Chinese Journal of Oceanology and Limnology Vol. 28 No. 4, P. 911-916, 2010 DOI: 10.1007/s00343-010-9061-y
Negative effects of Phaeocystis globosa on microalgae* LIU Jiesheng (刘洁生)†, VAN RIJSSEL Marion††, YANG Weidong (杨维东)†, PENG Xichun (彭喜春)†††, LÜ Songhui (吕颂辉)†††, WANG Yan (王艳)†††, CHEN Jufang (陈菊芳)†††, WANG Zhaohui (王朝晖)†, QI Yuzao (齐雨藻)†,** †
College of Life Sciences and Technology, Jinan University, Guangzhou 510632, China
††
Department of Marine Biology, University of Groningen, P.O. Box 14, 9750AA Haren, Netherlands
†††
Research Center for Harmful Algae and Aquatic Environments, Jinan University, Guangzhou 510632, China
Received Apr. 2, 2009; revision accepted Aug. 20, 2009 © Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag Berlin Heidelberg 2010
Abstract The potential allelopathic effects of the microalga, Phaeocystis globosa Scherffel, on three harmful bloom algae, Prorocentrum donghaiense Lu, Chattonella marina (Subrahmanyan) Hara et Chihara and Chattonella ovata Hara et Chihara were studied. The growth of C. marina and C. ovata was markedly reduced when the organisms were co-cultured with P. globosa or cultured in cell-free spent medium. Haemolytic extracts from P. globosa cells in the senescence phase had a similar inhibitory effect on the three harmful bloom algae. However, P. globosa had less influence on the brine shrimp, Artemia salina. These results indicate that P. globosa may have an allelopathic effect on microalgae, which would explain the superior competitive abilities of P. globosa. Because the addition of the haemolytic toxins from P. globosa had similar effects on algae as spent media, these compounds may be involved in the allelopathic action of P. globosa. Keyword: Phaeocystis globosa; allelopathy; haemolytic toxin
1 INTRODUCTION Since 1997, harmful algal blooms of Phaeocystis have frequently occurred in the coastal waters of the southeast Oceans in China, which has had a great impact on the local aquaculture industry (Huang et al., 1999; Shen et al., 2004). Phaeocystis is a genus of the family Prymnesiophyta (or Haptophyta) that has eurythermal and euryhaline characteristics and is widely distributed from polar to temperate seas (Lancelot et al., 1987; Gibson et al., 1990; Wassmann et al., 1990; Chen et al., 1999; Verity et al., 2007). Huang et al. (1999) described the Chinese bloom forming Phaeocystis species, which was later identified as P. globosa by ribosomal DNA sequence analysis (Chen et al., 2002). Haemolytic toxins that were responsible for massive fish kills have also been isolated from a harmful bloom of Phaeocystis (He et al., 1999). In previous studies conducted in our laboratory, P. globosa cells grown under nutrient limited conditions such as N- and Fe-limitation were found to have significantly higher haemolytic activity than those grown under non-limited conditions (Liu et al., 2006). The induction of
haemolytic substances under nutrient-limited conditions indicates that these compounds may be involved in an allelopathic response to adverse conditions as has been described for other prymnesiophytes (Johansson et al., 1999; Granèli et al., 2003). The production of toxic compounds is common, but not universal among harmful algal blooms (HABs). Accordingly, the reasons for the synthesis of these toxins by organisms comprising HABs are still the subject of debate (Smayda, 1997; Cembella, 2003; Legrand et al., 2003; Gross, 2003). Possible functions include the repulsion of grazers and inhibition of co-occurring species of phytoplankton (Turner et al., 1997). Allelopathy, which is the chemical interaction between plants that often takes the form of stimulation or growth inhibition, has recently been found to be an operating principle among * Supported by the NSFC-Guangdong Province Association Foundation (No. U0733006), the National Natural Science Foundation of China (No. 30970502), and the State Key Laboratory of Marine Environmental Science (Xiamen University MEL0403) ** Corresponding author:
[email protected]
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marine microalgae as well (especially among the species that comprise HABs) both in-vitro and in-situ (Chan et al., 1980; Maestrini et al., 1981, Arzul et al., 1999; Jin et al., 2003). Indeed, allelopathy appears to be a key factor that promotes the dominance of species that form HABs over other algal species. The main objective of this study was to provide more information regarding the action of P. globosa upon nearby competitors. To accomplish this, the effects of living P. globosa, culture filtrate and isolated haemolytic compounds on three HAB algae, Prorocentrum donghaiense Lu, Chattonella marina (Subrahmanyan) Hara et Chihara and Chattonella ovta Hara et Chihara, were investigated.
2 MATERIALS AND METHODS 2.1 Algal batch cultures Phaeocystis globosa Sherffell, Prorocentrum donghaiense Lu, Chattonella marina (Subrahmanyan) Hara et Chihara and Chattonella ovta Hara et Chihara were obtained from the Algal Culture Collection of the Research Center for Red Tide and Aquatic Environment of Jinan University in Guangzhou, China. The strains were maintained in the laboratory in artificial seawater supplemented with f/2 medium. In this study, all strains were grown as batch cultures in 16 Erlenmeyer flasks that each contained 1.2 L of f/2 medium. All media used were filter-sterilized through 0.22 µm Millipore filters. The cultures were grown at 221°C for 15 days in an ACI (Artificial Climate Incubator) that contained cool-white fluorescent tubes that provided 200 μmol m-2 s-1 under a 12/12 h light/dark cycle. Growth was monitored using the optical density measured at 680 nm (OD680) after homogenizing the subsamples. Additionally, the cell density was determined by counting the microalgal cells using an Olympus CKX41 inverted microscope. 2.2 Preparation of haemolytic extracts P. globosa cells were collected during the stationary phase (1.73×106 cells/ml) and then re-suspended in a mixed solution of menthol, chloroform and water (13:7:5, v/v), after which they were vigorously agitated in an ultrasonic bath at 4°C. The fractions were then evaporated to dryness (25°C, 0.1 Mpa) in a rotary evaporator and subsequently re-dissolved in 70% methanol and stored until use. One ml of methanol extract was equivalent to 1 liter of extracted culture.
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2.3 Biological tests The effects of live P. globosa on HAB algae were studied in co-cultures. Briefly, 90 ml of batch cultures of P. globosa in the stationary phase (1.73×106 cells/ml) were added to 60 ml cultures of C. marina or C. ovata that were in the logarithmic phase. The cultures were then enriched with inorganic nutrients to reach the concentration required for f/2 medium. The initial densities of C. marina and C. ovata were 4.3×103 cells/ml and 2.1×103 cells/ml, respectively. The cell densities of the two HAB algae in the bialgal cultures were determined daily by counting triplicate 0.1 ml aliquots of the samples under an inverted microscope. The influence of the cell-free filtrate from P. globosa cultures in the stationary phase (1.73×106 cells/ml) on P. donghaiense, C. marina and C. ovata was evaluated using the method developed by Gentien et al. (1990). Briefly, 60 ml of P. donghaiense, C. marina and C. ovata cultures were added to flasks containing 90 ml of the cell-free filtrate of P. globosa cultures, which was obtained by filtration through 0.2 µm pore-size filters. The cultures were then enriched with inorganic nutrients to reach the concentrations required for f/2 medium. Next, 0.1 ml aliquots of the cultures were collected at a set time each day and the microalgae in the aliquots were enumerated using an inverted microscope. All tests were conducted in triplicate and culture diluted with 90-ml of f/2 medium was used as a control. The effects of haemolytic extract of P. globosa on the three HAB algae were evaluated by adding haemolytic toxin (90 μl) from P. globosa to 60 ml cultures of C. marina, P. donghaiense and C. ovata. Briefly, 90 ml of f/2 medium was added to the cultures, after which the growth of the three HAB algae was evaluated by counting the cells under an inverted microscope. Cultures amended with 90 ml of f/2 medium without haemolytic extract were used as controls. The toxicity of the extracts against the brine shrimp, Artemia salina, was tested using cysts purchased from the South China Sea Institute of Oceanology, Chinese Academy of Science. Briefly, the cysts were allowed to hatch by incubation for 48 h in seawater with a salinity of 20 that had been passed through a 0.22 μm Millipore membrane filter. The seawater containing the hatched brine shrimp was continuously aerated and maintained at a
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temperature of 28°C using an ACI that contained cool-white fluorescent tubes that provided 200 μmol·m-2·s-1. The toxicity of the algal cultures, cell-free filtrates and haemolytic extracts of P. globosa against A. salina in the senescence phase was evaluated using the standard ARTOX procedure (Lush et al., 1996). Briefly, the extracts were diluted to a concentration of 1‰ in f/2 media. Ten nauplii of A. salina were then added into 1 ml of the test solutions containing f/2 with a salinity of 20 and cultured in 6×4 well plates The mortality of the nauplii was then recorded at 6 h, 12 h and 24 h. One milliliter of f/2 with a salinity of 20 was used as a control.
3 RESULTS 3.1 Effects of living P. globosa on other HAB microalgae Due to the similar cell size of P. donghaiense and P. globosa, it was difficult to evaluate the growth of P. donghaiense by counting cells under a microscope. Therefore, only the effects of P. globosa on the growth of C. marina and C. ovata in bialgal cultures were investigated. After one day of contact between the algae, the growth of the C. marina was reduced when compared with the control. At day 2, the cell density of C.marina was only about 7.84×103 cells/ml, which was much lower than that of control (about 1.04×104 cells/ml, Fig.1). The cell densities of C. ovata were not affected growing in the presence of P. globosa for up to two days. However, at day 3, growth was completely halted and the cell numbers were lower than that on day 2. Specifically, the co-culture contained approximately 3.28 ×103 cells/ml, half the amount of cells in the control (6.86×103 ells/ml) after three days of culture (Fig.2).
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Fig.2 Growth of C. ovata in the presence of live P. globosa
3.2 Effect of the cell-free filtrate of P. globosa on the three HAB algae The effects of cell-free filtrate from P. globosa cultures on P. donghaiense, C. marina and C. ovata cells are shown in Fig.3, 4 and 5. During the five days of culture in the cell-free filtrate of P. globosa culture, the cell densities of P. donghaiense, C. marina and C. ovata all increased. However, the increases in the cell density of the three HAB algae were all lower than the increases in the corresponding control. Additionally, negative effects became apparent after three days and P. donghaiense was most severely affected. When compared with live Phaeocystis, the filtrate was less effective and its effects were slower.
Fig.3 Growth of P. donghaiense in the presence of cell-free filtrate from P. globosa
3.3 Effect of haemolytic extract of P. globosa on the three HAB algae
Fig.1 Growth of C. marina in the presence of live P. globosa
All cultures evaluated in this study showed a drop or little change in cell density when cultured in the presence of P. globosa haemolytic extracts for five days. Specifically, the initial cell density of
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Fig.4 Growth of C. marina in the presence of cell-free filtrate from P. globosa
Fig.5 Growth of C. ovata in the presence of cell-free filtrate from P. globosa
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Fig.7 Growth of C. marina in f/2 medium containing haemolytic toxin
Fig.8 Growth of C. ovata in f/2 medium containing haemolytic toxin
C. ovata which gathered and deposited after three days of culture and remained at a density of 1.94×103 cells/ml, with P. donghaiense being the most severely affected and C. ovata the least affected. All cultures stopped growing and were lysed in the presence of the hemolysin (Figs.9&10). 3.4 Toxicity of P. globosa toward Artemia salina
Fig.6 Growth of P. donghaiense in f/2 medium containing haemolytic toxin
P. donghaiense, C. marina and C. ovata was about 2.7×104, 1.2×103 and 1.9×103 cells/ml, respectively. After five days of culture, the cell densities in controls without extracts increased to 1.1×105, 7.4×103 and 6.9×103 cells/ml, respectively. However, the density of P. donghaiense and C. marina with extracts decreased to 3.1×103 and 1.2×102 cells/ml, respectively.
The cultures and cell-free filtrates of P. globosa were not toxic to A. salina. Additionally, the solution containing the haemolytic substance induced only 10% mortality of A. salina after 24 h of culture, while none of the A. salina died in the controls.
4 DISCUSSION The growth of two Chattonella species was hampered by the presence of live Phaeocystis cells, which indicates that Phaeocystis has a competitive advantage over Chattonella. It is likely that this effect was due to competition for available nutrients or allelopathy. However, sufficient nutrients were
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Fig.9 Morphological changes in P. donghaiense induced by haemolytic toxins produced by P. globosa (sequence a-b-c)
Fig.10 Morphological changes in C. marina induced by haemolytic toxins produced by P. globosa (sequence a-b-c-d-e)
added to the co-cultures to prevent nutrient limitation, and the filtrate from Phaeocystis cultures exhibited similar inhibitory effects on the growth of P. donghaiense, C. marina and C. ovata. Therefore, it is reasonable to assume that allelopathy of Phaeocystis was primarily responsible for its competitive advantage. In extracts of P. globosa during harmful bloom events the major haemolytic compound identified was 1-heptadecadienoyl-3digalatosyl-glyceroll where the acyl group was polyunsaturated (He et al., 1999). Unsaturated acyl groups were also found to be part of a similar haemolytic compound of the prymnesiophyte Chrysochromulina polylepis Manton et Parke (1-acyl-3-digalatosyl-glycerol, Yasumoto et al., 1990). Additionally, a haemolytic glycolipid was isolated and identified in Prymnesium parvum Carter extracts as well as from the culture medium (He et al. 1996). Harmful algae often contain toxic compounds that are not involved in allelopathy, while other substances produced by these algae are allelopathic (Legrand et al., 2003). The filtrate of the Phaeocystis culture affected the three microalgae, but to a lesser extent than the live cultures. For some algae,
including Phaeocystis pouchetii (van Rijssel et al., 2007), live algae are more harmful than the filtrate. This may be because the toxin is bound to the Phaeocystis cells or because the cells add a compound that enhances the effect of the excreted compound. Because the filtrate has a negative effect on the microalgae, it is clear that something excreted by Phaeocystis is harmful to other algae. To determine if haemolytic glycolipids were the bioactive compounds, haemolytic extracts of Phaeocystis were added to the algae. The addition of the extracts produced similar results as the live cultures. These findings imply that hemolytic toxins may play an important role in the allelopathic action of P. globosa. However, other allelopathic materials may also be present. Indeed, neither culture nor spent medium influenced the brine shrimp and the extracts were only mildly toxic toward the brine shrimp, indicating that the causative agents are not very potent toxins.
5 CONCLUSION The results of this study indicate that P. globosa have an allelopathic effect on other algae; however, it
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is not clear if this effect is mediated by glycolipids. Additionally, Chinese P. globosa appear to differ from P. pouchetti in that do not contain haemolytic components within the cell (Stabell et al., 1999). However, the effects of P. globosa were comparable to those of Chrysochromulina polylepis (Yasumoto et al. 1990). References Arzul G, Seguel M, Guzman L, Erard-Le Denn E. 1999. Comparison of allelopathic properties in three toxic Alexandrium species. J. Exp. Mar. Biol. Ecol., 232(2): 285-295. Cembella A D. 2003. Chemical ecology of eukaryotic microalgae in marine ecosystems. Phycologia, 42(4): 420-447. Chan A T, Andersen R J, Le Blanc M J, Harrison P J. 1980. Algal plating as a tool for investigating allelopathy among marine microalgae. Mar. Biol., 59(1): 7-13. Chen J F, Xu N, Jiang T J, Wang Y, Wang Z H, Qi Y Z. 1999. A report of Phaeocystis globosa Scherffel bloom in coastal water of Southeast China. J. Jinan Univ. (Natural Science), 20(3): 124-129. (in Chinese with English abstract) Chen Y Q, Wang N, Zhang P, Zhou H, Qu L H. 2002. Molecular evidence identifies bloom- forming Phaeocystis (Prymnesiophyta) from coastal waters of southeast China as Phaeocystis globosa. Biochem. Syst. Ecol., 30(1): 15-22. Gentien P, Arzul G. 1990. A theoretical case of competition based on the ectocrine production by Gyrodinium cf. aureolum. In: Granèli E, Sundströn B, Edler L, Anderson D M, eds. Toxic Marine Phytoplankton. Elsevier Science Publisher, New York (USA). p. 161-164. Gibson J A E, Garrick R C, Burton H R, McTaggart A R. 1990. Dimethylsulfide and the alga Phaeocystis pouchetii in Antarctic coastal waters. Mar. Biol., 104(2): 339-346. Granéli E, Johansson N. 2003. Increase in the production of allelopathic substances by Prymnesium parvum cells grown under N- or P-deficient conditions. Harmful Algae, 2(2): 135-145. Gross E M. 2003. Allelopathy of aquatic autotrophs. Crit. Rev. Plant Sci., 22: 313-339. He J W, Shi Z X, Zhang Y H, Liu Y D, Jiang T J, Yin Y W, Qi Y Z. 1999. Morphological characteristics and toxins of Phaeocystis cf. pouchetii (Prymnesiophyceae). Oceanol. Limnol. Sin., 30(2): 172-179. (in Chinese with English abstract) He J W, Chen M H, He Z R. 1996. Isolation and characterization of toxins from the phytoflagellate Prymnesium parvum. Acta Hydrobiol. Sin., 20(1): 41-48. (in Chinese with English abstract) Huang C J, Dong Q X, Zheng L. 1999. Taxonomic and ecological studies on a large scale Phaeocystis pouchetii bloom in the southeast cast of China during late 1997. Ocean. Limnol. Sin., 30(6): 581-589. (in Chinese with English abstract) Johansson N, Granéli E. 1999. Cell density, chemical composition and toxicity of Chrysochromulina polylepis (Haptophyta) in relation to different N:P supply ratios. Mar. Biol., 135(2): 209-217.
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