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Variability in the allelopathic action of the Alexandrium tamarense species complex along the coast of China
Harmful Algae 47 (2015) 17–26
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Variability in the allelopathic action of the Alexandrium tamarense species complex along the coast of China Jian Chen a,c, Qian Ye a, Hai-Feng Gu b, Hong-Ye Li a, Song-Hui Lv a, Jie-Sheng Liu a,*, Wei-Dong Yang a,* a
College of Life Science and Technology, Key Laboratory of Aquatic Eutrophication and Control of Harmful Algal Blooms of Guangdong Higher Education Institute, Jinan University, Guangzhou 510532, China Third Institute of Oceanography, SOA, Xiamen 361005, China c Zhongshan Entry-Exit Inspection and Quarantine Bureau Technology Center, Zhongshan 528403, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 May 2014 Received in revised form 14 May 2015 Accepted 14 May 2015 Available online 7 June 2015
Members of the Alexandrium tamarense species complex are some of the most important toxigenic dinoflagellates and are also widely distributed along the coast of China. A. tamarense and another wellknown toxic raphidophycean alga, Chattonella marina, are usually found in the same sea areas, sometimes at the same place and time. In this study, under laboratory co-culture conditions, we found that most of the 15 A. tamarense strains from the South China Sea and the East China Sea had significant inhibition on the growth of C. marina, while most of the A. tamarense strains were inhibited to various extents by C. marina. These results suggest that there is a complex reciprocal inhibitory effect between A. tamarense and C. marina, which might be caused by their allelopathic potency, the intrinsic growth rate of the algal species and the sensitivity of the target alga to the allelochemicals, etc. However, no strict correlations were observed between the allelopathic actions and the geographical distribution. The allelopathic potency observed in co-culture did not correlate with the hemolytic activity of the extracellular toxins obtained by the solvent extraction method (p > 0.05) but did correlate with the hemolytic activity obtained by the dialysis method (p < 0.01). These results indicate that various strains of A. tamarense could produce diverse hemolytic substances with different compositions and properties, which might be responsible for the variability in A. tamarense allelopathic action. ß 2015 Elsevier B.V. All rights reserved.
Keywords: Alexandrium tamarense species complex Chattonella marina Allelopathy Hemolytic toxins
1. Introduction Members of the Alexandrium tamarense species complex, including A. tamarense (Lebour) Balech, Alexandrium fundyense Balech and Alexandrium catenella (Whedon and Kofoid) Balech, are some of the most important toxigenic dinoflagellates (Lilly et al., 2007; Gu et al., 2013). Recently, based on morphology data, ITS/ 5.8S genetic distances, ITS2 compensatory base changes, mating incompatibilities, toxicity, the sxtA gene, and rDNA phylogenies, John et al. (2014) amended this species into the five following species: (1) Group I, A. fundyense; (2) Group II, Alexandrium mediterraneum; (3) Group III, A. tamarense; (4) Group IV, Alexandrium pacificum; and (5) Group V, Alexandrium australiense. Along the coast of China, A. tamarense is widely distributed from
* Corresponding author. Tel.: +86 020 85228470/+86 020 85223686; fax: +86 020 85225183. E-mail addresses: [email protected], [email protected] (W.-D. Yang). http://dx.doi.org/10.1016/j.hal.2015.05.008 1568-9883/ß 2015 Elsevier B.V. All rights reserved.
the Bohai Sea to the South China Sea. Blooms of toxic Alexandrium spp. have also been reported in the Yellow Sea, the coastal waters adjacent to the Changjiang River estuary and some bays in the South China Sea (Wang et al., 2006a, 2011; Song et al., 2009; Gu et al., 2013; Zou et al., 2014). Furthermore, A. tamarense blooms have been found to be associated with paralytic shellfish poisoning (PSP) toxin contamination in the cultivated shellfish in China (Wang et al., 2011; Chen et al., 2013; Jiang et al., 2013). The wide distribution of the species and their implication in harmful algal blooms (HABs) and PSP events have attracted increasing attention in China, and many studies have been performed on the nutritional characteristics, toxin composition, phylogenetics and toxicity of this genus (Chen et al., 1999; Yan et al., 2003; Wu et al., 2005; Fang et al., 2006; Tang et al., 2006; Gu et al., 2013; Jiang et al., 2013). As with most dinoflagellate species, Alexandrium tends to have lower nutrient uptake affinities and maximum growth rates than other microalgae (Smayda, 1997; Frango´pulos et al., 2004). Allelopathic effects have been proposed to exert an important role in the formation and maintenance of Alexandrium blooms
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(Fistarol et al., 2004; Tillmann et al., 2008; Hattenrath-Lehmann and Gobler, 2011). Within the Alexandrium genus, lytic activity (usually exhibiting allelochemical activity) has been detected in all of the species tested; however, the strength of the lytic activity varies considerably among different species and strains of the same species from different geographical origins (Tillmann et al., 2009; Hattenrath-Lehmann and Gobler, 2011). An increasing number of studies have demonstrated significant intraspecific genotypic and phenotypic variability within unicellular algal populations (Tillmann et al., 2009; Kremp et al., 2012; Lebret et al., 2012; Dia et al., 2014). Microalgae from different strains of the same species, especially dinoflagellates and bacillariophyta, display various phenotypes and genotypes, leading to the variety in nutrient uptake, photosynthesis capacity and growth rate (Kremp et al., 2012). These capabilities facilitate HAB formation and adaptation to changing environments (Alpermann et al., 2010; Lebret et al., 2012). Several studies have shown that Alexandrium tamarense strains from the South China Sea and East China Sea exhibited strong allelopathic effects on Prorocentrum donghaiense, Chattonella marina and Heterosigam akashiwo (Wang et al., 2006b; You et al., 2006; Yang et al., 2010; Yin et al., 2010). However, at least 148 HAB species, such as Noctiluca scintillans, Prorocentrum minimum, P. mians, Gonyaulax polyedra, Skeletonema costatum, Mesodinium rubrum, Trichodesmium sp., and C. marina, have been found in China to date, of which 44 have caused HAB events in different sea areas (Zhu et al., 1997). These data imply that there might be a complicated reciprocal interaction between A. tamarense and target alga cells and that various strains of A. tamarense might exhibit different allelopathic potency. Nevertheless, due to the limited number of strains examined in these previous studies (Wang et al., 2006b; You et al., 2006; Yang et al., 2010; Yin et al., 2010), the variability in the allelopathic potency of A. tamarense strains in the China Sea remains unclear. Biotic interactions in phytoplankton such as allelopathy may be both complex and dynamic and seem to be determined by concrete group characteristics in target and donor species (Fistarol et al., 2004). The co-existing competitors may differ in their responses to allelopathy, and the involved target and donor species might both exhibit allelopathic properties (Prince et al., 2008). In some cases, the allelopathic effect may be reciprocal (Legrand et al., 2003). Although rarely studied, the growth of Alexandrium tamarense has been shown to be inhibited by other microalgae, such as Chattonella marina, one of the most noxious HAB organisms (Tiffany et al., 2001; Wang et al., 2006c; Yang et al., 2011). In recent years, this species also frequently forms HABs in the South China Sea, causing serious damage to fish farming by secreting ichthyotoxic toxins (Wang et al., 2006c; Yang et al., 2011). Furthermore, A. tamarense and C. marina cells were found in the
same sea areas along the coast of China, sometimes at the same place and time (Lv and Qi, 1992). Yang et al. (2011) found that C. marina cell-free filtrate could significantly inhibit the growth of A. tamarense, suggesting that A. tamarense cells can also be targets of other allelochemicals. That is, there may be a reciprocal allelopathic interaction between the two species. However, it is unknown whether there are differences in the reciprocal allelopathic interaction or response to other phytoplanktonderived allelochemicals among different A. tamarense strains. To date, few papers have investigated the allelopathic action (Poulson et al., 2010; Lelong et al., 2011), and information currently available regarding the reciprocal allelopathic interactions is very scarce (Legrand et al., 2003; Jang et al., 2007). Here, we report the variability in allelopathic effects of Alexandrium tamarense and potential reciprocal allelopathic interactions with other algae in the China Sea. The allelopathic effects of 15 A. tamarense strains from the South China Sea and East China Sea on Chattonella marina were investigated and the hemolytic activity was determined. Additionally, allelopathic effects between samples from the South China Sea and the East China Sea were compared, and the potential effects of the geographic origin on the allelopathic actions of the algal strains were discussed as well. 2. Materials and methods 2.1. Culture conditions The 15 Alexandrium tamarense strains used in this study are listed in Table 1. The one Chattonella marina strain used in the study was collected from the South China Sea. All of the strains were grown as batch cultures in Erlenmeyer flasks containing f/2 medium, which was filter-sterilized through 0.22-mm filters (Millipore). The cultures were grown at 20 1 8C in an artificial climate incubator. Cool-white fluorescent tubes provided an irradiance of 68 mmol photons/(m2 s) in a 12:12 h light:dark regime. 2.2. The co-culture experiment Cultures containing 75 ml of Alexandrium tamarense at a cell density of 6.0 106 cells/l were incubated in triplicate and mixed with an equal volume of Chattonella marina culture at 6.0 106 cells/l. For the controls, the same volume of f/2 medium was added. Nutrients were added to make sure that at least f/2 concentrations were present in the bialgal cultures. The co-culture experiment lasted for six days. Cell densities of the bi-algal cultures were counted daily under an inverted microscope using triplicate subsamples of 0.1 ml. The growth rate of the A. tamarense strains in
Table 1 Strains of the Alexandrium tamarense species complex used in this study. Strain
Collection/origin (if known)
Supplier/Isolator
ATDH01 ATDH02 ATDH05 ATDH17 ATDH23 ATMJ01 ATMJ02 ATXM08 ATDY03 ATDY07 CCMP1493 CCMP1598 ATHKQ HK9301 ATGX02
Germinated from cyst, the East China Sea, China, 2001 Germinated from cyst, the East China Sea, China, 2001 Germinated from cyst, the East China Sea, China, 2001 Germinated from cyst, the East China Sea, China, 2001 Germinated from cyst, the East China Sea, China, 2001 Germinated from cyst, the East China Sea, Min Chiang, China, 2000 Germinated from cyst, the East China Sea, Min Chiang, China, 2000 Germinated from cyst, the East China Sea, Xiamen, China, 2004 Germinated from cyst, South China sea, Daya Bay, China, 2010 Germinated from cyst, South China sea, Daya Bay, China, 2010 South China sea, Daya Bay, China South China Sea, Daya Bay, China South China Sea, Hongkong, China South China Sea, Hongkong, China Germinated from cyst, South China Sea, Guangxi, China, 2000
Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu NCMA (formerly the CCMP), USA NCMA Isolator is unknown Dr. D.Z. Wang of Xiamen University, China Prof. H.F. Gu
J. Chen et al. / Harmful Algae 47 (2015) 17–26
monoculture (A. tamarense control) was evaluated during the six days of culture. The allelopathic effect can be expressed as the EC50 concentration (i.e., the Alexandrium cell density yielding a 50% decline in target cell density), % of control and other parameters (Tillmann et al., 2007, 2009; Tillmann and Hansen, 2009; Xu et al., 2015). To highlight the inhibition of A. tamarense and C. marina on the growth of target cells, reciprocal inhibition between A. tamarense and C. marina in co-culture was expressed as the inhibition rate (IR) using the following equation (Wu et al., 2012; Gao et al., 2015): The cell density of the control group The cell density of the co-culture group 100% IR% ¼ The cell density of the control group Given the reciprocal effect of the two algal species and the difference in the intrinsic growth rate of the various Alexandrium tamarense strains, allelopathic potency and response of A. tamarense towards Chattonella marina were evaluated in terms of the inhibition effect on the first day after co-culture. The inhibition effect of each A. tamarense strain was expressed as the inhibition rate of A. tamarense on the growth of C. marina, whereas the growth response of various strains of A. tamarense to the coculture with C. marina was evaluated as the inhibitory effect of C. marina on the growth of A. tamarense. Based on the allelopathic potency and response of A. tamarense towards C. marina, hierarchical cluster analyses were conducted to group the 15 A. tamarense strains using the Average Linkage (between groups) function in SPSS 18 (SPSS, Inc., Chicago, IL, USA). 2.3. Extraction of extracellular toxins from A. tamarense cultures Extracellular toxins from Alexandrium tamarense were collected by two methods as described by Yang et al. (2010) and Gentien et al. (2007). In Method A (dialysis), 1 l of A. tamarense culture in exponential phase at a density of approximately 6.0 106 cell/l was centrifuged at 3000 g for 10 min. The cell-free supernatant was filtered through 0.22-mm filters (Millipore). Then, NaOH was added to adjust the pH to 10–11, which caused some milk-white precipitate to appear in the solution. After standing for 1 h, the precipitate was collected by centrifugation, dissolved in 100 ml of 1 M HCl, and dialyzed in 0.1 mM HCl for 3 days. According to the previous findings (Yamasaki et al., 2008; Ma et al., 2011), some amphipathic extracellular substances with large molecular weights may be responsible for the allelopathic action of A. tamarense. Additionally, Emura et al. (2004) isolated a protein-like hemolytic compound with a molecular weight of more than 10 kDa by dialysis from Alexandrium taylori. Thus, in this study, a dialysis bag with a cut-off value of 1 kDa was used to isolate possible allelochemicals or hemolytic substances. The dialysate was cryodesiccated and dissolved in 1 ml methanol to obtain the crude toxin (He et al., 1996). In Method B (solvent extraction), cell-free supernatants were obtained as described in method A and then filtered through 0.22mm filters (Millipore). Methanol/chloroform (100 ml:50 ml) was added to the filtrate and mixed. After phase separation, the lower phase was collected and evaporated in a water bath at 60 8C to remove the solvent. The residue was dissolved in 1 ml methanol to obtain the crude toxin (Gentien et al., 2007). 2.4. Hemolytic activity analysis The hemolytic test was performed according to the protocol of Peng et al. (2005). A certain amount of algal extract (0.1 ml) from the Alexandrium tamarense cultures in triplicate was diluted with phosphate buffered saline (PBS, pH 7.4) to a total volume of 0.4 ml
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and then added to 1.6 ml of 0.5% rabbit blood in PBS. After incubation at 37 8C for 30 min, intact blood cells were removed by centrifugation and hemolytic activity was detected spectrophotometrically by measuring the absorbance at 540 nm (10 mm cuvette). Methanol was used as a blank control and digitonin (Sigma, USA) as a standard. The standard curve was generated with 0.5–1.75 mg/ml digitonin solutions and related hemolytic percentages. A hemolytic unit (HU) was defined as the concentration of toxin that caused 50% lysis during the incubation. Similar to the assessment of allelopathic action, a hierarchical cluster analysis (Average Linkage, between groups) was performed with SPSS 18 to group the 15 A. tamarense strains according to hemolytic activity. 2.5. Statistical analysis All of the data in the current study are given as the mean values SD (n = 3). Hierarchical cluster analysis (Average Linkage, between groups) using SPSS 18 was used to group the different Alexandrium tamarense strains in terms of the reciprocal inhibition rate between A. tamarense and Chattonella marina on the first day after co-culture or hemolytic activity. Student’s t-test was used to compare differences in hemolytic activity between the extracellular toxins obtained by the solvent extraction method and that obtained by the dialysis method after testing for homogeneity of variance. Taking into account the potential correlation between the hemolytic activities of the extracellular toxins obtained by the two extraction methods, a partial correlation analysis was performed to evaluate the correlation between the hemolytic activity and the allelopathic potency of A. tamarense strains using SPSS 18.0. The allelopathic potency of A. tamarense was evaluated in terms of the inhibition rate on the first day after co-culture. 3. Results 3.1. Effects of A. tamarense on the growth of C. marina The effects of the different Alexandrium tamarense strains on the growth of Chattonella marina are presented in Table 2 and Fig. 1. The 15 strains exhibited high variation in allelopathic action on C. marina. Most of the A. tamarense strains inhibited the growth of C. marina to various degrees. The IR of C. marina 6 d after coculturing with A. tamarense ranged from 1.79% 7.10% to 100.00% 0.00% and significantly differed between the strains. For example, in the co-cultures with ATDH23, ATDY03, ATMJ02, ATMJ01, ATXM08 and ATGX02, the cell densities of C. marina decreased to zero after 6 days, suggesting strong inhibition of the six strains on C. marina. However, CCMP1598 and HK9301 exhibited a weaker inhibitory effect on C. marina. There was no significant difference in cell density between the monoculture of C. marina and co-cultures with CCMP1598 or HK9301 after 6 days (p > 0.05). Additionally, we noted that various strains of A. tamarense exhibited different changes in IR over time. For example, the IR of ATHKQ against C. marina steadily increased over the experimental period, while the IR of ATDH17 against C. marina increased during the first 3 days but decreased thereafter. During the experiment, the pH in both the monocultures and the co-cultures was monitored daily, and few changes (from 8.8 to 9.0) were observed. Furthermore, the growth of C. marina at pH 8.5 and 9 was very similar (data not shown). Based on the inhibitory effect of Alexandrium tamarense on the growth of Chattonella marina on the first day after co-culture, the 15 A. tamarense strains can be divided into 3 hierarchical groups as shown in Fig. 2. The inhibitory activities from weak to strong were as follows: (1) Weak (W)—ATDH05, ATDH17, ATDH01, ATDY07, CMP1493, HK9301, ATHKQ, and CCMP1598; (2) Moderate (M)— ATDH02 and ATMJ01; and (3) Strong (S)—ATDY03, ATGX02, ATDH23, ATXM08, and ATMJ02.
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J. Chen et al. / Harmful Algae 47 (2015) 17–26
Table 2 Reciprocal inhibition between the Alexandrium tamarense species complex and Chattonella marina in co-culture. Strain
Growth rate (d1)
IR (%)
1d
2d
3d
ATDH01
0.36 0.01
AT CM
10.09 7.10 20.73 2.66
20.91 9.25 23.66 1.74
48.56 3.05 25.46 0.77
44.63 3.39 24.52 2.49
60.72 3.58 21.50 9.96
62.91 5.51 15.36 9.65
ATDH02
0.28 0.01
AT CM
17.99 4.16 49.88 8.16
27.27 1.93 66.76 4.11
30.17 4.88 75.99 3.08
25.22 2.13 83.91 2.20
22.38 6.81 92.38 0.62
24.48 6.40 95.35 0.15
ATDH05
0.36 0.01
AT CM
13.88 1.10 25.47 10.41
39.49 0.77 35.08 4.51
59.93 0.51 40.98 0.36
38.53 3.05 50.28 0.85
50.44 1.24 52.61 3.18
53.67 4.95 40.13 5.54
ATDH17
0.10 0.01
AT CM
1.27 5.31 25.94 2.67
17.08 6.11 35.92 5.25
32.83 1.11 38.33 6.83
42.44 7.01 27.07 4.88
49.51 2.49 24.39 0.29
54.00 6.02 15.38 0.75
ATDH23
0.42 0.02
AT CM
14.80 5.75 79.94 4.48
34.89 5.21 86.71 2.86
29.08 10.08 91.33 2.23
33.85 6.08 97.75 1.48
24.73 5.78 100.00 0.00
15.98 8.04 100.00 0.00
ATDY03
0.24 0.01
AT CM
29.07 4.02 72.60 0.35
35.04 1.08 89.99 2.47
26.94 2.30 93.37 0.09
14.85 3.56 99.29 0.14
28.74 1.96 99.92 0.15
1.18 1.69 100.00 0.00
ATDY07
0.35 0.01
AT CM
31.00 3.50 22.08 7.70
35.77 4.48 45.29 3.89
45.92 1.04 50.24 7.25
55.41 0.99 52.39 2.67
66.71 2.52 51.41 4.51
76.78 2.38 42.26 6.90
HK9301
0.11 0.01
AT CM
2.42 3.28 13.09 6.25
16.32 5.01 14.94 3.30
7.68 3.20 11.96 7.18
17.05 3.66 13.37 2.45
33.95 3.61 3.83 2.88
52.04 3.84 8.90 4.57
ATHKQ
0.20 0.20
AT CM
17.86 6.80 14.32 7.65
25.52 5.22 20.83 2.63
19.84 2.55 37.45 1.31
24.01 2.70 43.44 2.59
40.20 7.37 58.89 3.91
47.79 1.26 59.79 1.25
ATMJ01
0.13 0.01
AT CM
9.43 5.71 51.58 7.25
25.36 4.36 68.94 1.95
20.00 4.53 83.24 0.55
20.08 0.75 91.74 1.37
3.21 3.05 98.76 0.27
13.64 2.85 99.57 0.20
ATMJ02
0.18 0.01
AT CM
19.79 1.57 89.42 3.27
12.14 3.62 98.60 0.17
16.89 6.62 99.73 0.25
20.64 0.60 100.00 0.00
1.53 1.17 100.00 0.00
4.17 5.22 100.00 0.00
ATXM08
0.34 0.02
AT CM
31.13 6.75 83.23 4.09
22.88 3.57 99.01 0.27
1.46 6.09 99.73 0.25
7.57 6.15 100.00 0.00
0.11 4.05 100.00 0.00
6.86 3.94 100.00 0.00
CCMP1493
0.25 0.01
AT CM
2.48 3.90 30.29 4.26
11.54 4.14 43.65 3.83
13.15 6.88 48.53 7.25
20.02 2.76 41.09 5.28
35.78 2.45 35.77 3.29
52.42 0.71 22.81 3.89
CCMP1598
0.25 0.01
AT CM
3.66 1.34 8.21 1.11
23.73 3.09 19.13 11.28
26.83 4.14 21.88 8.85
41.10 3.67 25.11 9.85
61.07 1.85 15.51 3.28
70.38 3.01 1.79 7.10
ATGX02
0.31 0.01
AT CM
38.81 9.13 74.57 6.45
26.05 11.33 85.35 6.45
34.09 4.30 92.01 1.86
33.10 4.39 97.92 0.67
32.92 7.41 99.75 0.25
27.35 9.29 100.00 0.00
4d
5d
6d
Reciprocal inhibition between A. tamarense and C. marina in co-culture was expressed as inhibition rate (IR) as described by Wu et al. (2012). AT indicates the inhibition rate of C. marina towards A. tamarense, while CM indicates the inhibition rate of A. tamarense towards C. marina. Data are given as mean values SD (n = 3).
3.2. Response of various A. tamarense strains to co-culturing with C. marina Similar to the inhibition of Chattonella marina by Alexandrium tamarense, different strains of A. tamarense exhibited various responses to co-culturing with C. marina (Fig. 1 and Table 2). The growth of ATDH01, ATDY07, ATDH17, ATHKQ, ATHK9301, CCMP1598 and CCMP1493 was significantly inhibited when cocultured with C. marina for six days, while the growth of ATXM08 and ATMJ02 was only slightly influenced. According to the inhibitory effect of C. marina on the growth of A. tamarense on the first day after co-culture, the 15 A. tamarense strains can be divided into 3 hierarchical groups as follows (Fig. 3): (1) Weak (W)—CCMP1493, HK9301, ATDH17, and CCMP1598; (2) Moderate (M)—ATMJ01, ATDH01, ATHKQ, ATDH05, ATDH23, ATDH02, and ATMJ02; and (3) Strong (S)—ATDY07, ATXM08, ATDY03, and ATGX02. 3.3. Hemolytic activity of A. tamarense A standard curve was obtained by plotting the hemolytic percentage vs. the concentration of digitonin. In terms of the standard curve, 1 HU was equal to 1.27 mg/ml of digitonin.
Hemolytic activities of the cell-free filtrates from cultures of different Alexandrium tamarense strains are shown in Fig. 4. There were significant differences in the hemolytic activity among the various Alexandrium tamarense strains. Except for ATMJ01, ATXM08, ATDY07 and CCMP1598, crude toxins obtained by the two methods exhibited different hemolytic activities (p < 0.05). The range of hemolytic activity obtained by the dialysis method was from 1.20 0.084 to 6.68 0.079 HU/1 106 cells, while that obtained by solvent extraction was from 1.81 0.091 to 6.41 0.49 HU/1 106 cells. According to the hemolytic activity of the extracellular toxins obtained by the dialysis method, the 15 Alexandrium tamarense isolates can be grouped into the following 3 hierarchical groups: (1) Group A (ATMJ01, CCMP1598, ATDY07, ATDH05, HK9301, ATDH01, ATDH02, CCMP1493, and ATHKQ); (2) Group B (ATDH23, ATXM08, ATDH17, ATDY03, and ATGX02); and (3) Group C (ATMJ02) (Fig. 5A). However, in accordance with their activities obtained by solvent extraction, the 15 Alexandrium tamarense strains were divided into another 3 groups as follows: (1) Group A (CCMP1493, CCMP1598, ATDY03, ATDH23, ATDH05, ATGX02, ATDH01, ATDH17, ATHKQ, ATMJ01, ATDH02, and ATDY07); (2) Group B (ATMJ02 and ATXM08); and (3) Group C (HK9301) (Fig. 5B).
J. Chen et al. / Harmful Algae 47 (2015) 17–26
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Fig. 1. Reciprocal inhibition between the Alexandrium tamarense species complex and Chattonella marina in co-culture. (A–O) Changes in cell density of A. tamarense in monoculture (&) and in mixed culture (&), and C. marina in monoculture (4) and in mixed culture (~). Data are given as mean values SD (n = 3). Vertical lines show the standard deviation.
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Fig. 1. (Continued ).
3.4. Correlation between the hemolytic activity and allelopathic action The partial correlation analysis revealed that the allelopathic potency observed in the co-cultures did not correlate
with the hemolytic activity of the extracellular toxin obtained by the solvent extraction method (r = S0.101, p > 0.05) but did correlate with the hemolytic property of the extracellular toxin obtained by the dialysis method (r = 0.823, p < 0.01).
J. Chen et al. / Harmful Algae 47 (2015) 17–26
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Fig. 2. Comparison of inhibitory effects of various A. tamarense strains on the growth of C. marina when co-culture. A hierarchical cluster analysis was conducted to group the 15 A. tamarense strains using Average Linkage (between groups) function in SPSS 18 in terms of the inhibitory effects of A. tamarense on the growth of C. marina on the first day after co-culture, where squared Euclidean distance was selected as the measurement. The abscissa means Rescaled Distance Cluster Combine.
Fig. 5. Hierarchical groups of the fifteen A. tamarense strains based on the hemolytic activity. (A) Hemolytic toxins obtained by dialysis method; (B) hemolytic toxins obtained by solvent extract method. Hierarchical cluster analyses were performed using Average Linkage (between groups) function in SPSS 18, where squared Euclidean distance was selected as the measurement. The abscissa means Rescaled Distance Cluster Combine. HU values are given in brackets.
Fig. 3. Comparison of the responses of various A. tamarense strains to co-culture with C. marina. A hierarchical cluster analysis was conducted to group the 15 A. tamarense strains using Average Linkage (between groups) function in SPSS 18 in terms of the inhibitory effect of C. marina against A. tamarense on the first day after co-culture, where squared Euclidean distance was selected as the measurement. The abscissa means Rescaled Distance Cluster Combine.
4. Discussion 4.1. Reciprocal inhibition between A. tamarense and C. marina Similar to the previous findings (Tillmann et al., 2009; Alpermann et al., 2010), our results show that most of the 15
Alexandrium tamarense strains have inhibitory effects on the growth of Chattonella marina, but significant differences were observed between strains. Additionally, most of the 15 A. tamarense strains were inhibited by C. marina to various degrees. That is, there is a reciprocal inhibition between A. tamarense and C. marina. Given the previous findings (Tillmann et al., 2009; Alpermann et al., 2010; Yang et al., 2011), it is likely that this reciprocal inhibition is mediated by allelochemicals from the two algal species. Nevertheless, some other sort of competition or interaction might also be present (Tillmann and Hansen, 2009). In the reciprocally allelopathic system, it is necessary for an algal species to continuously increase allelochemicals to inhibit target alga and vice versa (Jang et al., 2007; Wang and Tang, 2008). However, the amount of allelochemicals is dependent on the cell
Fig. 4. Relative hemolytic activities of cell-free filtrates from different strain cultures of A. tamarense. Data are given as mean values SD (n = 3). Vertical lines show the standard deviation. Significant differences in hemolytic activity between the extracts obtained by Method A and Method B are marked with an asterisk (*) (t-test, p < 0.05).
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density of the donor cells (Legrand et al., 2003; Wang and Tang, 2008; Tillmann and Hansen, 2009). In this study, an Alexandrium tamarense strain with high allelochemical potency towards Chattonella marina will actively reduce the amount of C. marina cells and therefore also reduce the amount of allelochemicals produced by C. marina during the six-day co-culture period, thus reducing the inhibition of C. marina against A. tamarense. This might explain the slight effect of C. marina towards ATMJ02 and ATXM08 and the potent inhibition against CCMP1598 and HK9301. On the first day after co-culture, the cell densities of C. marina cocultured with ATMJ02 and ATXM08 were only 11% and 17%, respectively, compared with the related controls (Fig. 1). Therefore, C. marina co-cultured with these two strains might produce a small amount of allelochemicals, thus exhibiting only a slight effect on the growth of ATMJ02 and ATXM08. The inhibition rates of C. marina against ATMJ02 and ATXM08 were only 4.17% and S6.86%, respectively, on the sixth day after co-culture (Table 2). In contrast, CCMP1598 and HK9301 exhibited a weaker inhibition on C. marina growth; consequently, a great amount of allelochemicals might be produced by C. marina (Table 2, Fig. 1). Therefore, these two A. tamarense strains are strongly inhibited. On the first day after co-culture, the inhibition rates of CCMP1598 and HK9301 against C. marina were 8.21% and 13.09%, respectively. Accordingly, the inhibition rates of C. marina against these two strains were 70.38% and 52.04%, respectively, six days after co-culture (Table 2). It is obvious that the allelopathic effect of one species is not only related to its own allelopathic potency but also influenced by the allelopathic potency of the target cells and vice versa in a reciprocally allelopathic system. The intrinsic growth rate of algae may also play an important role in the reciprocally allelopathic system. It should be noted that the inhibition rate of ATHKQ against Chattonella marina steadily increased over the experimental time frame, though the inhibition rate was only 14.32% on the first day after co-culture (Table 2). In contrast, the inhibition rate of ATDH17 against C. marina was 25.94% on the first day but only 15.38% after 6 days. The different intrinsic growth rates of the strains might be at least partly responsible for this discrepancy (Table 2). In any case, even a small supplement in cell density should benefit Alexandrium tamarense or C. marina in gaining dominance in co-cultures. On the first day after co-culture, the inhibition rates of C. marina against ATHKQ and ATDH17 were 17.86% and 1.27% (Table 2), respectively, indicating that ATHKQ exhibits a stronger response to co-culture with C. marina than ATDH17. However, ATHKQ possesses a relatively higher growth rate (0.20 d1), which partially covers a loss in cell density induced by C. marina, and thus, its inhibition against C. marina steadily increases over the time frame. Similarly, ATDH05 exhibits a stronger response to co-culture with C. marina than ATDH17; meanwhile, the two A. tamarense strains display a similar inhibition on C. marina growth the first day after co-culture. However, the relatively higher growth rate of ATDH05 (0.36 d1) might make it produce more allelochemicals with time, thus exhibiting a stronger inhibition on C. marina compared with ATDH17 after 6 days (Table 2). Nonetheless, ATDH05 possesses a higher growth rate and exhibits a stronger inhibition on the growth of C. marina and a weaker response to co-culture with C. marina than ATHKQ the first day after co-culture. Its inhibition rate against C. marina is less than that of ATHKQ six days after co-culture, suggesting that another sort of competition might also be present in reciprocal inhibition or some allelochemicals can be degraded in bi-algal cultures (Wang and Tang, 2008; Tillmann and Hansen, 2009). Taking into account the complex reciprocal effect of the two algal species and difference in intrinsic growth rate of the A. tamarense strains, we compared the allelopathic potency and response of each A. tamarense strain towards C. marina in terms of the inhibition effect the first day after co-culture.
From Fig. 2, in terms of the inhibitory rate of Alexandrium tamarense on the growth of Chattonella marina on the first day after co-culture, the 15 A. tamarense strains can be divided into different hierarchical groups. Similarly, based on the inhibitory rate of C. marina on A. tamarense, the A. tamarense strains can also be divided into 3 hierarchical groups (Fig. 3). However, the two hierarchical groups do not match each other, and no close associations between them were observed. For example, ATDY03, ATGX02, ATDH23, ATXM08 and ATMJ02 exhibited a strong inhibition on the growth of C. marina on the first day after coculture (Fig. 2). However, C. marina displays a potent inhibition on ATXM08, ATDY03 and ATGX02 (Fig. 3) but exerts a moderate inhibition on ATDH23 and ATMJ02 (Fig. 3). Furthermore, compared with ATXM08, ATDY03 and ATGX02, ATMJ02 has a lower growth rate (Table 2). Obviously, these results cannot be explained by the reciprocal effect of the two algal species or difference in the intrinsic growth rate of A. tamarense, suggesting that A. tamarense strains respond differently to co-culture with C. marina. Poulson et al. (2010) reported that the physiological state and cell concentration of competitors were important determinants of allelopathy in Karenia brevis, with lag phase cells being more sensitive to allelopathic effects than later growth stages in Skeletonema grethae. Similarly, Arzul et al. (1999) found that the allelopathic potency of Alexandrium on Gymnodinium mikimotoi and Chaetoceros gracile increased with the concentration and age of the cultures. Diluted filtrates from A. tamarense completely suppressed the growth of C. gracile and G. mikimotoi, except when using a dilution filtrate from exponential growth phase (EP) cultures, which had no observable effect (Arzul et al., 1999). In this study, all of the A. tamarense strains had the same initial cell density and sufficient nutrients were present in the bi-algal cultures. Therefore, it is likely that there are significant differences among the various A. tamarense strains in response to allelochemicals from C. marina. However, further studies examining the effects of cell-free supernatants from C. marina cultures on A. tamarense strains are needed to confirm our presumption. Interestingly, based on the comparison of the lytic activity of A. ostenfeldii, Alexandrium catenella and A. minutum, Tillmann et al. (2008) demonstrated that the intraspecific and intrapopulation clonal variability in lytic potency within Alexandrium tamarense might be even higher than the variability among strains of different Alexandrium species. However, it remains unclear whether the coexisting history of the algal strains have some effects on the allelopathy and whether there is a boundary in the allelopathic potency between different populations. Taking into account the origin of the target species Chattonella marina mentioned above, we compared the allelopathic effects between population samples from the South China Sea and the East China Sea. It might be more ideal to compare the allelopathic actions between samples from the South China Sea (or the East China Sea) and the Bohai Sea (or the Yellow Sea). However, we found that strains from the Yellow Sea and the Bohai Sea are both restricted to Alexandrium fundyense (Group I), whereas samples from the East China Sea and the South China Sea all belong to Alexandrium pacificum (Group IV) (Zou et al., 2014). Various species of A. tamarense may result in different phenotypes including toxin production, growth rate and allelopathy (Scholin et al., 1994; Murata et al., 2012). Thus, only samples from the South China Sea and the East China Sea are compared in our study. Based on the inhibitory effect, the 15 Alexandrium tamarense strains can be divided into 3 hierarchical groups. However, the hierarchical groups do not match the geographical origin. Strains isolated from the same geographical origin are not always clustered into one clade, while strains from different geographical origins are often clustered into one clade. For example, CCMP1598,
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CCMP1493, ATDY07 and ATDY03 from Daya Bay in the South China Sea scatter into two groups. Furthermore, ATDH01, ATDH17, ATDH05, ATDH02 and ATDH23 from the East China Sea scatter into three groups. Similarly, the hemolytic activity also varies considerably among the isolates from the same geographical origin, and strains isolated from the same geographical origin are not always clustered into one clade. This demonstrates that there is no significant boundary in allelopathy between the various populations and that the history of co-existence may have a limited effect on the allelopathy studied here. However, allelopathy is generally considered to be especially effective in stress situations (Reigosa et al., 1999), and changes in the growth rate under stress can have a significant influence on the allelopathic action (Legrand et al., 2003). Thus, further studies should be carried out in the future to authenticate the presumption using more strains, stress and/or limiting growth conditions. Additionally, it is of note that the A. tamarense strains assayed in this study included some vegetative cells that were collected at different times and some strains that germinated from cysts. These may, to a certain extent, influence the outcome of these mixed growth experiments. 4.2. Allelochemicals in A. tamarense Though many studies have been performed, the nature of allelochemicals involved in the allopathic action of Alexandrium tamarense remains unclear (Emura et al., 2004; Grane´li et al., 2008; Yamasaki et al., 2008; Ma et al., 2011). Some large amphipathic extracellular substances, independent of known phycotoxins, PSP toxins and spirolides, have been recommended to be responsible for the action (Tillmann and John, 2002; Emura et al., 2004; Tillmann et al., 2007; Yamasaki et al., 2008; Ma et al., 2011). In our study, to completely remove the algal cells and cell debris that cannot be removed by centrifugation, cell-free supernatants were filtered through 0.22-mm filters. Therefore, some of the macromolecule allelochemicals described by Ma et al. (2009) and Yamasaki et al. (2008) might have been be lost; however, strong hemolytic activities were still found, suggesting the chemical diversity expressed in the lytic substances. In fact, some hemolytic substances of approximately 1 kDa have been found in our filtrates with HPLC–MALDI–MS (data not shown), though their fine structures have not been identified. Additionally, we found that the allelopathic effects observed in co-culture are correlated with the hemolytic properties of the toxins obtained by dialysis (p < 0.01). Taking into account the cut-off value of the dialysis bag (1 kDa), it is reasonable to speculate that some large extracellular hemolytic substances greater than 1 kDa might be at least partly responsible for the allelopathic effects of A. tamarense. Furthermore, we found that these substances are resistant to acids and bases and can withstand temperatures of 75 8C without denaturation, suggesting their non-proteinaceous properties. Additionally, crude toxins obtained by solvent extraction exhibit strong hemolytic activities with significant differences between the Alexandrium tamarense strains. However, the partial correlation analysis failed to identify close associations between the allelopathic actions and hemolytic activities of the extracellular toxins obtained by the solvent extraction method (p > 0.05), suggesting that various A. tamarense strains could produce diverse hemolytic substances with different properties and profiles (Arzul et al., 1999; Tillmann et al., 2008). Similarly, Tillmann et al. (2008) found qualitative differences in the composition of allelochemicals from different Alexandrium species and strains. It is likely that multiple allelochemicals might target and differentially affect different phytoplankton (Ma et al., 2009; Poulson et al., 2010; Prince et al., 2010; HattenrathLehmann and Gobler, 2011).
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5. Conclusion Our results show that most of the 15 Alexandrium tamarense strains from the China Sea have an allelopathic effect on Chattonella marina with significant differences. In contrast, different A. tamarense strains exhibit various responses to co-culture with C. marina. There is a complicated reciprocal inhibitory effect between A. tamarense and C. marina, which might involve allelopathic potency, the intrinsic growth rate of algal species and sensitivity to the allelochemicals, etc. However, there is no close correlation between the phenotype (hemolytic activity and allelopathic action) and geographical distribution. Various A. tamarense strains could therefore produce diverse hemolytic substances with different properties and profiles, which might be responsible for the variability in allelopathic actions of A. tamarense. Acknowledgements This work was supported by the National Basic Research Program of China (973 Program) (2010CB428702), the National Natural Science Foundation of China (41230961, 41176088) and the Natural Science Foundation of Guangdong Province (8251063201000001). [SS] References Alpermann, T.J., Tillmann, U., Beszteri, B., Cembella, A.D., John, U., 2010. Phenotypic variation and genotypic diversity in a planktonic population of the toxigenic marine dinoflagellate Alexandrium tamarense (dinophyceae). J. Phycol. 46 (1), 18–32. Arzul, G., Seguel, M., Guzman, L., Erard-LeDenn, E., 1999. Comparison of allelopathic properties in three toxic Alexandrium species. J. Exp. Mar. Biol. Ecol. 232, 285–295. Chen, J.H., Yu, R.C., Gao, Y., Kong, F.Z., Wang, Y.F., Zhang, Q.C., Kang, Z.J., Yan, T., Zhou, M.J., 2013. Tracing the origin of paralytic shellfish toxins in scallop Patinopecten yessoensis in the northern Yellow Sea. Food Addit. Contam. A 30 (11), 1933–1945. Chen, Y.Q., Qiu, X.Z., Qu, L.H., Zeng, L.M., Qi, Y.Z., Zheng, L., 1999. Analysis of molecular biogeographic marker on red tide toxic Alexandrium tamarense in the South China Sea. Oceanol. Limnol. Sin. 30 (1), 45–51 (in Chinese, with English abstract). Dia, A., Guillou, L., Mauger, S., Bigeard, E., Marie, D., Valero, M., Destombe, C., 2014. Spatiotemporal changes in the genetic diversity of harmful algal blooms caused by the toxic dinoflagellate Alexandrium minutum. Mol. Ecol. 23, 549–560. Emura, A., Matsuyama, Y., Oda, T., 2004. Evidence for the production of a novel proteinaceous hemolytic exotoxin by dinoflagellate Alexandrium taylori. Harmful Algae 3 (1), 29–37. Fang, Q., Gu, H.F., Lan, D.Z., Huang, Y.H., 2006. Effect of temperature, salinity and nutrient on the growth of different strains of Alexandrium tamarense. Mar. Sci. Bull. 25 (6), 20–25 (in Chinese, with English abstract). Frango´pulos, M., Guisande, C., deBlas, E., Maneiro, I., 2004. Toxin production and competitive abilities under phosphorus limitation of Alexandrium species. Harmful Algae 3, 131–139. Fistarol, G.O., Legrand, C., Selander, E., Hummert, C., Stolte, W., Grane´li, E., 2004. Allelopathy in Alexandrium spp.: effect on a natural plankton community and on algal monocultures. Aquat. Microb. Ecol. 35, 45–56. Gao, H.J., Song, Y.H., Lv, C.J., Chen, X.M., Yu, H.B., Peng, J.F., Wang, M., 2015. The possible allelopathic effect of Hydrilla verticillata on phytoplankton in nutrientrich water. Environ. Earth Sci., http://dx.doi.org/10.1007/s12665-015-4316-8. Gentien, P., Lunven, M., Lazure, P., Youenou, A., Crassous, M.P., 2007. Motility and auto toxicity in Karenia mikimotoi (Dinophyceae). Phil. Trans. R. Soc. B 362, 1937–1946. Grane´li, E., Weberg, M., Salomon, P.S., 2008. Harmful algal blooms of allelopathic microalgal species: the role of eutrophication. Harmful Algae 8 (1), 94–102. Gu, H.F., Zeng, N., Liu, T.T., Yang, W.D., Mu¨ller, A., Krock, B., 2013. Morphology, toxicity, and phylogeny of Alexandrium (Dinophyceae) species along the coast of China. Harmful Algae 27, 68–81. Hattenrath-Lehmann, T.K., Gobler, C.J., 2011. Allelopathic inhibition of competing phytoplankton by North American strains of the toxic dinoflagellate, Alexandrium fundyense: Evidence from field experiments, laboratory experiments, and bloom events. Harmful Algae 11, 106–116. He, J.W., Chen, M.H., He, Z.R., 1996. Isolation and characterization of toxins from the phytoflagellate Prymnesium parvum. Acta Hydrobiol. Sin. 20, 41–48 (in Chinese, with English abstract). Jang, M.H., Ha, K., Takamura, N., 2007. Reciprocal allelopathic responses between toxic cyanobacteria (Microcystis aeruginosa) and duckweed (Lemna japonica). Toxicon 49, 727–733.
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Harmful Algae journal homepage: www.elsevier.com/locate/hal
Variability in the allelopathic action of the Alexandrium tamarense species complex along the coast of China Jian Chen a,c, Qian Ye a, Hai-Feng Gu b, Hong-Ye Li a, Song-Hui Lv a, Jie-Sheng Liu a,*, Wei-Dong Yang a,* a
College of Life Science and Technology, Key Laboratory of Aquatic Eutrophication and Control of Harmful Algal Blooms of Guangdong Higher Education Institute, Jinan University, Guangzhou 510532, China Third Institute of Oceanography, SOA, Xiamen 361005, China c Zhongshan Entry-Exit Inspection and Quarantine Bureau Technology Center, Zhongshan 528403, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 May 2014 Received in revised form 14 May 2015 Accepted 14 May 2015 Available online 7 June 2015
Members of the Alexandrium tamarense species complex are some of the most important toxigenic dinoflagellates and are also widely distributed along the coast of China. A. tamarense and another wellknown toxic raphidophycean alga, Chattonella marina, are usually found in the same sea areas, sometimes at the same place and time. In this study, under laboratory co-culture conditions, we found that most of the 15 A. tamarense strains from the South China Sea and the East China Sea had significant inhibition on the growth of C. marina, while most of the A. tamarense strains were inhibited to various extents by C. marina. These results suggest that there is a complex reciprocal inhibitory effect between A. tamarense and C. marina, which might be caused by their allelopathic potency, the intrinsic growth rate of the algal species and the sensitivity of the target alga to the allelochemicals, etc. However, no strict correlations were observed between the allelopathic actions and the geographical distribution. The allelopathic potency observed in co-culture did not correlate with the hemolytic activity of the extracellular toxins obtained by the solvent extraction method (p > 0.05) but did correlate with the hemolytic activity obtained by the dialysis method (p < 0.01). These results indicate that various strains of A. tamarense could produce diverse hemolytic substances with different compositions and properties, which might be responsible for the variability in A. tamarense allelopathic action. ß 2015 Elsevier B.V. All rights reserved.
Keywords: Alexandrium tamarense species complex Chattonella marina Allelopathy Hemolytic toxins
1. Introduction Members of the Alexandrium tamarense species complex, including A. tamarense (Lebour) Balech, Alexandrium fundyense Balech and Alexandrium catenella (Whedon and Kofoid) Balech, are some of the most important toxigenic dinoflagellates (Lilly et al., 2007; Gu et al., 2013). Recently, based on morphology data, ITS/ 5.8S genetic distances, ITS2 compensatory base changes, mating incompatibilities, toxicity, the sxtA gene, and rDNA phylogenies, John et al. (2014) amended this species into the five following species: (1) Group I, A. fundyense; (2) Group II, Alexandrium mediterraneum; (3) Group III, A. tamarense; (4) Group IV, Alexandrium pacificum; and (5) Group V, Alexandrium australiense. Along the coast of China, A. tamarense is widely distributed from
* Corresponding author. Tel.: +86 020 85228470/+86 020 85223686; fax: +86 020 85225183. E-mail addresses: [email protected], [email protected] (W.-D. Yang). http://dx.doi.org/10.1016/j.hal.2015.05.008 1568-9883/ß 2015 Elsevier B.V. All rights reserved.
the Bohai Sea to the South China Sea. Blooms of toxic Alexandrium spp. have also been reported in the Yellow Sea, the coastal waters adjacent to the Changjiang River estuary and some bays in the South China Sea (Wang et al., 2006a, 2011; Song et al., 2009; Gu et al., 2013; Zou et al., 2014). Furthermore, A. tamarense blooms have been found to be associated with paralytic shellfish poisoning (PSP) toxin contamination in the cultivated shellfish in China (Wang et al., 2011; Chen et al., 2013; Jiang et al., 2013). The wide distribution of the species and their implication in harmful algal blooms (HABs) and PSP events have attracted increasing attention in China, and many studies have been performed on the nutritional characteristics, toxin composition, phylogenetics and toxicity of this genus (Chen et al., 1999; Yan et al., 2003; Wu et al., 2005; Fang et al., 2006; Tang et al., 2006; Gu et al., 2013; Jiang et al., 2013). As with most dinoflagellate species, Alexandrium tends to have lower nutrient uptake affinities and maximum growth rates than other microalgae (Smayda, 1997; Frango´pulos et al., 2004). Allelopathic effects have been proposed to exert an important role in the formation and maintenance of Alexandrium blooms
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(Fistarol et al., 2004; Tillmann et al., 2008; Hattenrath-Lehmann and Gobler, 2011). Within the Alexandrium genus, lytic activity (usually exhibiting allelochemical activity) has been detected in all of the species tested; however, the strength of the lytic activity varies considerably among different species and strains of the same species from different geographical origins (Tillmann et al., 2009; Hattenrath-Lehmann and Gobler, 2011). An increasing number of studies have demonstrated significant intraspecific genotypic and phenotypic variability within unicellular algal populations (Tillmann et al., 2009; Kremp et al., 2012; Lebret et al., 2012; Dia et al., 2014). Microalgae from different strains of the same species, especially dinoflagellates and bacillariophyta, display various phenotypes and genotypes, leading to the variety in nutrient uptake, photosynthesis capacity and growth rate (Kremp et al., 2012). These capabilities facilitate HAB formation and adaptation to changing environments (Alpermann et al., 2010; Lebret et al., 2012). Several studies have shown that Alexandrium tamarense strains from the South China Sea and East China Sea exhibited strong allelopathic effects on Prorocentrum donghaiense, Chattonella marina and Heterosigam akashiwo (Wang et al., 2006b; You et al., 2006; Yang et al., 2010; Yin et al., 2010). However, at least 148 HAB species, such as Noctiluca scintillans, Prorocentrum minimum, P. mians, Gonyaulax polyedra, Skeletonema costatum, Mesodinium rubrum, Trichodesmium sp., and C. marina, have been found in China to date, of which 44 have caused HAB events in different sea areas (Zhu et al., 1997). These data imply that there might be a complicated reciprocal interaction between A. tamarense and target alga cells and that various strains of A. tamarense might exhibit different allelopathic potency. Nevertheless, due to the limited number of strains examined in these previous studies (Wang et al., 2006b; You et al., 2006; Yang et al., 2010; Yin et al., 2010), the variability in the allelopathic potency of A. tamarense strains in the China Sea remains unclear. Biotic interactions in phytoplankton such as allelopathy may be both complex and dynamic and seem to be determined by concrete group characteristics in target and donor species (Fistarol et al., 2004). The co-existing competitors may differ in their responses to allelopathy, and the involved target and donor species might both exhibit allelopathic properties (Prince et al., 2008). In some cases, the allelopathic effect may be reciprocal (Legrand et al., 2003). Although rarely studied, the growth of Alexandrium tamarense has been shown to be inhibited by other microalgae, such as Chattonella marina, one of the most noxious HAB organisms (Tiffany et al., 2001; Wang et al., 2006c; Yang et al., 2011). In recent years, this species also frequently forms HABs in the South China Sea, causing serious damage to fish farming by secreting ichthyotoxic toxins (Wang et al., 2006c; Yang et al., 2011). Furthermore, A. tamarense and C. marina cells were found in the
same sea areas along the coast of China, sometimes at the same place and time (Lv and Qi, 1992). Yang et al. (2011) found that C. marina cell-free filtrate could significantly inhibit the growth of A. tamarense, suggesting that A. tamarense cells can also be targets of other allelochemicals. That is, there may be a reciprocal allelopathic interaction between the two species. However, it is unknown whether there are differences in the reciprocal allelopathic interaction or response to other phytoplanktonderived allelochemicals among different A. tamarense strains. To date, few papers have investigated the allelopathic action (Poulson et al., 2010; Lelong et al., 2011), and information currently available regarding the reciprocal allelopathic interactions is very scarce (Legrand et al., 2003; Jang et al., 2007). Here, we report the variability in allelopathic effects of Alexandrium tamarense and potential reciprocal allelopathic interactions with other algae in the China Sea. The allelopathic effects of 15 A. tamarense strains from the South China Sea and East China Sea on Chattonella marina were investigated and the hemolytic activity was determined. Additionally, allelopathic effects between samples from the South China Sea and the East China Sea were compared, and the potential effects of the geographic origin on the allelopathic actions of the algal strains were discussed as well. 2. Materials and methods 2.1. Culture conditions The 15 Alexandrium tamarense strains used in this study are listed in Table 1. The one Chattonella marina strain used in the study was collected from the South China Sea. All of the strains were grown as batch cultures in Erlenmeyer flasks containing f/2 medium, which was filter-sterilized through 0.22-mm filters (Millipore). The cultures were grown at 20 1 8C in an artificial climate incubator. Cool-white fluorescent tubes provided an irradiance of 68 mmol photons/(m2 s) in a 12:12 h light:dark regime. 2.2. The co-culture experiment Cultures containing 75 ml of Alexandrium tamarense at a cell density of 6.0 106 cells/l were incubated in triplicate and mixed with an equal volume of Chattonella marina culture at 6.0 106 cells/l. For the controls, the same volume of f/2 medium was added. Nutrients were added to make sure that at least f/2 concentrations were present in the bialgal cultures. The co-culture experiment lasted for six days. Cell densities of the bi-algal cultures were counted daily under an inverted microscope using triplicate subsamples of 0.1 ml. The growth rate of the A. tamarense strains in
Table 1 Strains of the Alexandrium tamarense species complex used in this study. Strain
Collection/origin (if known)
Supplier/Isolator
ATDH01 ATDH02 ATDH05 ATDH17 ATDH23 ATMJ01 ATMJ02 ATXM08 ATDY03 ATDY07 CCMP1493 CCMP1598 ATHKQ HK9301 ATGX02
Germinated from cyst, the East China Sea, China, 2001 Germinated from cyst, the East China Sea, China, 2001 Germinated from cyst, the East China Sea, China, 2001 Germinated from cyst, the East China Sea, China, 2001 Germinated from cyst, the East China Sea, China, 2001 Germinated from cyst, the East China Sea, Min Chiang, China, 2000 Germinated from cyst, the East China Sea, Min Chiang, China, 2000 Germinated from cyst, the East China Sea, Xiamen, China, 2004 Germinated from cyst, South China sea, Daya Bay, China, 2010 Germinated from cyst, South China sea, Daya Bay, China, 2010 South China sea, Daya Bay, China South China Sea, Daya Bay, China South China Sea, Hongkong, China South China Sea, Hongkong, China Germinated from cyst, South China Sea, Guangxi, China, 2000
Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu Prof. H.F. Gu NCMA (formerly the CCMP), USA NCMA Isolator is unknown Dr. D.Z. Wang of Xiamen University, China Prof. H.F. Gu
J. Chen et al. / Harmful Algae 47 (2015) 17–26
monoculture (A. tamarense control) was evaluated during the six days of culture. The allelopathic effect can be expressed as the EC50 concentration (i.e., the Alexandrium cell density yielding a 50% decline in target cell density), % of control and other parameters (Tillmann et al., 2007, 2009; Tillmann and Hansen, 2009; Xu et al., 2015). To highlight the inhibition of A. tamarense and C. marina on the growth of target cells, reciprocal inhibition between A. tamarense and C. marina in co-culture was expressed as the inhibition rate (IR) using the following equation (Wu et al., 2012; Gao et al., 2015): The cell density of the control group The cell density of the co-culture group 100% IR% ¼ The cell density of the control group Given the reciprocal effect of the two algal species and the difference in the intrinsic growth rate of the various Alexandrium tamarense strains, allelopathic potency and response of A. tamarense towards Chattonella marina were evaluated in terms of the inhibition effect on the first day after co-culture. The inhibition effect of each A. tamarense strain was expressed as the inhibition rate of A. tamarense on the growth of C. marina, whereas the growth response of various strains of A. tamarense to the coculture with C. marina was evaluated as the inhibitory effect of C. marina on the growth of A. tamarense. Based on the allelopathic potency and response of A. tamarense towards C. marina, hierarchical cluster analyses were conducted to group the 15 A. tamarense strains using the Average Linkage (between groups) function in SPSS 18 (SPSS, Inc., Chicago, IL, USA). 2.3. Extraction of extracellular toxins from A. tamarense cultures Extracellular toxins from Alexandrium tamarense were collected by two methods as described by Yang et al. (2010) and Gentien et al. (2007). In Method A (dialysis), 1 l of A. tamarense culture in exponential phase at a density of approximately 6.0 106 cell/l was centrifuged at 3000 g for 10 min. The cell-free supernatant was filtered through 0.22-mm filters (Millipore). Then, NaOH was added to adjust the pH to 10–11, which caused some milk-white precipitate to appear in the solution. After standing for 1 h, the precipitate was collected by centrifugation, dissolved in 100 ml of 1 M HCl, and dialyzed in 0.1 mM HCl for 3 days. According to the previous findings (Yamasaki et al., 2008; Ma et al., 2011), some amphipathic extracellular substances with large molecular weights may be responsible for the allelopathic action of A. tamarense. Additionally, Emura et al. (2004) isolated a protein-like hemolytic compound with a molecular weight of more than 10 kDa by dialysis from Alexandrium taylori. Thus, in this study, a dialysis bag with a cut-off value of 1 kDa was used to isolate possible allelochemicals or hemolytic substances. The dialysate was cryodesiccated and dissolved in 1 ml methanol to obtain the crude toxin (He et al., 1996). In Method B (solvent extraction), cell-free supernatants were obtained as described in method A and then filtered through 0.22mm filters (Millipore). Methanol/chloroform (100 ml:50 ml) was added to the filtrate and mixed. After phase separation, the lower phase was collected and evaporated in a water bath at 60 8C to remove the solvent. The residue was dissolved in 1 ml methanol to obtain the crude toxin (Gentien et al., 2007). 2.4. Hemolytic activity analysis The hemolytic test was performed according to the protocol of Peng et al. (2005). A certain amount of algal extract (0.1 ml) from the Alexandrium tamarense cultures in triplicate was diluted with phosphate buffered saline (PBS, pH 7.4) to a total volume of 0.4 ml
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and then added to 1.6 ml of 0.5% rabbit blood in PBS. After incubation at 37 8C for 30 min, intact blood cells were removed by centrifugation and hemolytic activity was detected spectrophotometrically by measuring the absorbance at 540 nm (10 mm cuvette). Methanol was used as a blank control and digitonin (Sigma, USA) as a standard. The standard curve was generated with 0.5–1.75 mg/ml digitonin solutions and related hemolytic percentages. A hemolytic unit (HU) was defined as the concentration of toxin that caused 50% lysis during the incubation. Similar to the assessment of allelopathic action, a hierarchical cluster analysis (Average Linkage, between groups) was performed with SPSS 18 to group the 15 A. tamarense strains according to hemolytic activity. 2.5. Statistical analysis All of the data in the current study are given as the mean values SD (n = 3). Hierarchical cluster analysis (Average Linkage, between groups) using SPSS 18 was used to group the different Alexandrium tamarense strains in terms of the reciprocal inhibition rate between A. tamarense and Chattonella marina on the first day after co-culture or hemolytic activity. Student’s t-test was used to compare differences in hemolytic activity between the extracellular toxins obtained by the solvent extraction method and that obtained by the dialysis method after testing for homogeneity of variance. Taking into account the potential correlation between the hemolytic activities of the extracellular toxins obtained by the two extraction methods, a partial correlation analysis was performed to evaluate the correlation between the hemolytic activity and the allelopathic potency of A. tamarense strains using SPSS 18.0. The allelopathic potency of A. tamarense was evaluated in terms of the inhibition rate on the first day after co-culture. 3. Results 3.1. Effects of A. tamarense on the growth of C. marina The effects of the different Alexandrium tamarense strains on the growth of Chattonella marina are presented in Table 2 and Fig. 1. The 15 strains exhibited high variation in allelopathic action on C. marina. Most of the A. tamarense strains inhibited the growth of C. marina to various degrees. The IR of C. marina 6 d after coculturing with A. tamarense ranged from 1.79% 7.10% to 100.00% 0.00% and significantly differed between the strains. For example, in the co-cultures with ATDH23, ATDY03, ATMJ02, ATMJ01, ATXM08 and ATGX02, the cell densities of C. marina decreased to zero after 6 days, suggesting strong inhibition of the six strains on C. marina. However, CCMP1598 and HK9301 exhibited a weaker inhibitory effect on C. marina. There was no significant difference in cell density between the monoculture of C. marina and co-cultures with CCMP1598 or HK9301 after 6 days (p > 0.05). Additionally, we noted that various strains of A. tamarense exhibited different changes in IR over time. For example, the IR of ATHKQ against C. marina steadily increased over the experimental period, while the IR of ATDH17 against C. marina increased during the first 3 days but decreased thereafter. During the experiment, the pH in both the monocultures and the co-cultures was monitored daily, and few changes (from 8.8 to 9.0) were observed. Furthermore, the growth of C. marina at pH 8.5 and 9 was very similar (data not shown). Based on the inhibitory effect of Alexandrium tamarense on the growth of Chattonella marina on the first day after co-culture, the 15 A. tamarense strains can be divided into 3 hierarchical groups as shown in Fig. 2. The inhibitory activities from weak to strong were as follows: (1) Weak (W)—ATDH05, ATDH17, ATDH01, ATDY07, CMP1493, HK9301, ATHKQ, and CCMP1598; (2) Moderate (M)— ATDH02 and ATMJ01; and (3) Strong (S)—ATDY03, ATGX02, ATDH23, ATXM08, and ATMJ02.
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Table 2 Reciprocal inhibition between the Alexandrium tamarense species complex and Chattonella marina in co-culture. Strain
Growth rate (d1)
IR (%)
1d
2d
3d
ATDH01
0.36 0.01
AT CM
10.09 7.10 20.73 2.66
20.91 9.25 23.66 1.74
48.56 3.05 25.46 0.77
44.63 3.39 24.52 2.49
60.72 3.58 21.50 9.96
62.91 5.51 15.36 9.65
ATDH02
0.28 0.01
AT CM
17.99 4.16 49.88 8.16
27.27 1.93 66.76 4.11
30.17 4.88 75.99 3.08
25.22 2.13 83.91 2.20
22.38 6.81 92.38 0.62
24.48 6.40 95.35 0.15
ATDH05
0.36 0.01
AT CM
13.88 1.10 25.47 10.41
39.49 0.77 35.08 4.51
59.93 0.51 40.98 0.36
38.53 3.05 50.28 0.85
50.44 1.24 52.61 3.18
53.67 4.95 40.13 5.54
ATDH17
0.10 0.01
AT CM
1.27 5.31 25.94 2.67
17.08 6.11 35.92 5.25
32.83 1.11 38.33 6.83
42.44 7.01 27.07 4.88
49.51 2.49 24.39 0.29
54.00 6.02 15.38 0.75
ATDH23
0.42 0.02
AT CM
14.80 5.75 79.94 4.48
34.89 5.21 86.71 2.86
29.08 10.08 91.33 2.23
33.85 6.08 97.75 1.48
24.73 5.78 100.00 0.00
15.98 8.04 100.00 0.00
ATDY03
0.24 0.01
AT CM
29.07 4.02 72.60 0.35
35.04 1.08 89.99 2.47
26.94 2.30 93.37 0.09
14.85 3.56 99.29 0.14
28.74 1.96 99.92 0.15
1.18 1.69 100.00 0.00
ATDY07
0.35 0.01
AT CM
31.00 3.50 22.08 7.70
35.77 4.48 45.29 3.89
45.92 1.04 50.24 7.25
55.41 0.99 52.39 2.67
66.71 2.52 51.41 4.51
76.78 2.38 42.26 6.90
HK9301
0.11 0.01
AT CM
2.42 3.28 13.09 6.25
16.32 5.01 14.94 3.30
7.68 3.20 11.96 7.18
17.05 3.66 13.37 2.45
33.95 3.61 3.83 2.88
52.04 3.84 8.90 4.57
ATHKQ
0.20 0.20
AT CM
17.86 6.80 14.32 7.65
25.52 5.22 20.83 2.63
19.84 2.55 37.45 1.31
24.01 2.70 43.44 2.59
40.20 7.37 58.89 3.91
47.79 1.26 59.79 1.25
ATMJ01
0.13 0.01
AT CM
9.43 5.71 51.58 7.25
25.36 4.36 68.94 1.95
20.00 4.53 83.24 0.55
20.08 0.75 91.74 1.37
3.21 3.05 98.76 0.27
13.64 2.85 99.57 0.20
ATMJ02
0.18 0.01
AT CM
19.79 1.57 89.42 3.27
12.14 3.62 98.60 0.17
16.89 6.62 99.73 0.25
20.64 0.60 100.00 0.00
1.53 1.17 100.00 0.00
4.17 5.22 100.00 0.00
ATXM08
0.34 0.02
AT CM
31.13 6.75 83.23 4.09
22.88 3.57 99.01 0.27
1.46 6.09 99.73 0.25
7.57 6.15 100.00 0.00
0.11 4.05 100.00 0.00
6.86 3.94 100.00 0.00
CCMP1493
0.25 0.01
AT CM
2.48 3.90 30.29 4.26
11.54 4.14 43.65 3.83
13.15 6.88 48.53 7.25
20.02 2.76 41.09 5.28
35.78 2.45 35.77 3.29
52.42 0.71 22.81 3.89
CCMP1598
0.25 0.01
AT CM
3.66 1.34 8.21 1.11
23.73 3.09 19.13 11.28
26.83 4.14 21.88 8.85
41.10 3.67 25.11 9.85
61.07 1.85 15.51 3.28
70.38 3.01 1.79 7.10
ATGX02
0.31 0.01
AT CM
38.81 9.13 74.57 6.45
26.05 11.33 85.35 6.45
34.09 4.30 92.01 1.86
33.10 4.39 97.92 0.67
32.92 7.41 99.75 0.25
27.35 9.29 100.00 0.00
4d
5d
6d
Reciprocal inhibition between A. tamarense and C. marina in co-culture was expressed as inhibition rate (IR) as described by Wu et al. (2012). AT indicates the inhibition rate of C. marina towards A. tamarense, while CM indicates the inhibition rate of A. tamarense towards C. marina. Data are given as mean values SD (n = 3).
3.2. Response of various A. tamarense strains to co-culturing with C. marina Similar to the inhibition of Chattonella marina by Alexandrium tamarense, different strains of A. tamarense exhibited various responses to co-culturing with C. marina (Fig. 1 and Table 2). The growth of ATDH01, ATDY07, ATDH17, ATHKQ, ATHK9301, CCMP1598 and CCMP1493 was significantly inhibited when cocultured with C. marina for six days, while the growth of ATXM08 and ATMJ02 was only slightly influenced. According to the inhibitory effect of C. marina on the growth of A. tamarense on the first day after co-culture, the 15 A. tamarense strains can be divided into 3 hierarchical groups as follows (Fig. 3): (1) Weak (W)—CCMP1493, HK9301, ATDH17, and CCMP1598; (2) Moderate (M)—ATMJ01, ATDH01, ATHKQ, ATDH05, ATDH23, ATDH02, and ATMJ02; and (3) Strong (S)—ATDY07, ATXM08, ATDY03, and ATGX02. 3.3. Hemolytic activity of A. tamarense A standard curve was obtained by plotting the hemolytic percentage vs. the concentration of digitonin. In terms of the standard curve, 1 HU was equal to 1.27 mg/ml of digitonin.
Hemolytic activities of the cell-free filtrates from cultures of different Alexandrium tamarense strains are shown in Fig. 4. There were significant differences in the hemolytic activity among the various Alexandrium tamarense strains. Except for ATMJ01, ATXM08, ATDY07 and CCMP1598, crude toxins obtained by the two methods exhibited different hemolytic activities (p < 0.05). The range of hemolytic activity obtained by the dialysis method was from 1.20 0.084 to 6.68 0.079 HU/1 106 cells, while that obtained by solvent extraction was from 1.81 0.091 to 6.41 0.49 HU/1 106 cells. According to the hemolytic activity of the extracellular toxins obtained by the dialysis method, the 15 Alexandrium tamarense isolates can be grouped into the following 3 hierarchical groups: (1) Group A (ATMJ01, CCMP1598, ATDY07, ATDH05, HK9301, ATDH01, ATDH02, CCMP1493, and ATHKQ); (2) Group B (ATDH23, ATXM08, ATDH17, ATDY03, and ATGX02); and (3) Group C (ATMJ02) (Fig. 5A). However, in accordance with their activities obtained by solvent extraction, the 15 Alexandrium tamarense strains were divided into another 3 groups as follows: (1) Group A (CCMP1493, CCMP1598, ATDY03, ATDH23, ATDH05, ATGX02, ATDH01, ATDH17, ATHKQ, ATMJ01, ATDH02, and ATDY07); (2) Group B (ATMJ02 and ATXM08); and (3) Group C (HK9301) (Fig. 5B).
J. Chen et al. / Harmful Algae 47 (2015) 17–26
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Fig. 1. Reciprocal inhibition between the Alexandrium tamarense species complex and Chattonella marina in co-culture. (A–O) Changes in cell density of A. tamarense in monoculture (&) and in mixed culture (&), and C. marina in monoculture (4) and in mixed culture (~). Data are given as mean values SD (n = 3). Vertical lines show the standard deviation.
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Fig. 1. (Continued ).
3.4. Correlation between the hemolytic activity and allelopathic action The partial correlation analysis revealed that the allelopathic potency observed in the co-cultures did not correlate
with the hemolytic activity of the extracellular toxin obtained by the solvent extraction method (r = S0.101, p > 0.05) but did correlate with the hemolytic property of the extracellular toxin obtained by the dialysis method (r = 0.823, p < 0.01).
J. Chen et al. / Harmful Algae 47 (2015) 17–26
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Fig. 2. Comparison of inhibitory effects of various A. tamarense strains on the growth of C. marina when co-culture. A hierarchical cluster analysis was conducted to group the 15 A. tamarense strains using Average Linkage (between groups) function in SPSS 18 in terms of the inhibitory effects of A. tamarense on the growth of C. marina on the first day after co-culture, where squared Euclidean distance was selected as the measurement. The abscissa means Rescaled Distance Cluster Combine.
Fig. 5. Hierarchical groups of the fifteen A. tamarense strains based on the hemolytic activity. (A) Hemolytic toxins obtained by dialysis method; (B) hemolytic toxins obtained by solvent extract method. Hierarchical cluster analyses were performed using Average Linkage (between groups) function in SPSS 18, where squared Euclidean distance was selected as the measurement. The abscissa means Rescaled Distance Cluster Combine. HU values are given in brackets.
Fig. 3. Comparison of the responses of various A. tamarense strains to co-culture with C. marina. A hierarchical cluster analysis was conducted to group the 15 A. tamarense strains using Average Linkage (between groups) function in SPSS 18 in terms of the inhibitory effect of C. marina against A. tamarense on the first day after co-culture, where squared Euclidean distance was selected as the measurement. The abscissa means Rescaled Distance Cluster Combine.
4. Discussion 4.1. Reciprocal inhibition between A. tamarense and C. marina Similar to the previous findings (Tillmann et al., 2009; Alpermann et al., 2010), our results show that most of the 15
Alexandrium tamarense strains have inhibitory effects on the growth of Chattonella marina, but significant differences were observed between strains. Additionally, most of the 15 A. tamarense strains were inhibited by C. marina to various degrees. That is, there is a reciprocal inhibition between A. tamarense and C. marina. Given the previous findings (Tillmann et al., 2009; Alpermann et al., 2010; Yang et al., 2011), it is likely that this reciprocal inhibition is mediated by allelochemicals from the two algal species. Nevertheless, some other sort of competition or interaction might also be present (Tillmann and Hansen, 2009). In the reciprocally allelopathic system, it is necessary for an algal species to continuously increase allelochemicals to inhibit target alga and vice versa (Jang et al., 2007; Wang and Tang, 2008). However, the amount of allelochemicals is dependent on the cell
Fig. 4. Relative hemolytic activities of cell-free filtrates from different strain cultures of A. tamarense. Data are given as mean values SD (n = 3). Vertical lines show the standard deviation. Significant differences in hemolytic activity between the extracts obtained by Method A and Method B are marked with an asterisk (*) (t-test, p < 0.05).
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density of the donor cells (Legrand et al., 2003; Wang and Tang, 2008; Tillmann and Hansen, 2009). In this study, an Alexandrium tamarense strain with high allelochemical potency towards Chattonella marina will actively reduce the amount of C. marina cells and therefore also reduce the amount of allelochemicals produced by C. marina during the six-day co-culture period, thus reducing the inhibition of C. marina against A. tamarense. This might explain the slight effect of C. marina towards ATMJ02 and ATXM08 and the potent inhibition against CCMP1598 and HK9301. On the first day after co-culture, the cell densities of C. marina cocultured with ATMJ02 and ATXM08 were only 11% and 17%, respectively, compared with the related controls (Fig. 1). Therefore, C. marina co-cultured with these two strains might produce a small amount of allelochemicals, thus exhibiting only a slight effect on the growth of ATMJ02 and ATXM08. The inhibition rates of C. marina against ATMJ02 and ATXM08 were only 4.17% and S6.86%, respectively, on the sixth day after co-culture (Table 2). In contrast, CCMP1598 and HK9301 exhibited a weaker inhibition on C. marina growth; consequently, a great amount of allelochemicals might be produced by C. marina (Table 2, Fig. 1). Therefore, these two A. tamarense strains are strongly inhibited. On the first day after co-culture, the inhibition rates of CCMP1598 and HK9301 against C. marina were 8.21% and 13.09%, respectively. Accordingly, the inhibition rates of C. marina against these two strains were 70.38% and 52.04%, respectively, six days after co-culture (Table 2). It is obvious that the allelopathic effect of one species is not only related to its own allelopathic potency but also influenced by the allelopathic potency of the target cells and vice versa in a reciprocally allelopathic system. The intrinsic growth rate of algae may also play an important role in the reciprocally allelopathic system. It should be noted that the inhibition rate of ATHKQ against Chattonella marina steadily increased over the experimental time frame, though the inhibition rate was only 14.32% on the first day after co-culture (Table 2). In contrast, the inhibition rate of ATDH17 against C. marina was 25.94% on the first day but only 15.38% after 6 days. The different intrinsic growth rates of the strains might be at least partly responsible for this discrepancy (Table 2). In any case, even a small supplement in cell density should benefit Alexandrium tamarense or C. marina in gaining dominance in co-cultures. On the first day after co-culture, the inhibition rates of C. marina against ATHKQ and ATDH17 were 17.86% and 1.27% (Table 2), respectively, indicating that ATHKQ exhibits a stronger response to co-culture with C. marina than ATDH17. However, ATHKQ possesses a relatively higher growth rate (0.20 d1), which partially covers a loss in cell density induced by C. marina, and thus, its inhibition against C. marina steadily increases over the time frame. Similarly, ATDH05 exhibits a stronger response to co-culture with C. marina than ATDH17; meanwhile, the two A. tamarense strains display a similar inhibition on C. marina growth the first day after co-culture. However, the relatively higher growth rate of ATDH05 (0.36 d1) might make it produce more allelochemicals with time, thus exhibiting a stronger inhibition on C. marina compared with ATDH17 after 6 days (Table 2). Nonetheless, ATDH05 possesses a higher growth rate and exhibits a stronger inhibition on the growth of C. marina and a weaker response to co-culture with C. marina than ATHKQ the first day after co-culture. Its inhibition rate against C. marina is less than that of ATHKQ six days after co-culture, suggesting that another sort of competition might also be present in reciprocal inhibition or some allelochemicals can be degraded in bi-algal cultures (Wang and Tang, 2008; Tillmann and Hansen, 2009). Taking into account the complex reciprocal effect of the two algal species and difference in intrinsic growth rate of the A. tamarense strains, we compared the allelopathic potency and response of each A. tamarense strain towards C. marina in terms of the inhibition effect the first day after co-culture.
From Fig. 2, in terms of the inhibitory rate of Alexandrium tamarense on the growth of Chattonella marina on the first day after co-culture, the 15 A. tamarense strains can be divided into different hierarchical groups. Similarly, based on the inhibitory rate of C. marina on A. tamarense, the A. tamarense strains can also be divided into 3 hierarchical groups (Fig. 3). However, the two hierarchical groups do not match each other, and no close associations between them were observed. For example, ATDY03, ATGX02, ATDH23, ATXM08 and ATMJ02 exhibited a strong inhibition on the growth of C. marina on the first day after coculture (Fig. 2). However, C. marina displays a potent inhibition on ATXM08, ATDY03 and ATGX02 (Fig. 3) but exerts a moderate inhibition on ATDH23 and ATMJ02 (Fig. 3). Furthermore, compared with ATXM08, ATDY03 and ATGX02, ATMJ02 has a lower growth rate (Table 2). Obviously, these results cannot be explained by the reciprocal effect of the two algal species or difference in the intrinsic growth rate of A. tamarense, suggesting that A. tamarense strains respond differently to co-culture with C. marina. Poulson et al. (2010) reported that the physiological state and cell concentration of competitors were important determinants of allelopathy in Karenia brevis, with lag phase cells being more sensitive to allelopathic effects than later growth stages in Skeletonema grethae. Similarly, Arzul et al. (1999) found that the allelopathic potency of Alexandrium on Gymnodinium mikimotoi and Chaetoceros gracile increased with the concentration and age of the cultures. Diluted filtrates from A. tamarense completely suppressed the growth of C. gracile and G. mikimotoi, except when using a dilution filtrate from exponential growth phase (EP) cultures, which had no observable effect (Arzul et al., 1999). In this study, all of the A. tamarense strains had the same initial cell density and sufficient nutrients were present in the bi-algal cultures. Therefore, it is likely that there are significant differences among the various A. tamarense strains in response to allelochemicals from C. marina. However, further studies examining the effects of cell-free supernatants from C. marina cultures on A. tamarense strains are needed to confirm our presumption. Interestingly, based on the comparison of the lytic activity of A. ostenfeldii, Alexandrium catenella and A. minutum, Tillmann et al. (2008) demonstrated that the intraspecific and intrapopulation clonal variability in lytic potency within Alexandrium tamarense might be even higher than the variability among strains of different Alexandrium species. However, it remains unclear whether the coexisting history of the algal strains have some effects on the allelopathy and whether there is a boundary in the allelopathic potency between different populations. Taking into account the origin of the target species Chattonella marina mentioned above, we compared the allelopathic effects between population samples from the South China Sea and the East China Sea. It might be more ideal to compare the allelopathic actions between samples from the South China Sea (or the East China Sea) and the Bohai Sea (or the Yellow Sea). However, we found that strains from the Yellow Sea and the Bohai Sea are both restricted to Alexandrium fundyense (Group I), whereas samples from the East China Sea and the South China Sea all belong to Alexandrium pacificum (Group IV) (Zou et al., 2014). Various species of A. tamarense may result in different phenotypes including toxin production, growth rate and allelopathy (Scholin et al., 1994; Murata et al., 2012). Thus, only samples from the South China Sea and the East China Sea are compared in our study. Based on the inhibitory effect, the 15 Alexandrium tamarense strains can be divided into 3 hierarchical groups. However, the hierarchical groups do not match the geographical origin. Strains isolated from the same geographical origin are not always clustered into one clade, while strains from different geographical origins are often clustered into one clade. For example, CCMP1598,
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CCMP1493, ATDY07 and ATDY03 from Daya Bay in the South China Sea scatter into two groups. Furthermore, ATDH01, ATDH17, ATDH05, ATDH02 and ATDH23 from the East China Sea scatter into three groups. Similarly, the hemolytic activity also varies considerably among the isolates from the same geographical origin, and strains isolated from the same geographical origin are not always clustered into one clade. This demonstrates that there is no significant boundary in allelopathy between the various populations and that the history of co-existence may have a limited effect on the allelopathy studied here. However, allelopathy is generally considered to be especially effective in stress situations (Reigosa et al., 1999), and changes in the growth rate under stress can have a significant influence on the allelopathic action (Legrand et al., 2003). Thus, further studies should be carried out in the future to authenticate the presumption using more strains, stress and/or limiting growth conditions. Additionally, it is of note that the A. tamarense strains assayed in this study included some vegetative cells that were collected at different times and some strains that germinated from cysts. These may, to a certain extent, influence the outcome of these mixed growth experiments. 4.2. Allelochemicals in A. tamarense Though many studies have been performed, the nature of allelochemicals involved in the allopathic action of Alexandrium tamarense remains unclear (Emura et al., 2004; Grane´li et al., 2008; Yamasaki et al., 2008; Ma et al., 2011). Some large amphipathic extracellular substances, independent of known phycotoxins, PSP toxins and spirolides, have been recommended to be responsible for the action (Tillmann and John, 2002; Emura et al., 2004; Tillmann et al., 2007; Yamasaki et al., 2008; Ma et al., 2011). In our study, to completely remove the algal cells and cell debris that cannot be removed by centrifugation, cell-free supernatants were filtered through 0.22-mm filters. Therefore, some of the macromolecule allelochemicals described by Ma et al. (2009) and Yamasaki et al. (2008) might have been be lost; however, strong hemolytic activities were still found, suggesting the chemical diversity expressed in the lytic substances. In fact, some hemolytic substances of approximately 1 kDa have been found in our filtrates with HPLC–MALDI–MS (data not shown), though their fine structures have not been identified. Additionally, we found that the allelopathic effects observed in co-culture are correlated with the hemolytic properties of the toxins obtained by dialysis (p < 0.01). Taking into account the cut-off value of the dialysis bag (1 kDa), it is reasonable to speculate that some large extracellular hemolytic substances greater than 1 kDa might be at least partly responsible for the allelopathic effects of A. tamarense. Furthermore, we found that these substances are resistant to acids and bases and can withstand temperatures of 75 8C without denaturation, suggesting their non-proteinaceous properties. Additionally, crude toxins obtained by solvent extraction exhibit strong hemolytic activities with significant differences between the Alexandrium tamarense strains. However, the partial correlation analysis failed to identify close associations between the allelopathic actions and hemolytic activities of the extracellular toxins obtained by the solvent extraction method (p > 0.05), suggesting that various A. tamarense strains could produce diverse hemolytic substances with different properties and profiles (Arzul et al., 1999; Tillmann et al., 2008). Similarly, Tillmann et al. (2008) found qualitative differences in the composition of allelochemicals from different Alexandrium species and strains. It is likely that multiple allelochemicals might target and differentially affect different phytoplankton (Ma et al., 2009; Poulson et al., 2010; Prince et al., 2010; HattenrathLehmann and Gobler, 2011).
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5. Conclusion Our results show that most of the 15 Alexandrium tamarense strains from the China Sea have an allelopathic effect on Chattonella marina with significant differences. In contrast, different A. tamarense strains exhibit various responses to co-culture with C. marina. There is a complicated reciprocal inhibitory effect between A. tamarense and C. marina, which might involve allelopathic potency, the intrinsic growth rate of algal species and sensitivity to the allelochemicals, etc. However, there is no close correlation between the phenotype (hemolytic activity and allelopathic action) and geographical distribution. Various A. tamarense strains could therefore produce diverse hemolytic substances with different properties and profiles, which might be responsible for the variability in allelopathic actions of A. tamarense. Acknowledgements This work was supported by the National Basic Research Program of China (973 Program) (2010CB428702), the National Natural Science Foundation of China (41230961, 41176088) and the Natural Science Foundation of Guangdong Province (8251063201000001). [SS] References Alpermann, T.J., Tillmann, U., Beszteri, B., Cembella, A.D., John, U., 2010. Phenotypic variation and genotypic diversity in a planktonic population of the toxigenic marine dinoflagellate Alexandrium tamarense (dinophyceae). J. Phycol. 46 (1), 18–32. Arzul, G., Seguel, M., Guzman, L., Erard-LeDenn, E., 1999. Comparison of allelopathic properties in three toxic Alexandrium species. J. Exp. Mar. Biol. Ecol. 232, 285–295. Chen, J.H., Yu, R.C., Gao, Y., Kong, F.Z., Wang, Y.F., Zhang, Q.C., Kang, Z.J., Yan, T., Zhou, M.J., 2013. Tracing the origin of paralytic shellfish toxins in scallop Patinopecten yessoensis in the northern Yellow Sea. Food Addit. Contam. A 30 (11), 1933–1945. Chen, Y.Q., Qiu, X.Z., Qu, L.H., Zeng, L.M., Qi, Y.Z., Zheng, L., 1999. Analysis of molecular biogeographic marker on red tide toxic Alexandrium tamarense in the South China Sea. Oceanol. Limnol. Sin. 30 (1), 45–51 (in Chinese, with English abstract). Dia, A., Guillou, L., Mauger, S., Bigeard, E., Marie, D., Valero, M., Destombe, C., 2014. Spatiotemporal changes in the genetic diversity of harmful algal blooms caused by the toxic dinoflagellate Alexandrium minutum. Mol. Ecol. 23, 549–560. Emura, A., Matsuyama, Y., Oda, T., 2004. Evidence for the production of a novel proteinaceous hemolytic exotoxin by dinoflagellate Alexandrium taylori. Harmful Algae 3 (1), 29–37. Fang, Q., Gu, H.F., Lan, D.Z., Huang, Y.H., 2006. Effect of temperature, salinity and nutrient on the growth of different strains of Alexandrium tamarense. Mar. Sci. Bull. 25 (6), 20–25 (in Chinese, with English abstract). Frango´pulos, M., Guisande, C., deBlas, E., Maneiro, I., 2004. Toxin production and competitive abilities under phosphorus limitation of Alexandrium species. Harmful Algae 3, 131–139. Fistarol, G.O., Legrand, C., Selander, E., Hummert, C., Stolte, W., Grane´li, E., 2004. Allelopathy in Alexandrium spp.: effect on a natural plankton community and on algal monocultures. Aquat. Microb. Ecol. 35, 45–56. Gao, H.J., Song, Y.H., Lv, C.J., Chen, X.M., Yu, H.B., Peng, J.F., Wang, M., 2015. The possible allelopathic effect of Hydrilla verticillata on phytoplankton in nutrientrich water. Environ. Earth Sci., http://dx.doi.org/10.1007/s12665-015-4316-8. Gentien, P., Lunven, M., Lazure, P., Youenou, A., Crassous, M.P., 2007. Motility and auto toxicity in Karenia mikimotoi (Dinophyceae). Phil. Trans. R. Soc. B 362, 1937–1946. Grane´li, E., Weberg, M., Salomon, P.S., 2008. Harmful algal blooms of allelopathic microalgal species: the role of eutrophication. Harmful Algae 8 (1), 94–102. Gu, H.F., Zeng, N., Liu, T.T., Yang, W.D., Mu¨ller, A., Krock, B., 2013. Morphology, toxicity, and phylogeny of Alexandrium (Dinophyceae) species along the coast of China. Harmful Algae 27, 68–81. Hattenrath-Lehmann, T.K., Gobler, C.J., 2011. Allelopathic inhibition of competing phytoplankton by North American strains of the toxic dinoflagellate, Alexandrium fundyense: Evidence from field experiments, laboratory experiments, and bloom events. Harmful Algae 11, 106–116. He, J.W., Chen, M.H., He, Z.R., 1996. Isolation and characterization of toxins from the phytoflagellate Prymnesium parvum. Acta Hydrobiol. Sin. 20, 41–48 (in Chinese, with English abstract). Jang, M.H., Ha, K., Takamura, N., 2007. Reciprocal allelopathic responses between toxic cyanobacteria (Microcystis aeruginosa) and duckweed (Lemna japonica). Toxicon 49, 727–733.
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