Allelopathic Effects of Alexandium spp. on Growth of Prorocentrum donghaiense Lu: Is It Toxin Dependent?

The allelopathic properties of three Alexandrium species: A tamarense (ADHK), A. minimus (AM-1) and A. catenella (ACDH), on one species of dinoflagellate Prorocentrum donghaiense were determined using bioassays of allelopathic test. Their culture filtrates were prepared at different growth phases of exponential (EP) and stationary one (SP), and were applied for the cultivation of P. donghaiense in the co-culture. Results revealed that all the culture filtrates presented growth inhibition on the co-cultured P. donghaiense, among which ADHK presented the most severe inhibition. Culture filtrates at SP showed stronger inhibition as compard to that in EP, and the dilution of the culture filtrates could alleviate their negative effects. Analysis on hemolytic activity presented that all of the Alexandrum species had hemolytic activity, which was consistent with their reported PSP toxins. It was thus speculated that the toxin-independent allelochemical(s) were involved in the noxious effects of Alexandrum spp. on P. donghaiense.


Introduction
Alga-released allelochemicals and algal toxins are suggested to be the main reasons responding for the phytoplankton succession in aquatic ecosystem by affecting some species more than others (Fistarol et al., 2005;Gross, 2003). However, few reports focused on discriminating the different roles of toxin-mediated allelopathy between toxic phytoplankton (TPP) and non-toxic phytoplankton (NTP) during the succession process. Granèli and Johansson (2003) found P. parvum exerted negative effects on the co-cultured microalgae by producing toxins, which was suggested to play an allelopathic role. It was reported that toxin-allelopathy might lead to the overturn of competitive exclusion among phytoplankton species. In addition, the toxin-allelopathy was favorable for the stable coexistence of non-toxic phytoplankton that otherwise not coexisted Roy and Chattopadhyay (2006).
Dinoflagellates of the genus Alexandrium are well known for their feature of the occurrence of paralytic shellfishpoisoning (PSP). Moreover, the potentiallytoxic allelopathic substances were suspected to have detrimental effects on surrounding organisms. The effect of toxins or allelopathic substances produced by Alexandrum in its ecological success had received great attention. Tillman and John (2002) had reported that the toxic effects of Alexandrium spp. on the co-cultured dinoflagellates were not due to PSP toxins, but were caused by the released extracellular substances. A. taylori was found to produce a kind of proteinacelous hemolytic exotoxin against mammalian erythrocytes, and this toxin did not belong to PSP toxins (Emura et al., 2003). Dinoflagellates species of Prorocentrum donghaiense and Alexandrium tamarense were identified as the causative species of the bloom occurred annuyally in East China Sea (Lu and Goebel, 2001). We thus chose P. donghaiense as the target organism to examine the effects of different Alexandrum species with or without PSP toxin on its growth. The purpose of the present study aimed at elucidating the role of toxins and allelochemical(s) independt of toxins in the succession of the bloom.

Microalgal Cultivation
Three Alexandrium species: Alexandrium tamarense, Alexandrium minutum, Alexandrium catenella and Prorocentrum donghaiense were obtained from the Algal Center of the Institute of Oceanography, Chinese Academy of Sciences (IOCAS) and kept in Ecological laboratory of Ocean University of China. P. donghaiense was isolated from a bloom in East China Sea and was provided by IOCAS. A. tamarense strain ADHK was isolated in 2003 from a bloom occurred in South China Sea in Hongkong, China. A. minutum strain AM-1 was isolated from coastal area of Taiwan, China. A. catenella strain ACDH was isolated from Yangzi River estuary in East China Sea. The toxic properties of ADHK, ACDH and AM-1 were 2.60, 1.61 and 0.36 pg eq .STX.cell -1 , respectively according to the previous reports (Chen et al., 2007). They were designated as strong, medium and weak toxicity respectively in the present study.
All the microalgae were cultured in modified f/2 medium (Guillard, 1975) at 18±0.5 °C in flasks under illumination condition of 70-mol m -2 s -1 and 12-h light: dark cycle, initial pH of 8.0, and salinity of 30. All flasks were shaken manually twice a day at set times. The microalga in exponential growth phases were used in the experiments. During the experiment, the microalga were inoculated into 250-mL flasks containing fresh f/2-enriched seawater and the total experimental volume was 150 mL.

Allelopathic Test
The allelopathic activity of the Alexandrium sp. cultures towards P. donghaiense was tested by the method developed by Gentien and Arzul (Gentien and Arzul, 1990).

Culture Filtrate Preparation
In order to verify the effects of the extracellular products, the cell-free culture filtrates of Alexantrium spp. were studied. The culture filtrates were prepared from A. tamarense, A. minutum and A. catenella at their expoential phase (EP) at an initial cell density of 1.0×10 4 cells mL -1 . The culture media were firstly gentlely filtered through 0.45-μm and then 0.20-μm Millipore membrane with the vacuum value below 30 Pa for the sterilizing process to eliminate the bacteria. This sterilize filtrate was replete with f/2 stock medium (Guillard, 1975) and used for P. donghaiense cultivation. The initially inoculating cell density of P. donghaiense was set at 1.0 ×10 4 mL -1 and the correspondingly fluorenscence (fsu) was 3.6. Its growth was determined by measuring fluorenscence using a Turner Designs Model 10-700R fluorometer (Wang and Tang, 2008) every 24 h. All other conditions and procedures in this experiment were the same as those described in 2.1, and the experiment lasted for 18 days.

Experimental Set-Up
The culture filtrates above prepared were divided into two groups: one was directly used for P. donghaiense cultivation and the other was diluted for 3 times and then applied for microalgal cultivation. All the other conditions and procedures in this experiment were the same as those described in 2.2.1. Another experiment was performed to determine if there's any difference of the extracellular products released by the microalge at different growth phases, and A. tamarense was used as an example. It was firstly cultivated to their late stationary phase (SP) (about 20 days according to the preliminary experiment) and then diluted to a cell density of 1.0 ×10 4 mL -1 enriched with f/2 medium to eliminate the possible nutrient limitation. This filtrate was used for P. donghaiense cultivation with an initial cell density of 1.0×10 4 mL -1 . The experiment lasted for 18 days. The growth rate was determined and the results were expressed as μ according to the method of Guillard (1975): t and t0 denoted the times when there were N and N0 cells L -1 respectively, t0 and N0 corresponding to the initial situation.

Hemolytic Analysis
The haemolytic property of the seawater in Alexandrium cultures was assayed according to the method of Arzul et al. (1999). The assays were performed on rabbit red blood cells, which were obtained from Beijing Solarbio Science & Technology Company. The rabbit blood cells was stored in the dark at 4 °C to avoid photolysis. The results of haemolysis were expressed in Haemolytic Units per litre (HU L -1 ).

Statistics Assay
Results were analyzed with SPSS 20.0 and Sigmaplot 12.0. The significance between experimential groups was analyzed by one-way ANOVA with significance set at P < 0.05.

Allelopathic Test
The cellular behaviours of P. donghaiense in the culture filtrates obtianed different strains of Alexandrium spp. were quite different (Fig 1). Little difference was observed between the group of control and that in the culture filtrate of A. catenella (P>0.05), but obvious growth inhibition was observed in groups of cultivated in culture filtrates of A. tamarense and A. minutum (P˂0.01) (Fig.1A). However, great changes were occurred in groups cultivated in the diluted culture filtrates (Fig.1B). The growth of P. donghaiense grown in filtrate of A. catenella was greatly increased and the arbitrary value of fluorescence was increased by about 35% as compared to the control (P<0.01). The growth inhibition in groups of culture filtrates of A. minutum and A. tamarense were not as severe as that in the groups without dilution. It seemed that the dilution could alleviate the growth inhibition. On possible explanation might be that the dilution decreease the concentraion of the substances existing in the culture filtrate, which might be allelochemical(s).  In order to further evaluate the possible effects of allelochemcial (s) produced in different growth phase, the culture filtrates prepared from exponential growth phase (EP) and stationary growth phase (SP) were applied for P. donghaiense cultivation. Little difference was observed during the first 9 days after inoculation between the treated goups and the control with and without dilution in either EP or SP groups. However, great difference was observed thereafter, and significant growth inhibition was found in group of SP. in this rgards, we speculated that more allelochemical(s) produced more that in EP. Dilution was also perfomed on culture filtrates at different growth phase.
When the culture filtrates prepared from different growth phases were diluted for 3 times for the microalgal cultivation, the microalgal growth differed obviously. Little difference was found in the first 8 days after inoculation (P>0.05) and thereafer, growth in Effects of culture filtrates obtained from different and great significance was observed thereafter. The microalgal growth in EP group was much higher than that in the SP group, inferring the existence of the potential allelochemical(s).  Figure 3 shows the hemolytic activities of filtrate of different Alexandrium strains cultures. ADHK presented the highest hemotytic activity, followed by ACDH and AM-1. This sequence was consistent with that of contents of STX reported by Chen et al. (2007).

Discussion
Allelopathy in Alexandrium spp. was first described by Hansen (1989) and so far, it has been demonstrated by many reports. However, little was known about the action of allelochemical involved in this process. Tillmann and John (2002) investigated the effects of A. tamarense on the other dinoflagellates and found that the observed toxicity did not correlate with PSP toxins in cells. Some Alexandrium strains that did not contain PSP toxins were allelopathic. Therefore, the allelopathy in Alexandrium spp. was attributed to unknown toxins. In addition to PSP toxins, dinoflagellates can produce hemolytic substances which might be helpful in their competition with other organism in natural envionments. In the present study, the allelopathic test was applied to determine the toxic effects of different Alexantrum species on growth of P. donghaiense. We found that the culture filtrates presented growth inhibition on P. donghaiesne, inferring the existence of allelochemical(s). When the culture filtrates were duluted, the growth inhibition was alleviated and even growth increment was observed on group of A. minimum. In fact, low levels of alleochemicals may stimulate the growth of target organisms, which was known as hormesis (Stebbing, 1982). The growth inhibtition or growth stimulation with different treatment further evidenced that the toxin-independent allelochemical(s) produced by Alexandrum spp. was involved in the interaction between them. On the other hand, the allelochemical(s) was found to perform differently in different growth phases, and those produced in stationary phase (SP) seemed more toxic as compared to those in exponential phase (EP).
Moreover, we also observed the existence of hemolytic activity in the three strains. The extracellular components produced by ADHK showed similar effect on P. donghaiense as ACDH, while AM-1 had less inhibiting effect. These observations paralleled the measured activity of hemolytic components, as well as the toxins (Chen et al., 2007) present in the exudates of the strains. Hemolytic activity is a common biochemical feature of Alexandrium species, but its relationship between the PSP toxins was controversial. For instance, Simonsen and Moestrup (1997) found that one A. tamarense strain contained low level of PSP toxins had strong hemolytic activity; and thus attributed the hemolytic activity to still unknown compounds. Hemolytic activity of Alexandrium species was also reported by Eschbach et al (2001). However, no one definitely bridged the gap between the allelopathy and hemolytic toxins in Alexandrium species. Results suggested an important role of hemolytic components in the interaction between Alexantrum spp. and P. donghaiense.
When combined considered results in the present study, we speculated the PSP-independent allelochemical(s) was involved in the toxicity exerted on P. donghaiense. However, since the consistence of the PSP toxins and hemolytic activity of different Alexantrum spp., we wondered if it was the combination of PSP toxins and allelochimcal(s) that was responsible for the overall phenomenon observed in the population dymanics? Further idenrification might be focus on this problem.