ALLELOPATHIC EFFECTS OF Prorocentrum

Thalassas, 31(1) · January 2015: 33-49 An International Journal of Marine Sciences

ALLELOPATHIC EFFECTS OF Prorocentrum donghaiense AND Karenia mikimotoi ON EACH OTHER UNDER DIFFERENT TEMPERATURE ANGLU SHEN(1,2), XIAOLI XING(3) & DAOJI LI(1)* *corresponding author: [email protected] (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 (3) College of Ocean Science and Engineering, Shanghai Maritime University, Shanghai 201306, China

ABSTRACT Allelopathic effects of Prorocentrum donghaiense and Karenia mikimotoi, which are the main dinoflagellates bloom species, were investigated by using cell-free filtrate of algal culture experiments on each other under different temperatures with several combinations of initial cell densities. The growth of P. donghaiense was dramatically suppressed in the K. mikimotoi culture filtrate at these initial cell densities at 16 ºC, 20 ºC, 24 ºC and 28 ºC compared with the control and the inhibitory effect of K. mikimotoi culture filtrate on the growth of P. donghaiense was ordered as 16 ºC > 20 ºC > 24 ºC > 28 ºC; the growth of K. mikimotoi was suppressed in the P. donghaiense culture filtrate with the highest initial cell density under four tempeartures compared with the control, and the inhibitory effect of P. donghaiense culture filtrate on the growth of K. mikimotoi was ordered as 16 ºC > 20 ºC > 28 ºC > 24 ºC , while the inhibitory effect of P. donghaiense culture filtrate was smaller with middle initial cell density at 20 ºC, 24 ºC and 28 ºC. Besides, there was little inhibitory effect even the stimulating effect at some treatment groups when the initial cell density was low. The results indicated that the allelochemicals released by K. mikimotoi was heat-sensitive and P. donghaiense was not heat-sensitive. The results also showed that the culture temperature and the initial cell density play key roles in allelopathic effects of P. donghaiense and K. mikimotoi. Key words: Allelopathic effects, temperature, initial cell density, Prorocentrum donghaiense and Karenia mikimotoi.

RESUMEN Se estudiaron los efectos alelopáticos de Prorocentrum donghaiense y Karenia mikimotoi, que son dos dinoflagelados de gran importancia en la formación de mareas rojas, mediante experimentos de cultivo de algas en filtrados libres de células. El crecimiento de P. donghaiense sufrió supresión radical en el filtrado procedente de cultivos de K. mikimotoi en todas las densidades celulares iniciales a las distintas temperaturas ensayadas: 16 ºC, 20 ºC, 24 ºC y 28 ºC cuando las comparamos con el control, y el efecto inhibitorio de los filtrados de K. mikimotoi sobre el crecimiento de P. donghaiense se ordena como sigue: 16 ºC > 20 ºC > 24 ºC > 28 ºC; el crecimiento de K. mikimotoi fue inhibido por los filtrados de cultivos de P. donghaiense a las densidades celulares mayores en las cuatro temperaturas ensayadas en comparación con el control, y la ordenación del efecto inhibidor de los cultivos de P. donghaiense sobre el crecimiento de K. mikimotoi se ordena: 16 ºC > 20 ºC > 28 ºC > 24 ºC , por otra parte los efectos de los filtrados de P. donghaiense fueron menos relevantes cuando las densidades celulares iniciales fueron intermedias, pero fueron significativos a 20 ºC, 24 ºC y 28 ºC. Por el contrario, hubo efecto inihibidores poco importantes o incluso estimulación cuando la densidad celular inicial fue baja. Los resultados inicaron que los aleloquímicos liberados por K. mikimotoi fueron sensibles al calor y los P. donghaiense no mostró sensibilidad a la temperatura elevada. Los resultados también mostraron que tanto la temperatura como las densidades celulares iniciales juegan un papel en los efectos alelopáticos de P. donghaiense y K. mikimotoi. Palabras clave: Efectos alelopáticos, temperatura, Densidad celular inicial, Prorocentrum donghaiense y Karenia mikimotoi. Thalassas, 31(1) · January 2015

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ANGLU SHEN, XIAOLI XING & DAOJI LI

INTRODUTION Allelopathy in phytoplankton is the study of the negative/positive effect of allelochemicals produced by certain algae on other algal groups/species (Granéli et al., 2008a). Dinoflagellates, prymnesiohpytes and raphidophytes are main marine groups which can produce the allelochemicals (Granéli and Hansen, 2006). For example, Karenia is a genus containing at least 12 species of marine dinoflagellates and most Karenia species produce a variety of toxins that can kill fish and other marine organisms when they bloom (Brand et al., 2012). Alexandrium is another genus includes at least 4 species (A. andersoni, A. catenella, A. minutum and A. tamarense) which can release allelochemicals (Arzul et al., 1999; Frangópulos et al., 2004; Fistarol et al., 2004a). Prymnesium parvum and Chrysochromulina polylepis are important toxic species belong to prymnesiohpytes which can produce prymnesin, fatty acids and haemolysin (Granéli and Hansen, 2006). Chattonella antiqua and Heterosigma akashiwo are main harmful bloom species (HABs) belong to raphidophytes and the chemical properties of their allelochemicals were not investigated yet (Yamasaki et al., 2007, 2010; Qiu et al., 2011). In addition, Fistarol et al. (2003) reported that the allelopathic effect of P. parvum which produecs toxins affected the whole phytoplankton community, resulting in a decrease in both chlorophyll a and carbon uptake, and inducing changes in the plankton community structure, killing other members of the marine food-web, and similar allelopathic effect of Alexandrium spp. on a natural plankton community had been reported (Fistarol et al., 2004a). Many abiotic and biotic factors known to affect allelopathy, such as light, pH are essential for the production of some HABs and the role of nutrient in algae allelopathy has been investigated detailedly (Granéli and Hansen, 2006; Granéli et al., 2008a). Toxcity in phytoplankton usually increases under nutrient limitation, for example, P. parvum and C. polylepis (Johansson and Granéli, 1999; Granéli and Johansson, 2003), Alexandrium (Guisande et al., 2002; Frangópulos et al., 2004). Not much is known about how changes in temperature affect phytoplankton allelopathy (Fistarol et al., 2004a; van Rijssel et al., 2007). On the other hand, the intensity of the effect of the allelochemicals on the target species can vary depending on some biotic factors such as species-specific allelopathic effect of donor species and target species, cell densities of donor species and target species and the growth phase of the donor species (Legrand et al., 2003; Granéli et al., 2008a; Granéli et al., 2008b). In addition, the strength of the allelochemicals effect on target species is strain-dependent, and different strains of the donor species have different effects on target species (Fistarol et al., 2004b; Figueredo et al., 2007). 34

Thalassas, 31(1) · January 2015

Allelopathy and cell contact are main factors affecting the growth interactions of phytoplankton (Qiu et al., 2011), for Prorocentrum donghaiense D. Lu, 2001, it was similar to the inhibitory effect of P. donghaiense on A. tamarense in the bi-algal cultures occurred mainly by cell contact (You, 2006), and P. donghaiense and Scrippsiella trochoidea interaction with each other in the bi-algal cultures mainly by releasing allelochemicals (Wang and Tang, 2008); for Karenia mikimotoi G. Hansen and Ø. Moestrup, 2000, it was similar to the inhibitory effect of K. mikimotoi on Heterocapsa circularisquama in the bi-algal cultures occurred mainly by cell contact (Uchida et al., 1999), and K. mikimotoi can release the hemolytic and cytotoxic compounds such as fatty acids, gymnodimine (gymnocin-A and gymnocin-B) and affect the target species by membrane damage (Satake et al., 2002; Satake et al., 2005; Legrand et al., 2003). However, the mechanism of the growth interactions between P. donghaiense and K. mikimotoi is not clear. Therefore, it is important to realize P. donghaiense and K. mikimotoi competition mechanism through allelopathy or cell contact or both of them. Cross culture of algae refers to the target algae culture in cell-free filtrate of the donor algae, which is a classic method for allelopathy and many phytoplankton allelopathic phenomenon were found in this way (Granéli and Johnsson, 2003; Fistarol et al., 2004a; Li, 2011). Furthermore, P. donghaiense and K. mikimotoi are major HABs in the East China Sea (ECS) and have occurred during the same time at times in the adjacent coastal waters of the ECS from late April to June since 2005 (Zhou et al., 2006; Li et al., 2009; Zhao, 2010), during these periods, the field’s surface sea temperature was approximately from 16 to 27 ºC, thus, it is very necessary to investigate the relationship between allelopathy and the temperature in the bloom seasonal succession. In the present study, the allelopathic effects of P. donghaiense and K. mikimotoi on each other by using cell-free filtrate of algal culture experiments under different temperatures with several combinations of initial cell densities of the two species were examined. RESULTS Prorocentrum donghaiense growth in the K. mikimotoi culture filtrate Figure 1 and 2 showed the growth of P. donghaiense at different initial cell densities (0.45 × 104 cells ml-1, 0.9 × 104 cells ml-1 and 1.8 × 104 cells ml-1) in the K. mikimotoi culture filtrate at 16 ºC and 20 ºC, respectively. The growths of P. donghaiense were dramatically suppressed from 4d to 12d (P < 0.01) at all initial cell densities.

ALLELOPATHIC EFFECTS OF Prorocentrum donghaiense AND Karenia mikimotoi ON EACH OTHER UNDER DIFFERENT TEMPERATURE

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Fig. 1. Figure 1: Growth of P. donghaiense in K. mikimotoi filtrate at 16 ºC. The filtrate was prepared at a density of 1.50 × 105 cells ml-1, and the initially inoculated 4 cell density of P. donghaiense were 0.45 × 104 cells ml-1 (A), 0.90 × 1024 cells ml-1 (B) and 1.80 × 104 cells ml-1 (C), respectively. ◆ Control; ◇ Growth of P.

donghaiense in K. mikimotoi filtrate. Data are given as mean values ± SD (n = 3). Vertical lines show the standard deviation. *P < 0.05: significant or **P < 0.01: extremely significant between experimental groups and the control at the same time. Thalassas, 31(1) · January 2015

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Growth of P. donghaiense in K. mikimotoi filtrate at 20 ºC. The filtrate was prepared at a density of 1.50 × 105 cells ml-1, and the initially inoculated cell density of P. donghaiense were 0.45 × 104 cells ml-1 (A), 0.90 × 104 cells ml-1 (B) and 1.80 × 104 cells ml-1 (C), respectively. ◆ Control; ◇ Growth

25 ± SD (n = 3). Vertical lines show the standard deviation. *P < 0.05: significant of P. donghaiense in K. mikimotoi filtrate. Data are given as mean values

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or **P < 0.01: extremely significant between experimental groups and the control at the same time. Thalassas, 31(1) · January 2015


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Growth of P. donghaiense in K. mikimotoi filtrate at 24 ºC. The filtrate was prepared at a density of 1.50 × 105 cells ml-1, and the initially inoculated cell density of P. donghaiense were 0.45 × 104 cells ml-1 (A), 0.90 × 104 cells ml-1 (B) and 1.80 × 104 cells ml-1 (C), respectively. ◆ Control; ◇ Growth

26 ± SD (n = 3). Vertical lines show the standard deviation. *P < 0.05: significant of P. donghaiense in K. mikimotoi filtrate. Data are given as mean values


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Growth of P. donghaiense in K. mikimotoi filtrate at 28 ºC. The filtrate was prepared at a density of 1.5 × 105 cells ml-1, and the initially inoculated cell density of P. donghaiense were 0.45 × 104 cells ml-1 (A), 0.90 × 104 cells ml-1 (B) and 1.80 × 104 cells ml-1 (C), respectively. ◆ Control; ◇ Growth of P. donghaiense in K. mikimotoi filtrate. Data are given as mean values 27 ± SD (n = 3). Vertical lines show the standard deviation. *P < 0.05: significant

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Figure 3 showed the growth of P. donghaiense at different initial cell densities (0.45 × 104 cells ml-1, 0.9 × 104 cells ml-1 and 1.8 × 104 cells ml-1) in the K. mikimotoi culture filtrate at 24 ºC. When the initial cell density of P. donghaiense was 0.45 × 104 cells ml-1, the growth of P. donghaiense was dramatically suppressed from 4d to 12d (P < 0.01). Similarly, when the initial cell density of P. donghaiense was 0.9× 104 cells ml-1, the growth of P. donghaiense was dramatically suppressed from 4d to 8d (P < 0.01) and then the inhibitory effect was decreased (P < 0.05) at 10d and 12d. Again, when the initial cell density of P. donghaiense was 1.8 × 104 cells ml-1, the growth of P. donghaiense was dramatically suppressed from 4d to 12d (P < 0.01, except 6d: P < 0.05). Figure 4 showed the growth of P. donghaiense at different initial cell densities (0.45 × 104 cells ml-1, 0.9 × 104 cells ml-1 and 1.8 × 104 cells ml-1) in the K. mikimotoi culture filtrate at 28 ºC. When the initial cell density of P. donghaiense was 0.45 × 104 cells ml-1, the growth of P. donghaiense was dramatically suppressed from 4d to 8d (P < 0.01), and then the inhibitory effect was decreased (10d: P < 0.05, 12d: P > 0.05). When the initial cell density of P. donghaiense was 0.9× 104 cells ml-1, the growth of P. donghaiense was dramatically suppressed at 4d (P < 0.01), and then growth of P. donghaiense was weakly suppressed at 6d, 8d and 12d (P < 0.05) and even the inhibitory effect was weak at 10d (P > 0.05). When the initial cell density of P. donghaiense was 1.8 × 104 cells ml-1, the growth of P. donghaiense was dramatically suppressed at 4d (P < 0.01), then growth of P. donghaiense was weakly suppressed from 6d to 12d (P < 0.05). According to these results, the growth of P. donghaiense was highly significantly suppressed under the 16 ºC, 20 ºC, 24 ºC and 28 ºC at all initial cell densities, adding that the initial cell density and temperature was higher and the inhibitory effect was lighter. Karenia mikimotoi growth in the P. donghaiense culture filtrate

Figure 6 showed the growth of K. mikimotoi at different initial cell densities (0.35 × 104 cells ml-1, 0.7 × 104 cells ml-1 and 1.4 × 104 cells ml-1) in the P. donghaiense culture filtrate at 20 ºC. When the initial cell density of K. mikimotoi was 0.35 × 104 cells ml-1, the growth of K. mikimotoi was weakly suppressed at 10d and 12d (P < 0.05, Fig. 6A). When the initial cell density of K. mikimotoi was 0.7 × 104 cells ml-1, the growth of K. mikimotoi was weakly suppressed at 10d and 12d, too(P < 0.05, Fig. 6B). When the initial cell density of K. mikimotoi was 1.4 × 104 cells ml-1, the growth of K. mikimotoi was dramatically suppressed from 6d to 12d (Fig.6C). Figure 7 showed the growth of K. mikimotoi at different initial cell densities (0.35 × 104 cells ml-1, 0.7 × 104 cells ml-1 and 1.4 × 104 cells ml-1) in the P. donghaiense culture filtrate at 24 ºC. When the initial cell density of K. mikimotoi was 0.35 × 104 cells ml-1, there was no difference between the treatment group and the control (Fig. 7A). When the initial cell density of K. mikimotoi was 0.7 × 104 cells ml-1, the growth of K. mikimotoi was suppressed at 10d and 12d (P < 0.01, Fig. 7B). When the initial cell density of K. mikimotoi was 1.4 × 104 cells ml-1, the growth of K. mikimotoi was dramatically suppressed from 6d to 12d (Fig.7C). Figure 8 showed the growth of K. mikimotoi at different initial cell densities (0.35 × 104 cells ml-1, 0.7 × 104 cells ml-1 and 1.4 × 104 cells ml-1) in the P. donghaiense culture filtrate at 28 ºC. When the initial cell density of K. mikimotoi was 0.35 × 104 cells ml-1, there was no difference between the treatment group and the control at first 8 days and the growth of K. mikimotoi was weakly suppressed at 12 d (Fig. 8A). When the initial cell density of K. mikimotoi was 0.7 × 104 cells ml-1, the growth of K. mikimotoi was suppressed at 10d and 12d (P < 0.05, Fig. 8B). When the initial cell density of K. mikimotoi was 1.4 × 104 cells ml-1, the growth of K. mikimotoi was suppressed from 6d to 12d (Fig.8C). DISCUSSION

Figure 5 showed the growth of K. mikimotoi at different initial cell densities (0.35 × 104 cells ml-1, 0.7 × 104 cells ml-1 and 1.4 × 104 cells ml-1) in the P. donghaiense culture filtrate at 16 ºC. When the initial cell density of K. mikimotoi was 0.35 × 104 cells ml-1, the growth of K. mikimotoi was better than the control from 6d to 12d (Fig. 5A). When the initial cell density of K. mikimotoi was 0.7 × 104 cells ml-1, there was no difference between the treatment group and the control (Fig. 5B). When the initial cell density of K. mikimotoi was 1.4 × 104 cells ml-1, the growth of K. mikimotoi was weakly suppressed at 8d (P < 0.05) and dramatically suppressed from 10d to 12d (P < 0.01).

Role of temperature in the culture filtrates of P. donghaiense and K. mikimotoi Seawater temperature including its variation is one of the key factors when the red tides occur, which can directly control the occurrence, development, succession and extinction of red tide (You, 2006). However, not much is known about how changes in temperature affect phytoplankton allelopathy. Sivonen (1990) reported that the optimum temperature for hepatotoxin production and growth of green Oscillatoria agardhii was 25 °C, red O. agardhii produced almost similar amounts of Thalassas, 31(1) · January 2015

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Growth of K. mikimotoi in P. donghaiense filtrate at 16 ºC. The filtrate was prepared at a density of 1.00 × 105 cells ml-1, and the initially inoculated cell density of K. mikimotoi were 0.35 × 104 cells ml-1 (A), 0.70 × 104 cells ml-1 (B) and 1.40 × 104 cells ml-1 (C), respectively. ◆ Control; ◇ Growth of K.

28 ± SD (n = 3). Vertical lines show the standard deviation. *P < 0.05: significant mikimotoi in P. donghaiense filtrate. Data are given as mean values

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Growth of K. mikimotoi in P. donghaiense filtrate at 20 ºC. The filtrate was prepared at a density of 1.00 × 105 cells ml-1, and the initially inoculated cell density of K. mikimotoi were 0.35 × 104 cells ml-1 (A), 0.70 × 104 cells ml-1 (B) and 1.40 × 104 cells ml-1 (C), respectively. ◆ Control; ◇ Growth of K. mikimotoi in P. donghaiense filtrate. Data are given as mean values 29 ± SD (n = 3). Vertical lines show the standard deviation. *P < 0.05: significant


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Growth of K. mikimotoi in P. donghaiense filtrate at 24 ºC. The filtrate was prepared at a density of 1.00 × 105 cells ml-1, and the initially inoculated cell density of K. mikimotoi were 0.35 × 104 cells ml-1 (A), 0.70 × 104 cells ml-1 (B) and 1.40 × 104 cells ml-1 (C), respectively. ◆ Control; ◇ Growth of K. mikimotoi in P. donghaiense filtrate. Data are given as mean values 30 ± SD (n = 3). Vertical lines show the standard deviation. *P < 0.05: significant

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Growth of K. mikimotoi in P. donghaiense filtrate at 28 ºC. The filtrate was prepared at a density of 1.00 × 105 cells ml-1, and the initially inoculated cell density of K. mikimotoi were 0.35 × 104 cells ml-1 (A), 0.70 × 104 cells ml-1 (B) and 1.40 × 104 cells ml-1 (C), respectively. ◆ Control; ◇ Growth of

31 ± SD (n = 3). Vertical lines show the standard deviation. *P < 0.05: significant K. mikimotoi in P. donghaiense filtrate. Data are given as mean values


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toxin at temperatures of 15 to 25 ºC and the lowest toxin production by both strains was at 30 ºC. Issa (1999) found that the accumulation of antibiotic substances released by Oscillatoria angustissima and Calothrix parietina in medium is temperature dependent and the highest production level of antibiotic were detected at 25 ºC. Hobson and Fallowfield (2003) reported that the highest concentration of toxin produced by Nodularia spumigena in lake water would occur under high temperature and high irradiance conditions. However, there was no difference in the effects of filtrate obtained from A. tamarense at 14 ºC and 20 ºC on the phytoplankton community growth rates, number of dead Scrippsiella trochoidea cells and temporary cysts forming (Fistarol et al., 2004b). The growth of P. donghaiense in S. trochoidea filtrate was greatly changed when the cellfree filtrate was pre-treated under different temperature conditions (maintained water bath at 30, 60 and 100 ºC, for 30mins) (Wang and Tang, 2008). In the present study, the inhibitory effect of K. mikimotoi culture filtrate on the growth of P. donghaiense was ordered as 16 ºC > 20 ºC > 24 ºC > 28 ºC under three initial cell densities at the end of experiment (Fig. 9), which showed the allelochemicals released by K. mikimotoi might be heat-sensitive and easily be decomposed under heat conditions. It has been reported that the allelochemicals produced by P. parvum are sensitive to temperature, too (Kvernstuen, 1993 cited in Larsen and Bryant, 1998). On the other hand, the inhibitory effect of P. donghaiense culture filtrate on the growth of K. mikimotoi was ordered as 16 ºC > 20 ºC > 28 ºC > 24 ºC under three initial cell densities at the end of the experiment (Fig. 10), which showed the allelochemicals released by P. donghaiense was not sensitive to heat. Similar result was reported by Wang and Tang (2008), the growth of S. trochoidea was significantly suppressed from 72 h till the end of the experiment in the pre-treated (30, 60 and 100 ºC) P. donghaiense filtrate. Role of initial cell density in the culture filtrates of P. donghaiense and K. mikimotoi Allelopathic effect depends on the cell concentration of the donor and the target species (Schmidt and Hansen, 2001; Tillmann and John, 2002; Tillmann, 2003) and the general pattern is that higher cell numbers of the donor species results in more magnified detrimental effects, and vice versa (Granéli et al., 2008b). The percentage of target species (Oxyrrhis marina) mortality was not only reduced by diluting the donor species (P. parvum) culture but also by increasing the target species concentration (Tillmann, 2003). The immobilisation effect of O. marina was strongly dependent on the Alexandrium spp. cell concentration (Tillmann and John, 2002). Wang et al. (2006) reported that the growth of P. donghaiense with 44


low initial cell density (1 × 104 cells ml-1) was considerably suppressed and all cells were killed by the end of the experiment in the A. tamarense culture filtrate and the growth of P. donghaiense with high initial cell density (1 × 105 cells ml-1) was significantly suppressed but no out-competement was observed. In the present study, allelopathic effect (AE) of K. mikimotoi culture filtrate on P. donghaiense (AE = (100 × (control-filtrate culture) / control, Fistarol et al., 2004a) was reduced by increasing the target species (P. donghaiense) initial cell density (Fig. 11A). However, AE of P. donghaiense culture filtrate on K. mikimotoi was low when the initial cell density of K. mikimotoi was 0.35 × 104 cells ml-1, and high relatively when the initial cell density of K. mikimotoi were 0.7 × 104 cells ml-1 and 1.4 × 104 cells ml-1, even AE was negative at 16 ºC and the explanation for this observation is that the allelochemicals released by P. donghaiense have a stimulatory effect on the growth of K. mikimotoi when the initial cell density were 0.35 × 104 and 0.7 × 104cells ml-1 at 16 ºC (Fig. 11B). It is an interesting result and further study will be examined on this aspect. In conclusion, the growth of P. donghaiense in the K. mikimotoi culture filtrate was suppressed in all experiment, the temperature and the initial cell density was higher and the inhibitory effect was lighter besides. The growth of K. mikimotoi was suppressed in the P. donghaiense culture filtrate with the highest initial cell density at four temperature treatments. It is an interesting result that there was little inhibitory effect when the initial cell density was low and even the stimulating effect at 16 ºC. In addition, the allelochemicals released by K. mikimotoi was heat-sensitive and P. donghaiense was not heat-sensitive according to the order of inhibitory effect. Actually, the phosphorus concentration in adjacent coastal waters of the ECS was low from late spring to early summer when P. donghaiense blooms or K. mikimotoi blooms occurred (Zhao, 2010), and allelochemicals in phytoplankton usually increases under phosphorus deficient conditions (Granéli and Johansson, 2003), therefore, allelopathic effects of two algae on each other under different temperatures in phosphorus limitation condition will be determined. MATERIALS AND METHODS Algal species and culture conditions Prorocentrum donghaiense was provided by Prof. Lu of the second institute of oceanography, State Oceanic Administration People’s Republic of China, in Hangzhou, China. Karenia mikimotoi was obtained from East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai, China. They were grown


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Fig. 9temperature. The filtrate was prepared at a density of 1.50 × 105 cells ml-1, and Growth of P. donghaiense in K. mikimotoi filtrate under the different the initially inoculated cell density of P. donghaiense were 0.45 × 104 cells ml-1 (A), 0.90 × 104 cells ml-1 (B) and 1.80 × 104 cells ml-1 (C), respectively.

◆ 16 ºC; ■ 20 ºC; ▲ 24 ºC; ● 28 ºC. Data are given 32 as mean values ± SD (n = 3). Vertical lines show the standard deviation.


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Growth of K. mikimotoi in P. donghaiense filtrate under the different temperature. The filtrate was prepared at a density of 1.00 × 105 cells ml-1, and the initially inoculated cell density of K. mikimotoi were 0.35 × 104 cells ml-1 (A), 0.7 × 104 cells ml-1 (B) and 1.4 × 104 cells ml-1 (C), respectively. ◆ 16 ºC;

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■ 20 ºC; ▲ 24 ºC; ● 28 ºC. 33 Data are given as mean values ± SD (n = 3).



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Figure 11: Allelopathic effect of K. mikimotoi on P. donghaiense (I) under the different temperature after 12 d of exposure to cell-free filtrate. The filtrate was prepared at a density of 1.5 × 105 cells ml-1, and the initially inoculated cell density of P. donghaiense were 0.45 × 104 cells ml-1 (A), 0.9 × 104 cells ml-1 (B) and 1.8 × 104 cells ml-1 (C), respectively. ◆ 16 ºC; ■ 20 ºC; ▲ 24 ºC; ● 28 ºC. Data are given as mean values ± SD (n = 3). Vertical lines show the standard deviation. Allelopathic effect of P. donghaiense on K. mikimotoi (II) under the different temperature after 12 d of exposure to cell-free filtrate. The filtrate was prepared at a density of 1.00 × 105 cells ml-1, and the initially inoculated cell density of K. mikimotoi were 0.35 × 104 cells ml-1 (A), 0.70 × 104 cells ml-1 (B) and 1.40 × 104 cells ml-1 (C), respectively. ◆ 16 ºC; ■ 20 ºC; ▲ 24 ºC; ● 28 ºC. Data are given as mean values ± SD (n = 3).

in modified f/2 medium (Guillard, 1975), which was based on autoclaved (121 ºC, 20 min) seawater, at 20 ºC and a light intensity of 65-70 µmol m-2s-1 under a 12:12 h light: dark cycle in illuminating incubators (MTI-201B, Rikakikai, Japan). All cultures were shaken manually twice daily at a set time. Effect of the culture filtrate of P. donghaiense on K. Fig. 11 mikimotoi 34 Culture filtrates were prepared from P. donghaiense culture by passing through a 0.7 µm GF/C glass

microfiber filter (Whatman International Ltd.) on a 47 mm polysulfone holder under gravity filtration when the cell density was 4.00 × 104 cells ml-1. The filtrate media were then enriched with f/2 medium. The prepared culture filtrates were used to cultivate K. mikimotoi. The experiments were conducted in 100 ml Erlenmeyer flasks containing 50 ml of K. mikimotoi cells in exponential growth phase (stock culture: 1.00 × 105 cells ml-1), which then were inoculated to a final cell density of 0.35 × 104 cells ml-1, 0.70 × 104 cells ml-1 and 1.40 × 104 cells ml-1 at 16 ºC, 20 ºC, 24 ºC and 28 ºC, respectively. Cell grown in monoculture with f/2 medium were used as controls. A 0.5 Thalassas, 31(1) · January 2015

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ml sample was collected and preserved in Lugol’s solution to monitor the growth of the microalgae by directly counting the cell numbers using a haemocytometer under an optical microscope (BX43, Olympus, Japan) at 0, 4, 6, 8, 10, and 12 d, respectively. Effect of the culture filtrate of K. mikimotoi on P. donghaiense Culture filtrates were prepared from K. mikimotoi culture by passing through a 0.7 µm GF/C glass microfiber filter (Whatman International Ltd.) on a 47 mm polysulfone holder under gravity filtration when the cell density was 1.50 × 105 cells ml-1. The filtrate media were then enriched with f/2 medium. The prepared culture filtrates were used to cultivate P. donghaiense. The experiments were conducted in 100 ml Erlenmeyer flasks containing 50 ml of P. donghaiense cells in exponential growth phase (stock culture: 5.00 × 104 cells ml-1), which then were inoculated to a final cell density of 0.45 × 104 cells ml-1, 0.90 × 104 cells ml-1 and 1.80 × 104 cells ml-1 at 16 ºC, 20 ºC, 24 ºC and 28 ºC, respectively. Cell grown in monoculture with f/2 medium were used as controls. A 0.5 ml sample was collected and preserved in Lugol’s solution to monitor the growth of the microalgae by directly counting the cell numbers using a haemocytometer under an optical microscope (BX43, Olympus, Japan) at 0, 4, 6, 8, 10, and 12 d, respectively. STATISTICAL ANALYSES Data were presented as mean ± SD of three replicates. Student’s t-test was used to examine the differences in growth between experimental groups and the control (P < 0.05: significant or P < 0.01: extremely significant). All analyses were conducted using Excel 2010 and PASW Statistics 18.0. ACKNOWLEDGEMENTS This work was supported by the Ministry of Science and Technology of China (2010CB951203), and the State Key Laboratory of Estuarine and Coastal Research in East China Normal University (2009KYYW03). REFERENCES Arzul G, Seguel M, Guzman L, Erard-LeDenn E (1999). Comparison of allelopathic properties in three toxic Alexandrium species. Journal of Experimental Marine Biology and Ecology, 232: 285-295. Brand LE, Campbell L, Bresnan E (2012). Karenia: The biology and ecology of a toxic genus. Harmful Algae, 14: 156-178. Figueredo CC, Giani A, Bird DF (2007). Does allelopathy contribute to Cylindrospermopsis raciborskii (cyanobacteria) 48


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Yamasaki Y, Nagasoe S, Tameishi M, Shikata T, Shimasaki Y, Oshima Y, Honjo T (2007). Allelopathic interactions between the bacillariophyte Skeletonema castatum and the raphidophyte Heterosigma akashiwo. Marine Ecology Progress Series, 339: 83-92. Yamasaki Y, Nagasoe S, Tameishi M, Shikata T, Zou Y, Jiang Z, Matsubara T, Shimasaki Y, Yamaguchi K, Oshima Y, Oda T, Honjo T (2010). The role of interactions between Prorocentrum minimum and Heterosigma akashiwo in bloom formation. Hydrobiologia 641: 33-44. You XH (2006). Studies on the growth of Prorocentrum donghaiense and Alexandrium tamarense under different environmental factor and the interspecific competition. Ocean University of China, Thesis of Master, Qingdao, China. Zhao DZ eds. (2010). Marine red tide disaster distribution in typical areas in China. Marine Press, Beijing. Zhou WH, Yin KD, Zhu DD (2006). Phytoplankton biomass and high frequency of Prorocentrum donghaiense harmful algal bloom in Zhoushan sea area in spring. Chinese Journal of Applied Ecology, 17(5): 887-893. (in Chinses with English abstract).

(Received: February, 26, 2014; Accepted: July, 23, 2014)


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