangense culture filtrate (open circles, thin solid line). Symbols represent treatment means SE.
During the C. polykrikoides red tides in Tongyoung, the phototrophic dinoflagellates Karenia mikimotoi, Prorocentrum donghaiense, Prorocentrum triestinum, Scrippsiella trochoidea, Polykrikos hartmanii, Gymnodinium spp., and heterotrophic dinoflagellate Gyrodinium spp., Protoperidinium spp., Pfiesteria-like dinoflagellates, and diatom Skeletonema costatum, Pseudonitzschia spp., Thalassiosira spp., Chaetoceros spp., and the naked ciliates (<50 mm) co-occurred. Over time, the abundance of C. polykrikoides in the control bottles slowly increased, while that in the presence of 800 cells ml1 of A. pohangense or equivalent filtrates rapidly decreased after 2 h, but slowly decreased thereafter (Fig. 3A). C. polykrikoides cells incubated with A. pohangense cells were completely removed within 24 h, while those with the A. pohangense filtrates were maintained after 6 h. The abundance of C. polykrikoides incubated with A. pohangense cells at 2, 6, 12, and 24 h was significantly lower than that at 0 h (p < 0.005, one-tailed t-test). Furthermore, the abundance of C. polykrikoides incubated with the A. pohangense filtrates at 2, 6, 12, and 24 h was significantly lower than that at 0 h (p < 0.005, one-tailed t-test). However, the abundance of A. pohangense in the experiment gradually increased over the 24-h incubation period (Fig. 3B).
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Among the other protists co-occurring with C. polykrikoides, the abundance of total diatoms in the control and experimental bottles followed a similar pattern. The abundance of total diatoms slightly decreased from ca. 70 to ca. 40 cells ml1 at 6 h, but increased to 170–290 cells ml1 in both the control and experimental bottles (Fig. 4A). However, the abundance of total phototrophic dinoflagellates excluding C. polykrikoides, in the control bottles increased, while those containing A. pohangense cells and equivalent filtrates slowly decreased until 6 h and were maintained or increased thereafter (Fig. 4B). The abundance of heterotrophic dinoflagellates in the control bottles slowly increased, while the abundance in bottles containing A. pohangense cells or equivalent filtrates decreased until 6 h but slightly increased thereafter (Fig. 5A). The abundance of total ciliates in both the control and experimental bottles decreased at 6 h and was almost zero at 24 h (Fig. 5B). 3.3. Ichthyotoxicity test During the experiments, the water temperature, salinity, pH, and dissolved oxygen in the experimental plastic boxes were 17– 20 C, 31–33, 7.3–8.1, and 5.9–7.9 mg l1, respectively. Over elapsed time, P. olivaceus juveniles in the control and experimental boxes (A. pohangense cells and equivalent filtrates) survived during the 72-h incubation period. During the experiment, no abnormal behaviors, such as disorientated swimming and/or turning upside down, were observed in the fish.
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4. Discussion The results of the present study clearly show that both cells and culture filtrates of A. pohangense are effective at controlling both cultured and natural populations of C. polykrikoides. The minimum A. pohangense cell concentrations causing 100% mortality of C. polykrikoides at 24 h and 48 h were 800 cells ml1 and 200 cells ml1, respectively. Thus, depending on the intensity of a C. polykrikoides bloom, A. pohangense cell concentrations between 200 and 800 cells ml1 may be used to mitigate the bloom. However, further field experiments are still required because these results were obtained from laboratory experiments. The minimum A. pohangense cell concentration producing culture filtrates in which all culture C. polykrikoides cells were dead within 72 h was 800 cells ml1. Thus, the culture filtrates of A. pohangense are less effective materials than A. pohangense cells. Within 24 h of 800 A. pohangense cells ml1 being introduced to natural populations of C. polykrikoides at 1500 cells ml1, all C. polykrikoides cells had been removed. A. pohangense cells can remove both cultured and natural populations of C. polykrikoides at similar rates. In land-based aquaculture, seawater is pumped up and stored in flow-through tanks for recirculation. Once seawater is pumped into a large seawater reservoir tank, the waters distribute into each fish-rearing tank. Thus, if dense C. polykrikoides cells in the tank are observed, the C. polykrikoides cells may be removed by the introduction of mass cultured A. pohangense (final concentration 200 cells ml1).
Fig. 6. Schematic diagrams showing differences between heterotrophic ciliates (A1–A3) and mixtrophic dinoflagellate Alexandrium pohangense (B1–B3) as grazers removing Cochlodinium polykrikoides (Cp) when mass-cultured grazers are introduced to waters containing Cp red-tide patches in aqua-tanks on land. (A1) Introduced ciliate grazers remove Cp cells in the first arrived red-tide patch. (A2) The ciliates starve to death after eliminating all Cp cells if no prey is supplied. (A3) New ciliate populations should be introduced to remove Cp cells in newly arrived Cp red-tide patch. (B1) Introduced Alexandrium removes Cp cells in the first arrived Cp red-tide patch. (B2) The Alexandrium cells are maintained for a long time after the elimination of all Cp cells. (A3) The maintained Alexandrium populations can remove Cp cells in a newly arrived Cp red tide patch.
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A. pohangense can grow fast on C. polykrikoides prey (maximum mixotrophic growth rate = 0.5 d1), but they can also grow slowly photosynthetically or be maintained without the addition of prey (phototrophic growth rate = 0.1 d1 at 20 C, 100 mE m2 s1; Lim et al., 2015a). Thus, it is much easier to increase or maintain A. pohangense populations than those of heterotrophic protist grazers, to which optimal prey should be constantly supplied. Some heterotrophic protist grazers have been shown to be effective grazers on C. polykrikoides, and have thus been suggested as biological control agents. Jeong et al. (2008) suggested that large-scale culture of the large naked ciliates Strombidinopsis jeokjo could be used to control C. polykrikoides red tides. This ciliate can be isolated from natural seawaters, and grows rapidly upon ingestion of C. polykrikoides (growth and ingestion rates = 0.72 d1 and 72 cells grazer1 d1). However, S. jeokjo is not easy to maintain because it needs to be fed with optimal prey every day. This heterotrophic ciliate divides more than once a day, but it starves to death within 2 days without prey. In natural environments, S. jeokjo feeds on C. polykrikoides cells until complete removal and grazes other organisms. Thus, S. jeokjo can grow continuously. However, in land-based aqua-tanks, S. jeokjo may starve to death within 2–3 days of the complete removal of C. polykrikoides. Thus, new populations of cultured S. jeokjo would need to be introduced when C. polykrikoides blooms again. However, A. pohangense is able to survive photosynthetically once C. polykrikoides has been eliminated. Thus, it is not necessary to introduce new populations of A. pohangense to remove newly arrived C. polykrikoides red tide patches (Fig. 6). A. pohangense may have an advantage over S. jeokjo in the maintenance of stock cultures for controlling C. polykrikoides in land-based aqua-tanks. Furthermore, using A. pohangense cultures to control C. polykrikoides red tides may be more cost-efficient than using S. jeokjo cultures because an additional system for S. jeokjo would be required to maintain. This method of controlling C. polykrikoides red-tide patches in land-based aqua-tanks may be cost-effective because populations of A. pohangense increase when feeding on C. polykrikoides. These larger populations can remove more C. polykrikoides cells. An A. pohangense culture with a concentration of 200 cells ml1 is able to remove all C. polykrikoides cells within 2 days in aqua-tanks when the initial concentration of C. polykrikoides is 1000 cells ml1 as in experiment 1. Based on the results of the present study (i.e., the growth rates of A. pohangense when feeding on red-tide blooming C. polykrikoides = 0.41 d1), the population of the A. pohangense culture is calculated to increase by 60% every day. Thus, theoretically the 1 m3 A. pohangense cultures of 4000 cells ml1 increase to 2.27, 5.15, and 11.7 m3 at the same concentration after 2, 4, and 6 days, respectively (Fig. 7A). In turn, these populations can remove all C. polykrikoides cells within 45, 103, and 234 m3 mixed waters, respectively (final A. pohangense and C. polykrikoides cell concentrations = 200 and ca. 1000 cells ml1, respectively, in the mixed waters of A. pohangense culture and C. polykrikoides water, Fig. 7B). The cost for the production of 1 m3 of A. pohangense stock culture is approximately 250 US dollars in Korea when calculated by summing all costs such as purchase of f/2 medium for cultivating C. polykrikoides as prey, labor, and electricity charge. The introduced 1 m3 of A. pohangense stock culture can remove C. polykrikoides cells in 20 m3 waters within 2 days in an aqua-tank. Thus, this method is cost-effective and could be used to remove C. polykrikoides cells in land-based aqua-tanks. The patterns in temporal variations in the abundances of total diatoms and ciliates in the experimental and control bottles after being with both A. pohangense cells and filtrates were similar, while those of the other phototrophic dinoflagellates and heterotrophic dinoflagellates were different. However, the abundance of heterotrophic dinoflagellates incubated with A.
Fig. 7. Calculated volumes of Alexandrium pohangense stock cultures and treatable waters containing Cochlodinium polykrikoides red-tide patches. (A) Volume (m3) of the Alexandrium stock cultures containing 4000 A. pohangense cells ml1 as a function of elapsed time following the addition of a 1 m3 Alexandrium stock culture to a population of Cochlodinium (1000 cells ml1), calculated with the assumption that the growth rate of A. pohangense feeding on C. polykrikoides is 0.41 day1 (see text). (B) Calculated volumes (m3) of treatable waters containing C. polykrikoides using A. pohangense culture (final concentration = 200 cells ml1), which can be obtained by a 1/20 dilution of the Alexandrium stock culture.
pohangense cells or equivalent filtrates almost maintained. Thus, introduced A. pohangense cells and filtrates do not cause marked adverse effects on total diatoms, heterotrophic dinoflagellates, and ciliates, but may cause some adverse effects on other phototrophic dinoflagellates. However, the dominant species in the other phototrophic dinoflagellates was Karenia mikimotoi, which is harmful. Thus, this method may have the advantage of removing other harmful species, although this still needs to be investigated. In the last few decades, several physical and chemical methods of controlling C. polykrikoides red tides, such as clay and dredged sediment disposal, and spraying with sodium hypochlorite (NaOCl), copper sulfate (CuSO4), and thiazolidinedione derivative (TD49), have been developed and some of these have been used in Korean waters (Haas, 1999; Jeong et al., 2002; Sengco and Anderson, 2004; Kim, 2010; Song et al., 2010; Baek et al., 2014; Ebenzer et al., 2014). However, these methods are not speciestargeted because they can kill diverse marine organisms and some of them produce unpleasant odors (Shumway et al., 2003; Kim, 2010; Jung et al., 2015). Thus, the use of biological mitigation methods can be a safer and more sustainable technique.
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5. Conclusions The present study demonstrated that: (1) A. pohangense can control both cultured and natural population of C. polykrikoides in the laboratory condition. Addition of A. pohangense cultures at a concentration of 800 cells ml1 killed most C. polykrikoides cells in both cultured and natural populations within 24 h. (2) Culture filtrates of 800 A. pohangense cells ml1 reduced 80% of cultured C. polykrikoides populations within 48 h. Thus, A. pohangense cells remove C. polykrikoides cells by feeding and/or releasing extracellular compounds. However, A. pohangense cells or filtrates did not affect the abundance of total diatoms and heterotrophic dinoflagellates in the natural waters. (3) Moreover, none of the olive flounder juveniles incubated with 2000 cells ml1 of A. pohangense for 3 days died. Based on the results of the present study, a 1 m3 stock culture of A. pohangense with 4000 cells ml1 is calculated to remove all C. polykrikoides cells in ca. 200 m3 mixed waters in 6 days. Therefore, this method of controlling C. polykrikoides populations may result as a safe and effective method that can be easily applied to land-based aqua-tanks. Acknowledgements This paper was supported by the Useful Dinoflagellate Program of Korea Institute of Marine Science and Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries (MOF) and Management of marine organisms causing ecological disturbance and harmful effect Program of KIMST and the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2015M1A5A1041806) award to HJJ.[SS] References Baek, S.H., Shin, K., Son, M., Bae, S.W., Cho, H., Na, D.H., Kim, Y.O., Kim, S.W., 2014. Algicidal effects of yellow clay and the thiazolidinedione derivative TD49 on the fish-killing dinoflagellate Cochlodinium polykrikoides in microcosm experiments. J. Appl. Phycol. 26, 2367–2378. Bostock, J., McAndrew, B., Rishards, R., Jauncey, K., Telfer, T., Lorenzen, K., Little, D., Ross, L., Handisyde, N., Gatward, I., Corner, R., 2010. Aquaculture: global status and trends. Philos. Trans. R. Soc. B 365, 2897–2912. Dorantes-Aranda, J.J., García-de la Parra, L.M., Alonso-Rodríguez, R., Morquecho, L., Voltolina, D., 2010. Toxic effect of the harmful dinoflagellate Cochlodinium polykrikoides on the spotted rose snapper Lutjanus guttatus. Environ. Toxicol. 25 (4), 319–326. Ebenzer, V., Lim, W.A., Ki, J., 2014. Effects of the algicides CuSO4 and NaOCl on various physiological parameters in the harmful dinoflagellate Cochlodinium polykrikoides. J. Appl. Phycol. 26, 2357–2365. Fukuyo, Y., Imai, I., Kodama, M., Tamai, K., 2002. Red tide and other harmful algal blooms in Japan. In: Tayler, F.R.J., Trainer, V.L. (Eds.), Harmful Algal Blooms in the PICES Region of the North Pacific. PICES Sci. Rep. No. 23 pp. 152. Gobler, C.J., Berry, D.L., Anderson, O.R., Burson, A., Koch, F., Rodger, B.S., Moore, L.K., Goleski, J.A., Allam, B., Bowser, P., Tang, Y., Nuzzi, R., 2008. Characterization, dynamics, and ecological impacts of harmful Cochlodinium polykrikoides blooms on eastern Long Island, NY, USA. Harmful Algae 7, 293–307. Griffith, A.W., Gobler, C.J., 2016. Temperature controls the toxicity of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides. Mar. Ecol. Prog. Ser. 545, 63–76. Guillard, R.R.L., Ryther, J.H., 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Grun. Can. J. Microbiol. 8, 229– 239. Haas, C.N., 1999. Disinfection, In: Letterman, R.D. (Ed.), Water Quality and Treatment a Handbook of Community Water Supplies. 5th edition McGraw-Hill, New York, pp. 14.1–14.6 American water works association. He, P., 2015. Harmful algal blooms. In: Sahoo, D., Seckbach, J. (Eds.), The Algae World. Springer, Netherlands, pp. 339–356. Imai, I., Kimura, S., 2008. Resistance of the fish-killing dinoflagellate Cochlodinium polykrikoides against algicidal bacteria isolated from the coastal Sea of Japan. Harmful Algae 7 (3), 360–367. Jeong, H.J., Kim, H.R., Kim, G.Y., Park, K.H., Kim, S.T., Yoo, Y.D., Song, J.Y., Kim, J.S., Seong, K.A., Yih, Y.H., Pae, S.J., Lee, C.H., Huh, M.D., Lee, S.H., 2002. NaOCl produced by electrolysis of natural seawater as a potential method to control marine red-tide dinoflagellate. Phycologia 41 (6), 643–656. Jeong, H.J., Kim, J.S., Yoo, Y.D., Kim, S.T., Kim, T.H., Park, M.G., Lee, C.H., Seong, K.A., Kang, N.S., Shim, J.H., 2003. Feeding by the heterotrophic dinoflagellate Oxyrrhis marina on the red-tide raphidophyte Heterosigma akashiwo: a potential
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