FORMING DINOFLAGELLATES Prorocentrum Donghaiense and Karenia Mikimotoi UNDER DIFFERENT TEMPERATURE

Home , Karenia (dinoflagellate), Prorocentrum donghaiense

Thalassas, 30(2) · June 2014: 33-45 An International Journal of Marine Sciences

GROWTH INTERACTIONS BETWEEN THE BLOOM- FORMING DINOFLAGELLATES Prorocentrum donghaiense AND Karenia mikimotoi UNDER DIFFERENT TEMPERATURE

ANGLU SHEN(1,2), XINGWEI YUAN(2), GUANGPENG LIU(1) & DAOJI LI(1)*

(1) State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai 200062, China (2) East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China

* corresponding author: [email protected].

ABSTRACT Growth interactions between Prorocentrum donghaiense and Karenia mikimotoi, which are major harmful algal blooms (HABs) species in the East China Sea (ECS), were investigated by using bi-algal cultures under different temperatures. When the culture temperature was 16 °C, the growth of P. donghaiense and K. mikimotoi in bi-algal cultures had influence or no influence on each other when the cell density ratio (P. donghaiense : K. mikimotoi) was 2:1 or 1:1, P. donghaiense was not affected by K. mikimotoi and K. mikimotoi was affected by P. donghaiense when the cell density ratio was 1:2 and just the opposite result when the cell density ratio was 1:4. When the culture temperature was 20 °C, the growth of K. mikimotoi was suppressed by P. donghaiense in bi-algal cultures under the case that the initial density ratios were 2:1 and 1:1, two microalgae had no influence on each other when the cell density ratio was 1:2 and the growth of P. donghaiense was suppressed by K. mikimotoi when the cell density ratio was 1:4. When the culture temperature was 24 °C, the growth of P. donghaiense was suppressed by K. mikimotoi under all initial density ratios and P. donghaiense cannot survive in bi-algal cultures when the initial cell density ratio was 1:4, the growth of K. mikimotoi was not suppressed by P. donghaiense under the case that the initial density ratios were 1:1, 1:2 and 1:4. When the culture temperature was 28 °C, the growth of P. donghaiense and K. mikimotoi had no influence on each other when the cell density ratios were 2:1 and 1:1, P. donghaiense cannot survive in bi-algal cultures when the initial cell density ratios were 1:2 and 1:4. In a word, P. donghaiense has a survivial strategy that is superior to that of K. mikimotoi in bi-algal cultures at low temperature (16 °C and 20 °C), K. mikimotoi has a survivial strategy that is superior to that of P. donghaiense in bi-algal cultures at higher temperature (24 °C and 28 °C) and K. mikimotoi became dominant when the initial cell density ratio was 1:4 under four temperatures. The results showed that the culture temperature and the initial cell density play key roles in the growth interactions between P. donghaiense and K. mikimotoi. Key words: Growth interaction, temperature, initial cell density, Prorocentrum donghaiense, Karenia mikimotoi.

RESUMEN Se estudiaron las interacciones en el crecimiento entre Prorocentrum donghaiense y Karenia mikimotoi, que son algas components mayoritarios de las floraciones tóxicas (HABs) en el Mar de China del Este (ECS), mediante el uso de cultivos bialgales a diferentes temperaturas. Cuando la temperature fue de 16 °C, hubo poca o ninguna influencia entre P. donghaiense y K. mikimotoi cuando las densidades celulares se econtraron en valores de 2:1 o 1:1 (P. donghaiense : K. mikimotoi). P. donghaiense no result afectado por K. mikimotoi y K. mikimotoi result afectada por P. donghaiense cuando la razón de densidades fue de 1:2 mientras que el resultado se invirtió cuando la razón de densidades estuvo en 1:4. Cuando el cultivo se realize a 20 °C, se suprimió el crecimiento de K. mikimotoi debido a la presencia de P. donghaiense con razones iniciales de densidad de 2:1 y 1:1, no hubo influencia de una microalga sobre la otra cuando la ratio inicial de densidad fue 1:2 y se inhibió el crecimiento de P. donghaiense debido al efecto de K. mikimotoi cuando la razón de densidad inicial fue 1:4. A 24 °C, el crecimiento de P. donghaiense fue suprimido por K. mikimotoi en todas las densidades inciales. P. donghaiense no pudo sobrevivir en cultivos bialgales cuando la densidad inicial fue de 1:4. Por su parte, el crecimiento de K. mikimotoi no se vió afectado por P. donghaiense cuando las densidades relativas iniciales fueron 1:1, 1:2 y 1:4. Cuando la temperatura de los cultivos se estableció en 28 °C, no hubo influencia mutual de P. donghaiense y K. mikimotoi en valores iniciales de 2:1 y 1:1, mientras que P. donghaiense no sobrevivió cuando las densidades celulares iniciales fueron 1:2 y 1:4. En resumen, P. donghaiense posee una estategia de supervivencia más favorable que K. mikimotoi en cultivos bialgales a bajas temperaturas (16 °C y 20 °C), mientras que la estrategia de supervivencia de K. mikimotoi es superior a la de P. donghaiense en las temperaturas superiores (24 °C y 28 °C). Además K. mikimotoi llegó a ser dominante cuando las densidades celulares iniciales fueron en la relación 1:4 en las cuatro temperaturas ensayadas. Los resultados demuestran que tanto la temperatura como las densidades relativas iniciales juegan papeles clave en las inteacciones de crecimiento entre P. donghaiense y K. mikimotoi. Palabras clave: Interacción de crecimiento, temperatura, densidad celular inicial, Prorocentrum donghaiense, Karenia mikimotoi

Thalassas, 30(2) · June 2014 33 ANGLu SHEN, XINGWEI YUAN, GUANGPENG LIU & DAOJI LI

INTRODUTION sustain their growth (Ou et al., 2008), K. mikimotoi can assimilate different kind of inorganic and phosphorus Prorocentrum donghaiense Lu (formerly named as sources as well as some organic phosphorus compounds, P. dentatum, Lu & Goebel 2001; Gómez, 2005), is a too (Lei & Lu, 2011). S. costatum blooms became dinoflagellate belonging to Prorocentrum, Procentraceae, dominate after June because of the nutrients have been Prorocentriada. It is a major HABs species near the supplied again from the Yangtze River Diluted Water Yangtze Estuary and the adjacent coastal waters of the ECS and the Taiwan Warm Current (Li et al., 2009; Zhao, during late April-May annually since 2000 (Lu & Goebel 2010). It seems to be the nutrient play an important role 2001; Lu et al., 2005; Tang et al., 2006). More than 120 in diatom-dinoflagellate blooms succession. However, P. donghaiense bloom events were recorded in ECS from P. donghaiense and K. mikimotoi blooms have been 2001 to 2010 (Zhao, 2010), and the scale of these blooms observed occurring in the same area from late April to ranged from several thousand to more than 10,000 km2 June, and the nutrient in seawater are in the same level of and lasted from several days to one month. It is estimated the two species, other factors (e.g. temperature, light and that the direct loss to fisheries caused by P. donghaiense so on) may play a leading role in the P. donghaiense and blooms is more than several million renminbi (RMB) K. mikimotoi blooms succession. every year (data from State Oceanic Administration in East China Sea Branch). Karenia mikimotoi Hansen Interactions between HABs species have been studied (formerly named as Gymnodinium mikimotoi, Hansen et widely by many authors. For example, S. costatum, a al., 2000; Gómez, 2005), is a dinoflagellate belonging typical diatom species, interactions with other species to Karenia, Kareniaceae, Gymnodiniida. It is a red- such as Gyrodinium instriatum (Nagasoe et al., 2006), P. tide species around the world including regions such as minimum (Tameishi et al., 2009), Heterosigma akashiwo the eastern North Atlantic, Japan, Europe, Australia, (Yamasaki et al., 2007, 2011), Chattonella marina (wang et South America, North Africa and China (reviewed by al., 2010) under a single temperature condition with some Gentien, 1998). Large scale K. mikimotoi blooms in the initial cell densities have been studied and these results seas of Hong Kong in 1998 resulted in the die-off of fish, showed that S. costatum has a competitive advantage causing enormous economic losses estimated at US$ 40 compared with others. P. donghaiense which interactions million (Wang et al., 2001; Lu & Hodgkiss, 2004). K. with Alexandrium tamarense (Wang et al., 2006; Yang et mikimotoi blooms occurred frequently near the adjacent al., 2010), Scrippsiella trochoidea (Wang & Tang, 2008), coastal waters of Zhejiang and Fujian province, China A. minutum (Yang et al., 2010) have been studied and since 2004 (Zhao, 2010). In addition, the hemolytic and the results indicated that P. donghaiense did not have a cytotoxic compounds released by K. mikimotoi have competitive advantage compared with others. K. mikimotoi been associated with ichthyotoxicity (Neely & Campbell, which interactions with H. circularisquama (Uchida et 2006; Satake et al., 2005) and the toxic substances in K. al., 1999), H. akashiwo (Zhao et al., 2009), P. micans mikimotoi can be transferred through marine food webs (Ji et al., 2011) have been studied and the results showed and harm humans (Chen et al., 2011). that K. mikimotoi did not have a competitive advantage compared with others, either. Interspecific competition Skeletonema costatum, P. donghaiense and K. between P. donghaiense and K. mikimotoi under 25 °C mikimotoi are major HABs species near the Yangtze River with some initial cell densities has been studied and the estuary and the ECS since the 21st century, the frequency results showed that the two microalgae had an influence of these three HABs occurrence for nearly half of all the on each other (Huang et al., 2009). However, most of these red tides (Zhao, 2010). Diatom blooms and dinoflagellate species form alternating blooms in various coastal waters blooms often appears alternatively in the bloom succession and few two species blooms occurred together except P. (diatom-dinoflagellate-diatom). S. costatum blooms donghaiense and K. mikimotoi. Therefore, it is important occurred in early spring when the nutrient (especially to realize the interactions between P. donghaiense and K. the phosphorus) were sufficient because S. costatum had mikimotoi under different conditions. a greater advantage in phosphate affinity and also had a higher phosphorus demand for growth, thus, the growth Temperature is a key factor that affects the total of S. costatum collapsed soon after phosphate depletion abundance of phytoplankton seasonal distribution (Zhao, (Ou et al., 2008). P. donghaiense blooms and K. mikimotoi 2010). The phytoplankton biomass, dominant species and blooms have been observed in the coastal waters of the community structure have been changed in the Taiwan ECS from late April to June since 2005 (Zhou et al., 2006; Strait and Yangtze Estuary under global Climate Changes Li et al., 2009; Zhao, 2010). P. donghaiense could make (Hong et al., 2005; Zhang, 2009). In this case, the red good use of the metabolized dissolved organic phosphorus tide succession will appear abnormal fluctuations, for in the water after the collapse of S. Costatum blooms to instance, the blooms of 2005 was unusual as it begin with

34 Thalassas, 30(2) · June 2014 GROWTH INTERACTIONS BETWEEN THE BLOOM-FORMING DINOFLAGELLATES Prorocentrum donghaiense AND Karenia mikimotoi UNDER DIFFERENT TEMPERATURE

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cells ml K2 K4 4 4 6 8

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0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Time (d) Time (d)

Figure 1: Growth of Prorocentrum donghaiense and Karenia mikimotoi in the bi-algal culture with four initial cell density ratios at 16°C: (A) 2:1; (B) 1:1; (C) 1:2; (D) 1:4. The initial cell density of P. donghaiense in the bi-algal culture were set at: 0.2 × 104 cells ml-1 (P1) and 0.4 × 104 cells ml-1(P2), and the initial cell density of K. mikimotoi in the bi-algal culture were set at: 0.8 × 104 cells ml-1 (K4); 0.4 × 104 cells ml-1 (K2) and 0.2 × 104 cells ml-1(K1). ♦ Growth of P. donghaiense in control; ● Growth of K. mikimotoi in control; ◊ Growth of P. donghaiense in the bi- algal culture; ○ Growth of K. mikimotoi in the bi- algal culture. Data are given as mean values ± SD (n = 3). Vertical lines show the standard deviation.

K. mikimotoi blooms, followed by P. donghaiense blooms mikimotoi in bi-algal culture was almost identical as in in Zhoushan islands area due to the higher temperature the mono-algal culture during the treatment time of 11 d and the bloom of 2008 was P. donghaiense blooms which and significantly suppressed at 12d (P = 0.0201 < 0.05). mainly occurred in Zhejiang province area because of The results showed the two microalgae had influence the lower temperature (< 21°C) compared to other years on each other under this cell density ratio (2:1). When (Zhao, 2010). Although both P. donghaiense and K. the initial cell densities of both P. donghaiense and K. mikimotoi have high potential to form blooms in later- mikimotoi were 0.2 × 104 cells ml-1 (Fig. 1B), the growth spring and early-summer near the Yangtze Estuary and of P. donghaiense in bi-algal culture was almost identical the adjacent coastal waters of the ECS and even occurred as in the mono-algal culture during the treatment time of together, growth interactions between two species under 11 d, and weakly suppressed at 12d (P = 0.1105 > 0.05). different temperatures in the process of blooms succession The growth of K. mikimotoi in bi-algal culture was almost have not been carried out. In the present study, the growth identical as in the mono-algal culture during the treatment interactions between P. donghaiense and K. mikimotoi time of 11 d, and weakly suppressed at 12d (P = 0.0775 by using bi-algal culture experiments under different > 0.05), too. The results showed the two microalgae had temperatures with several combinations of initial cell no influence on each other under this cell density ratio densities of the two species were examined. (1:1). When the initial cell densities of P. donghaiense and K. mikimotoi were 0.2 × 104 cells ml-1 and 0.4 × RESULTS 104 cells ml-1, respectively (Fig. 1C), the growth of P. donghaiense in bi-algal culture and in the mono-algal Fig. 1. Microalgal growth in bi-algal culture at 16 °C culture were alike during the treatment time of 11 d, and Shen et al. the cell densities decreased at 12d (P = 0.2873 > 0.05). Fig. 1 showed the growth of P. donghaiense and K. The growth of K. mikimotoi in bi-algal culture and in mikimotoi in bi-algal cultures with different initial cell the mono-algal culture were alike during the treatment densities at 16 °C. When the initial cell densities of P. time of 11 d, and the cell densities decreased at 12d (P donghaiense and K. mikimotoi were 0.4 × 104 cells ml-1 = 0.0052 < 0.05). The results showed P. donghaiense and 0.2 × 104 cells ml-1, respectively (Fig. 1A), the growth was not affected by K. mikimotoi and K. mikimotoi was of P. donghaiense in bi-algal culture was almost identical affected by P. donghaiense under this cell density ratio as in the mono-algal culture during the treatment time of (1:2). When the initial cell densities of P. donghaiense and 9 d, and the growth of P. donghaiense was significantly K. mikimotoi were 0.2 × 104 cells ml-1 and 0.8 × 104 cells suppressed at 12d (P = 0.0074 < 0.05). The growth of K. ml-1, respectively (Fig. 1D), the growth of P. donghaiense

Thalassas, 30(2) · June 2014 35 ANGLu SHEN, XINGWEI YUAN, GUANGPENG LIU & DAOJI LI

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0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Time (d) Time (d)

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0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Time (d) Time (d) Figure 2: Growth of Prorocentrum donghaiense and Karenia mikimotoi in the bi-algal culture with four initial cell density ratios at 20°C: (A) 2:1; (B) 1:1; (C) 1:2;

-1 -1 (D) 1:4. The initial cell density of P. donghaiense in the bi-algal culture were set at: 0.2 × 104 cells ml (P1) and 0.4 × 104 cells ml (P2), and the initial cell density of K. mikimotoi in the bi-algal culture were set at: 0.8 × 104 cells ml-1 (K4); 0.4 × 104 cells ml-1 (K2) and 0.2 × 104 cells ml-1(K1). ♦ Growth of P. donghaiense in control; ● Growth of K. mikimotoi in control; ◊ Growth of P. donghaiense in the bi- algal culture; ○ Growth of K. mikimotoi in the bi- algal culture. Data are given as mean values ± SD (n = 3). Vertical lines show the standard deviation.

in bi-algal culture was almost identical as in the mono- by P. donghaiense under this cell density ratio (2:1). When algal culture during the treatment time of 11 d, but the the initial cell densities of both P. donghaiense and K. growth of P. donghaiense was significantly suppressed at mikimotoi were 0.2 × 104 cells ml-1 (Fig. 2B), the growth 12d (P = 0.0197 < 0.05), the cell density of P. donghaiense of P. donghaiense in bi-algal culture was almost the same was about 55% of that in mono-algal culture. The growth as in the mono-algal culture during the treatment time of of K. mikimotoi in bi-algal culture was almost the same as 9 d, and the cell density decreased at 12d (P = 0.0791 > in the mono-algal culture during 12 d and the cell density 0.05). The growth of K. mikimotoi in bi-algal culture was was a little less than the mono-algal culture (P = 0.0721 almost the same as in the mono-algal culture during 11d > 0.05). The results showed P. donghaiense was affected and suppressed at 12d (P = 0.0086 < 0.05). The results by K. mikimotoi and K. mikimotoi was not affected by P. showed P. donghaiense was not affected by K. mikimotoi donghaiense under this cell density ratio (1:4). and K. mikimotoi was affected by P. donghaiense under this cell density ratio (1:1). When the initial cell densities Microalgal growth in bi-algal culture at 20 °C of P. donghaiense and K. mikimotoi were 0.2 × 104 cells -1 4 -1 ml and 0.4 × 10 cells ml , respectively (Fig. 2C), the Fig. 2 showed the growth of P. donghaiense and K. growth of P. donghaiense in bi-algal culture was alike mikimotoi in bi-algal cultures with different initial cell to that in the mono-algal culture during 12d (12d: P = densities at 20 °C. When the initial cell densities of P. 0.0583 > 0.05). The growth of K. mikimotoi in bi-algal donghaiense and K. mikimotoi were 0.4 × 104 cells ml-1 culture was alike to that in the mono-algal culture during and 0.2 × 104 cells ml-1, respectively (Fig. 2A), the growth 12d (12d: P = 0.4763 > 0.05), too. The results showed of P. donghaiense in bi-algal culture was almost the same P. donghaiense and K. mikimotoi had no influence on as in the mono-algal culture during the treatment timeFig. 2. each other in bi-algal culture under this cell density ratio of 11 d, and the growth of P. donghaiense was weakly (1:2). When initial cell densities of P. donghaiense and suppressed at 12d (P = 0.2726 > 0.05) and the cell densityShen et al. K. mikimotoi were 0.2 × 104 cells ml-1 and 0.8 × 104 cells of P. donghaiense was notably higher than the cell density ml-1, respectively (Fig. 2D), the growth of P. donghaiense K. mikimotoi from 6d to 11d. The growth of K. mikimotoi in bi-algal culture was significantly suppressed from 7d in bi-algal culture and in the mono-algal culture were to 12d (P = 0.0121, 0.0148, 0.0016, 0.0116, 0.0289, 0.0248, alike during the treatment time of 11 d, and the growth respectively). The growth of K. mikimotoi in bi-algal of K. mikimotoi was suppressed at 12d (P = 0.0049 culture was better than in the mono-algal culture during < 0.05). The results showed P. donghaiense was not 12 d. The results showed P. donghaiense was affected affected by K. mikimotoi and K. mikimotoi was affected by K. mikimotoi and K. mikimotoi was not affected by P.

36 Thalassas, 30(2) · June 2014 GROWTH INTERACTIONS BETWEEN THE BLOOM-FORMING DINOFLAGELLATES Prorocentrum donghaiense AND Karenia mikimotoi UNDER DIFFERENT TEMPERATURE

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Figure 3: Growth of Prorocentrum donghaiense and Karenia mikimotoi in the bi-algal culture with four initial cell density ratios at 24°C: (A) 2:1; (B) 1:1; (C) 1:2; (D) 1:4. The initial cell density of P. donghaiense in the bi-algal culture were set at: 0.2 × 104 cells ml-1 (P1) and 0.4 × 104 cells ml-1(P2), and the initial cell

density of K. mikimotoi in the bi-algal culture were set at: 0.8 × 104 cells ml-1 (K4); 0.4 × 104 cells ml-1 (K2) and 0.2 × 104 cells ml-1(K1). ♦ Growth of P. donghaiense in control; ● Growth of K. mikimotoi in control; ◊ Growth of P. donghaiense in the bi- algal culture; ○ Growth of K. mikimotoi in the bi- algal culture. Data are given as mean values ± SD (n = 3). Vertical lines show the standard deviation. donghaiense under this cell density ratio (1:4), and even mikimotoi was not affected by P. donghaiense under this the growth of K. mikimotoi was better than control maybe cell density ratio (1:1). When the initial cell densities of by the stimulation of P. donghaiense. P. donghaiense and K. mikimotoi were 0.2 × 104 cells ml-1 and 0.4 × 104 cells ml-1, respectively (Fig. 3C), the growth Microalgal growth in bi-algal culture at 24 °C of P. donghaiense in bi-algal culture was suppressed from 9-12d (P = 0.0036, 0.0095, 0.0116, 0.0090, respectively). Fig. 3 showed the growth of P. donghaiense and K. The growth of K. mikimotoi in bi-algal culture was almost mikimotoi in bi-algal cultures with different initial cell the same as in the mono-algal culture during 12d (12d: densities at 24 °C. When the initial cell densities of P. P = 0.0534 > 0.05). The results showed P. donghaiense donghaiense and K. mikimotoi were 0.4 × 104 cells ml-1 was affected by K. mikimotoi and K. mikimotoi was not 4 -1 and 0.2 × 10 cells ml , respectively (Fig. 3A), the growth affected by P. donghaiense under this cell density ratio of P. donghaiense in bi-algal culture was almost identical (1:2). When the initial cell densities of P. donghaiense and as in the mono-algal culture during the treatment time of K. mikimotoi were 0.2 × 104 cells ml-1 and 0.8 × 104 cells 11 d, and the growth of P. donghaiense was significantly ml-1, respectively (Fig. 3D), the growth of P. donghaiense suppressed at 12d (P = 0.0111 < 0.05). The growth of K. in bi-algal culture was suppressed from 8-9d (P = 0.0096 mikimotoi in bi-algal culture and in the mono-algal culture and 0062) and all P. donghaiense were killed at 10d. The were alike during 12d (12d: P = 0.2943 > 0.05). The results growth of K. mikimotoi in bi-algal culture was as almost showed P. donghaiense was affected by K. mikimotoi and identical as in the mono-algal culture during 12d (12d: K. mikimotoi was not affected by P. donghaiense under P = 0.4075 > 0.05). The results showed P. donghaiense this cell density ratio (2:1). When the initial cell densities was affected by K. mikimotoi and K. mikimotoi was not of both P. donghaiense and K. mikimotoi were 0.2 Fig.× 3. affected by P. donghaiense under this cell density ratio 104 cells ml-1 (Fig. 3B), the growth of P. donghaiense in (1:4). bi-algal culture was almost the same as in the mono-algalShen et al. culture during the treatment time of 8 d, and significantly Microalgal growth in bi-algal culture at 28°C suppressed from 9-12d (P = 0.0055, 0023, 0499, 0445, respectively). The growth of K. mikimotoi in bi-algal Fig. 4 showed the growth of P. donghaiense and K. culture and in the mono-algal culture were alike during mikimotoi in bi-algal cultures with different initial cell 11d and the cell density was higher than the mono-algal densities at 28 °C. When the initial cell densities of P. culture at 12d (P = 0.0294 < 0.05). The results showed donghaiense and K. mikimotoi were 0.4 × 104 cells ml-1 P. donghaiense was affected by K. mikimotoi and K. and 0.2 × 104 cells ml-1, respectively (Fig. 4A), the growth

Thalassas, 30(2) · June 2014 37 ANGLu SHEN, XINGWEI YUAN, GUANGPENG LIU & DAOJI LI

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Cell density (10 5 2 0 0 2 4 6 8 10 12 0 Time (d) 0 2 4 6 8 10 12 Time (d) Figure 4: Growth of P. donghaiense and K. mikimotoi in the bi-algal culture with four initial cell density ratios at 28°C: (A) 2:1; (B) 1:1; (C) 1:2; (D) 1:4. The initial cell density of P. donghaiense in the bi-algal culture were set at: 0.2 × 104 cells ml-1 (P1) and 0.4 × 104 cells ml-1(P2), and the initial cell density of K. mikimotoi in the bi-algal culture were set at: 0.8 × 104 cells ml-1 (K4); 0.4 × 104 cells ml -1 (K2) and 0.2 × 104 cells ml-1(K1). ♦ Growth of P. donghaiense in control; ● Growth of K. mikimotoi in control; ◊ Growth of P. donghaiense in the bi- algal culture; ○ Growth of K. mikimotoi in the bi- algal culture. Data are given as mean values ± SD (n = 3). Vertical lines show the standard deviation.

of P. donghaiense in bi-algal culture was alike to that in 104 cells ml-1 and 0.8 × 104 cells ml-1, respectively (Fig.

the mono-algal culture during the treatment time of 12 d 4D), the growth of P. donghaiense in bi-algal culture were (12d: P = 0.3974 > 0.05). The growth of K. mikimotoi in suppressed from 6-9d (P = 0.0195, 0.0105, 0.0120, 0.0246, bi-algal culture was alike to that in the mono-algal culture respectively) and all P. donghaiense were killed at 10d. during 11d and the cell density weakly increased at 12d The growth of K. mikimotoi in bi-algal culture and in the (P = 0.1308 > 0.05). The results showed P. donghaiense mono-algal culture were alike during 12d (12d: P = 0.1102 and K. mikimotoi had no influence on each other in > 0.05). The results showed P. donghaiense was affected bi-algal culture under this cell density ratio (2:1). When by K. mikimotoi and K. mikimotoi was not affected by P. the initial cell densities of both P. donghaiense and K. donghaiense under this cell density ratio (1:4). mikimotoi were 0.2 × 104 cells ml-1 (Fig. 4B), the growth of P. donghaiense in bi-algal culture and in the mono- DISCUSSION algal culture were alike during 12d (12d: P = 0.2803 > 0.05). The growth of K. mikimotoi in bi-algal culture and Role of temperature in competition between P. in the mono-algal culture were alike during 12d (12d: P donghaiense and K. mikimotoi

= 0.3836 > 0.05). The results showed P. donghaiense and K. mikimotoi had no influence on each other in bi-algal Nutrients, temperature, salinity, light and so on are culture under this cell density ratio (1:1). When the initial important environmental factors related to microalgae cell densities of P. donghaiense and K. mikimotoi were growth, and affect the population dynamics of individual 4 -1 4 -1 0.2 × 10 cells ml and 0.4 × 10 cells ml , respectively species and species succession in the field (Karentz & (Fig. 4C), the growth of P. donghaiense in bi-algal Smayda, 1984). In the field, P. donghaiense blooms have culture was suppressed from 10d (P = 0.0096 < 0.05)Fig. 4. been observed in May near the adjacent coastal waters of and all P. donghaiense were killed at 11d. The growth the north area such as Zhoushan Islands and K. mikimotoi of K. mikimotoi in bi-algal culture were almost the sameShen et al.blooms have been observed in June near the adjacent as in the mono-algal culture during 12d and the cell coastal waters of the south area such as Dongtou Islands density was higher than control at 6-10d (P = 0.0082, (Zhao, 2010). The optimal temperature for growth of P. 0.0125, 0.0039, 0.0091, 0.0153, respectively). The results donghaiense was 18-21°C (Wang, 2003) and long-term showed P. donghaiense was affected by K. mikimotoi monitoring data of Zhoushan Islands showed that P. and K. mikimotoi was not affected by P. donghaiense donghaiense blooms became dominate when the surface under this cell density ratio (1:2). When the initial cell sea temperature (SST) was below 20 °C and the population densities of P. donghaiense and K. mikimotoi were 0.2 × of P. donghaiense collapsed rapidly when the SST was

38 Thalassas, 30(2) · June 2014 GROWTH INTERACTIONS BETWEEN THE BLOOM-FORMING DINOFLAGELLATES Prorocentrum donghaiense AND Karenia mikimotoi UNDER DIFFERENT TEMPERATURE

Table 1: Estimated parameters from equations in mono-algal and bi-algal cultures of P. donghaiense (PD) and K. mikimotoi (KM) with different temperature and initial cell density ratio at 16 °C.

Carrying Interaction rate Initial cell density Growth rate, r capacity, K A or B ratio (PD:KM) (Divisions day-1) a or b (Cells ml-1) (ml cell-1 s-1)

PD 168,777 0.380 5.166 1.16 × 10-5 2:1 KM 71,190,036 0.437 5035.376 3.09 × 10-5

PD 37,312,465 0.335 2469.654 2.22 × 10-5 1:1 KM 71,190,036 0.437 5255.911 3.23 × 10-5

PD 37,312,465 0.335 1152.001 1.03 × 10-5 1:2 KM 208,201,053 0.477 16099.570 3.69 × 10-5

PD 37,312,465 0.335 1265.701 1.14 × 10-5 1:4 -5 KM 422,396 0.415 27.089 2.66 × 10

above 23 °C (Wang & Huang, 2003; Zhao, 2010). The al., 2008) and have different thermal stability. In general, optimal temperature for the growth of K. mikimotoi was allelopathic substance(s) have been degraded easily in 23 °C (Yao et al., 2007). In this study, P. donghaiense has high temperature environment (higher than that of the an advantage in the competition with K. mikimotoi when suitable algae growth temperature) which will diminish the sea water temperature is low (16-20°C). K. mikimotoi the toxic effects on the target organisms, on the other has an advantage in the competition with P. donghaiense hand, more allelopathic substance(s) will been released by when the sea water temperature is high (24-28°C). This algae in higher temperature environment (during suitable results are very similar in the rules of P. donghaiense or algae growth temperature). Further study is needed to K. mikimotoi blooms occurrence, which showed that 80% determine the effect of the culture filtrate of K. mikimotoi PD-blooms occurred blow 22 °C and 74% KM-blooms and P. donghaiense on each other. occurred above 22 °C in the ECS from 2001 to 2010 (Unpublished data). Therefore, Temperature plays a very Role of initial cell density in competition between P. important role in the process of dinoflagellate blooms donghaiense and K. mikimotoi succession. However, not much is known about how changes in temperature affect phytoplankton allelopathy. Allelopathy and cell contact are important factors in For example, the haemolytic activity of Phaeocystis the competition between phytoplankton species (Granéli pouchetii at 4 °C was approximately half the activity & Johansson, 2003; Granéli & Hansen, 2006; Uchida et measured at 15 °C (van Rijssel et al., 2007). In addition, al., 1995, 1999; Uchida, 2001). The allelopathic effect there was no difference between the effects of filtrate and lethal effect by cell contact which depends on the obtained from A. tamarense at 14 °C and 20 °C on S. cell density of the donor and the target species (Uchida et trochoidea or Heterocapsa trquetra (Fistarol et al., al., 1999; Schmidt & Hansen, 2001). Uchida et al. (1999) 2004). Wang & Tang, 2008 reported that the pre-treated had reported that H. circularisquama would become (maintained water bath at 30, 60 and 100 °C, for 30mins) a temporary cyst after 59 contacts with K. mikimotoi S. trochoidea filtrate inhibited P. donghaiense growth and K. mikimotoi would be killed after 19 contacts with during the first 120 h and then significantly increased its H. circularisquama. Thus, the initial cell densities of growth and ordered by 100 °C > 60 °C > 30 °C > control both toxic algae and its target seem to be important by the end, but the same pre-treated P. donghaiense for the outcome of the interaction (Granéli & Hansen, filtrate inhibited S. trochoidea growth from 72 h till the 2006). For instance, when the initial cell density of P. end of the experiment. There are different allelopathic donghaiense was low (1.0 × 104 cells ml-1), the growth of substance(s) of different harmful algal species (Granéli et P. donghaiense was dramatically suppressed and finally

Thalassas, 30(2) · June 2014 39 ANGLu SHEN, XINGWEI YUAN, GUANGPENG LIU & DAOJI LI

Table 2: Estimated parameters from equations in mono-algal and bi-algal cultures of P. donghaiense (PD) and K. mikimotoi (KM) with different temperature and initial cell density ratio at 20 °C.

Carrying Interaction rate Initial cell density Growth rate, r capacity, K A or B ratio (PD:KM) (Divisions day-1) a or b (Cells ml-1) (ml cell-1 s-1)

PD 68,471 0.661 0.520 5.02 × 10-6 2:1 KM 33,968,107 0.464 496.275 6.78 × 10-6

-6 PD 53,083 0.700 0.408 5.38 × 10 1:1 KM 33,968,107 0.464 368.615 5.04 × 10-6

-6 PD 53,083 0.700 0.744 9.81 × 10 1:2 -5 KM 287,232 0.492 7.165 1.23 × 10

-6 PD 53,083 0.700 0.431 5.69 × 10 1:4 -5 KM 227,434 0.718 3.783 1.19 × 10

all P. donghaiense cells were killed by A. tamarense density ratio of P. donghaiense and K. mikimotoi was 2:1 (the initial cell density was 0.28 × 104 cells ml-1) in at 16 and 20°C, and the higher the temperature, the less bi-algal cultures, and when the initial cell density of obvious competitive advantage (Fig. 1, 2, 3, and 4). Thus, P. donghaiense was high (1.0 × 105 cells ml-1) and the it serves as an important role of the initial cell density in initial cell density of A. tamarense was unchanged, the competition between P. donghaiense and K. mikimotoi. growth of P. donghaiense was significantly suppressed compared to the control, but no outcompetement was Growth simulation in bi-algal cultures observed in the end (Wang et al., 2006). K. mikimotoi became dominant when the initial cell density ratios of The growth of P. donghaiense and K. mikimotoi in H. akashiwo and K. mikimotoi were 1:1 and 1:4, and H. bi-algal cultures with different temperature and initial cell akashiwo overcame K. mikimotoi when the initial cell density ratio was simulated using the following equations density ratios of H. akashiwo and K. mikimotoi was 4:1 (Uchida et al., 1999): (Zhao et al., 2009). When the initial cell densities of S. 2 -1 4 -1 -1 -1 castatum and H. akashiwo were 10 cells ml and 10 dx dt = rxx [(1 - x) Kx ] - Axy = rxx [1 - (x + ay) Kx ] (1) -1 -1 -1 -1 cells ml , respectively, the growth of H. akashiwo was dy dt = ryy [(1 - y) Ky ] - Bxy = ryy [1- (bx + y) Ky ] (2) virtually the same as in both bi-algal and mono-algal cultures, and the growth of S. castatum was remarkably Here, x and y are the cell densities of P. donghaiense and suppressed in bi-algal cultures, when the initial cell K. mikimotoi, respectively. The rx and Kx are the growth 4 densities of S. castatum and H. akashiwo were 10 cells rate and carrying capacity of P. donghaiense, and ry and Ky ml-1 and 102 cells ml-1, the growth of the two species were are the growth rate and carrying capacity of K. mikimotoi completely reversed (Yamasaki et al., 2007). The similar when each species is cultured in a mono-algal culture. results were observed in other species competitions such A measures the degree of inhibition of P. donghaiense as H. akashiwo and P. minimum (Yamasaki et al., 2010), by K. mikimotoi, and B vice versa, the parameters A and -1 -1 H. akashiwo and Chattonella antiqua (Qiu et al., 2011). B are calculated from Eqs. A = arx Kx and B = bry Ky , In the present study, K. mikimotoi became dominant respectively. Parameters a and b are non-dimensional, when the initial cell density ratio of P. donghaiense indicating the degree of inhibition by the other species and K. mikimotoi was 1:4 at 16, 20, 24 and 28°C, when compared to its self-interference. respectively, and the higher the temperature, the more obvious competitive advantage (Fig. 1, 2, 3, and 4). P. Eqs. (1) and (2) can be approximated with the donghaiense became dominant when the initial cell following equations:

40 Thalassas, 30(2) · June 2014 GROWTH INTERACTIONS BETWEEN THE BLOOM-FORMING DINOFLAGELLATES Prorocentrum donghaiense AND Karenia mikimotoi UNDER DIFFERENT TEMPERATURE

Table 3: Estimated parameters from equations in mono-algal and bi-algal cultures of P. donghaiense (PD) and K. mikimotoi (KM) with different temperature and initial cell density ratio at 24 °C.

Carrying Interaction rate Initial cell density Growth rate, r capacity, K A or B ratio (PD:KM) (Divisions day-1) a or b (Cells ml-1) (ml cell-1 s-1)

-6 PD 36,366,180 0.255 1158.081 8.12 × 10 2:1 KM 46,106,434 0.512 779.840 8.66× 10-6

-5 PD 111,739 0.378 5.849 1.98 × 10 1:1 KM 46,106,434 0.512 1828.736 2.03 × 10-5

-6 PD 111,739 0.378 0.620 2.10 × 10 1:2 KM 2,402,741 0.411 52.796 9.03 × 10-6

PD 111,739 0.378 -- -- 1:4 KM 410,206 0.575 -- --

Note: “--” out-competement was observed in bi-algal culture and the interaction rate cannot be estimated.

-1 -1 -1 (Lnxi+1 - Lnxi-1) (ti+1 - ti-1) = rx - rxxi Kx - rxayi Kx (3) K. mikimotoi will co-exist in bi-algal cultures under -1 -1 -1 (Lnyi+1 - Lnyi-1) (ti+1 - ti-1) = ry–rybxi Ky –ryyi Ky (4) most experimental conditions. Several previous studies have used this model to examine the growth interactions

Here, xi and yi denote the observed cell densities at time ti between P. donghaiense or K. mikimotoi and other for P. donghaiense and K. mikimotoi, respectively. When species. Uchida et al. (1999) reported that the degree each species is cultured in a mono-algal culture, a = b of inhibition on K. mikimotoi by H. circularisquama = 0 can be set. The logistic parameters are estimated by was three times larger than the degree of inhibition Eqs. (3) and (4) using the mono-algal culture data. The on H. circularisquama by K. mikimotoi and indicated parameters a and b are calculated using bi-algal culture that K. mikimotoi was killed by cell contact with H. data directly from Eqs. (3) and (4). circularisquama and H. circularisquama was suppressed by K. mikimotoi because of allelopathic substances. The values of all parameters used in the growth Wang et al. (2006) reported that the inhibitory strength simulations are shown in Table 1, 2, 3 and 4. The carrying of A. tamarense on P. donghaiense was 17 times higher capacities for P. donghaiense (Kx) were lower than the than the inhibitory strength of P. donghaiense on A. carrying capacity for K. mikimotoi (Ky) except some tamarense when the initial cell density ratio was 3.6:1 treatments at 16 °C. The growth rates of P. donghaiense and the inhibitory strength of A. tamarense on P.

(rx) were higher at 20 °C (rx = 0.661 and 0.700 divisions donghaiense was 8 times higher than the inhibitory -1 day ) than others temperature groups (rx = 0.255 ~ 0.489 strength of P. donghaiense on A. tamarense when the divisions day-1), and the growth rates of K. mikimotoi initial cell density ratio was 36:1. The growth interactions

(ry) were higher than P. donghaiense (rx) except some between P. donghaiense and S. trochoidea was reported treatments at 20 °C, the values of ry were estimated and the results showed that the inhibitory strength of approximately from 0.411 to 0.806 divisions day-1. The S. trochoidea on P. donghaiense were 43 and 24 times parameters A and B were very close in most of the higher than the inhibitory strength of P. donghaiense on treatment groups and the ratio of A and B were mostly S. trochoidea when the initial cell density ratio were 1.9:1 around 1 (Table 1, 2, 3 and 4), for example, the value of A and 19:1, respectively (Wang & Tang, 2008). Therefore, for P. donghaiense was 5.38 × 10-6 ml cell-1 s-1 and B for the growth interactions between two different species K. mikimotoi was 5.04 × 10-6 ml cell-1 s-1 when the initial appear to be interspecies-specific and the mechanisms cell density ratio was 1:1 at 20 °C (Table 2). The model behind those interactions (allelopathy or cell contact simulation that was based on the results of these bi-algal and even two of them) can vary from species to species culture experiments predicted that P. donghaiense and (Tameishi et al., 2009). However, in the present study,

Thalassas, 30(2) · June 2014 41 ANGLu SHEN, XINGWEI YUAN, GUANGPENG LIU & DAOJI LI

Table 4: Estimated parameters from equations in mono-algal and bi-algal cultures of P. donghaiense (PD) and K. mikimotoi (KM) with different temperature and initial cell density ratio at 28 °C.

Carrying Interaction rate Initial cell density Growth rate, r capacity, K A or B ratio (PD:KM) (Divisions day-1) a or b (Cells ml-1) (ml cell-1 s-1)

PD 189,667 0.300 6.318 9.99 × 10-6 2:1 KM 216,068 0.680 3.001 9.94× 10-6

-6 PD 54,681 0.489 5.628 1.98 × 10 1:1 KM 216,068 0.680 4.68 2.03 × 10-5

PD 54,681 0.489 -- -- 1:2 KM 243,240 0.629 -- --

PD 54,681 0.489 -- -- 1:4 KM 218,329 0.806 -- --

Note: “--” out-competement was observed in bi-algal culture and the interaction rate cannot be estimated.

the growth patterns of the two species as predicted In conclusions, results of the present study demonstrated using the model (Uchida et al., 1999) are not necessarily that growth interactions between P. donghaiense and K. similar to those analyzed based on student’s t-test. For mikimotoi under different temperatures indicated that P. instance, when P. donghaiense and K. mikimotoi in donghaiense has an advantage at low temperature and K. bi-algal culture at 20 °C, P. donghaiense was not affected mikimotoi has an absolutely advantage at high temperature by K. mikimotoi in bi-algal culture when the cell density and out-competements were observed in bi-algal cultures by ratios were 2:1, 1:1 and 1:2, respectively (Fig. 2), when P. the end when the initial cell density ratio of P. donghaiense donghaiense and K. mikimotoi in bi-algal culture at 24 °C, and K. mikimotoi was 1:2 or 1:4 at 24°C and 28°C. In addition, P. donghaiense were affected by K. mikimotoi in bi-algal it serves as an important role in the initial cell density in culture in all cell density ratios (Fig. 3). P. donghaiense competition between P. donghaiense and K. mikimotoi. In has a survival strategy superior to that of K. mikimotoi fact, the phosphorus concentration in the adjacent coastal when the temperature was 20 °C and K. mikimotoi has a waters of the ECS was low when P. donghaiense blooms or survival strategy superior to that of P. donghaiense when K. mikimotoi blooms occurred (Zhao, 2010), thus, the growth the temperature was 24 °C. The causes of this result interactions between P. donghaiense and K. mikimotoi under may have the following two aspects. On the one hand, different temperatures in phosphorus limitation condition the parameters, which can be calculated, were used to will be determined. Allelopathy of harmful dinoflagellate model predictions are based on results obtained under may play a key role in the growth dynamics of blooms and fixed environmental conditions of short-term (12d) at the effects of the culture filtrate of P. donghaiense and K. which time the two species can coexist. On the other mikimotoi on each species under different environmental hand, the carrying capacity for P. donghaiense (Kx) or conditions will be conducted, too.

K. mikimotoi (Ky) was considerably larger than the cell density of P. donghaiense or K. mikimotoi in bi-algal MATERIALS AND METHODS cultures, which indicated that the two species can survive normally under this environmental condition. Further Seawater preparation study is necessary to determine whether the degree of inhibition between P. donghaiense and K. mikimotoi Seawater was collected from the East China Sea near under long-term (>12d). Furthermore, it is necessary Shengshan Islands, Zhoushan city, Zhejiang province to better integrate parameters for interactions between (30°45′N, 122°50′E), whose pH and salinity were 8.0 and phytoplankton species into existing models for more 28, respectively. This seawater was used in the cultures accurate prediction of interspecific competition. for all experiments of the present study.

42 Thalassas, 30(2) · June 2014 GROWTH INTERACTIONS BETWEEN THE BLOOM-FORMING DINOFLAGELLATES Prorocentrum donghaiense AND Karenia mikimotoi UNDER DIFFERENT TEMPERATURE

Algal species and culture conditions ml-1, subsamples were diluted 4 × to 10 × with fresh f/2 medium before counting. P. donghaiense was provided by Prof. Lu of the second institute of oceanography, State Oceanic Administration, STATISTICAL ANALYSES in Hangzhou, China. K. mikimotoi was obtained from East China Sea Fisheries Research Institute, Chinese Data were presented as mean ± SD of three replicates. Academy of Fishery Sciences, Shanghai, China. They Student’s t-test was used to examine the difference in were grown in modified f/2 medium (Guillard, 1975), growth between experimental groups and the control (P which was based on autoclaved (121 °C, 20 min) seawater, < 0.05: significant or P < 0.01: extremely significant). All at 20°C and a light intensity of 65-70 µmol m-2s-1 under analyses were conducted using Excel 2010 and PASW a 12:12 h light: dark cycle in illuminating incubators Statistics 18.0. (MTI-201B, RIKAKIKAI, JAPAN). All cultures were shaken manually twice daily at a set time. The microalgae ACKNOWLEDGEMENTS were cultivated to the exponential growth phase for use. During the experiment, cultures were maintained This work was supported by the Ministry of Science in 100 ml Erlenmeyer flasks containing 50 ml fresh f/2 and Technology of China (2010CB951203), and the State enriched seawater. These microalgal cultures were under Key Laboratory of Estuarine and Coastal Research in the different temperatures (16°C , 20°C, 24°C and 28°C) East China Normal University (2009KYYW03). and different initial cell densities (0.2 × 104 cells ml-1, 0.4 × 104 cells ml-1 and 0.8 × 104 cells ml-1), which called REFERENCES “monocultures”, and were used as controls throughout the experiment. A 0.5 ml sample was collected and Chen Y, Yan T, Yu RC, Zhou J (2011). Toxic effects of Karenia preserved in Lugol’s solution to minitor the growth of mikimotoi extracts on mammalian cells. 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44 Thalassas, 30(2) · June 2014 GROWTH INTERACTIONS BETWEEN THE BLOOM-FORMING DINOFLAGELLATES Prorocentrum donghaiense AND Karenia mikimotoi UNDER DIFFERENT TEMPERATURE

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(Received: October, 10, 2013; Accepted: January, 24, 2014)

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