Fig. 7 Haemolytic activity observed in mesocosm samples (mesocosms day 24) incubated at different light intensities. Bars represent standard deviation close examination of trends in densities of the other plankton groups revealed that these were unlikely to be the haemolytic agents. The non-motile P. pouchetii cells present in colonies were responsible for haemolytic activity, although it cannot be excluded that motile P. pouchetii cells (almost negligible in their biomass contribution) were also able to produce lytic substances. Hae-molytic activity was absent during the diatom blooms that preceded the P. pouchetii blooms.
From the relationship between cell numbers and haemolytic activity, an EC50 cell density was estimated to 1.86 x 107 P. pouchetii cells l_1. This value compares favourably with the EC50 densities of other algae that produce harmful algal blooms (Table 3). However, values for the other algae were measured with cell extracts where the EC50 values expressed on a biovolume basis indicate an internal toxicity level relevant for the assessment of grazing effects. The values in this mesocosms study were obtained with live cells, whereas P. pouchetii extracts contained only one percent of the total activity measured with live samples. Consistent with this, low haemolytic values were reported earlier for methanol extracts of P. pouchetii (Stabell et al. 1999) and extracts of P. pouchetii cells were not inhibitory to growth of yeast cells. Fractions derived from P. pouchetii culture water containing the PUA 2-trans-4-trans-decadienal, however, blocked sea urchin embryo development as well as the growth of yeast cells (Hansen et al. 2003, 2004).
Approximately 14% of the haemolytic activity in whole-mesocosm samples was present in GF/F filtrate, while almost no activity was extracted from the broken cells. It is unlikely that the bulk of the activity was bound to the cell membranes because a substantial part of the cell debris was still present in the extract (own observation). An alternative explanation would be that the live P. pouchetii cells produce an unstable component which greatly enhances haemolysis. In accordance with this is the observation that haemolytic activity did not accumulate over time in the meso-cosms, but followed daily variations of chlorophyll a. A mechanism that involves physical contact between Phaeocystis colony and the blood cell could also explain the activity of live Phaeocystis.
During high-light conditions an increased mortality of cod larvae incubated in seawater from a P. pouchetii bloom has been observed (Eilertsen and Raa 1995; Aanesen etal. 1998). Similarly, light enhanced the eVect of P. pouchetii-contain-
ing mesocosm samples on blood cells and may have been caused by the same toxic principle that killed the cod larvae with their exposed gills. It is tempting to speculate on the mechanism involved. Perhaps the haemolytic compound itself reacts in a light dependent manner. Light dependent hae-molytic cytotoxins were recently identified in raphidophyte cultures (Kuroda et al. 2005). An alternative explanation is the following. During the mesocosms blooms the PUA that was identified earlier for P. pouchetii (Hansen et al. 2004) may have been converted into more toxic derivates by ROS produced by living cells: a cascade reaction sequence described earlier in diatoms (Juttner 2001). This scenario provides an explanation for the action of living cells as well as the eVect of light. The light-dependent production of liable ROS such as superoxide seems to be a common feature among microalgae, including prymnesiophytes (Marshall et al. 2005).
Haemolytic activity displayed by P. pouchetii was almost exclusively related to active cells and not to compounds within the cells. This situation seems to be diVerent from the haemolytic glycolipids extracted from P. globosa isolated from ichthyotoxic blooms in Chinese coastal waters (He et al. 1999), the glycolipids from the prymesiophyte Chrysochromulina polylepis (Yasumoto et al. 1990), or the poly ethers found for Prymnesium species (Legrand et al. 2003, and references therein). Because of the low haemo-lytic activity of the cell extracts, it is doubtful if predators on P. pouchetii in the mesocosm would experience negative eVects after consumption of this prey, although healthy colonies are avoided by some copepods (Estep et al. 1990). Organisms such as bacteria and phytoplankton that co-occur with Phaeocystis, however, may be inhibited by this lytic action. If so, actively growing P. pouch-etii colonies in this way further improve their competitive advantage, perhaps contributing to subsequent dominance.
Haemolytic activities observed in the nutrient-enriched mesocosms were higher than those measured in the control bag and values to be expected during blooms in the field. From the data it was possible to estimate the lysis rate at ambient temperature in a natural bloom, based on cell densities or chlorophyll a present. The
24 h of exposure to 7 pmol photons m2 s-1 was on the same order of magnitude as the average daily received illumination in the mesocosms and the field (cf. Nejstgaard et al. 2006). Cell densities reported during P. pouchetii blooms of 1-2 x 107 cells per litre (Schoemann et al. 2005) would lead to a daily lysis rate of 12-29%, whereas the reported Chl a values between 5-10 ug l-1 would lead to a 16-29% lysis rate. These rates indicate that unprotected cells like the blood cells used in this study, would lyse within days during a P. pouchetii bloom. Live P. pouchetii colonies are highly haemolytic and the mechanism seems to be fundamentally diVerent from the haemolytic harmful algal bloom species studied so far.
Acknowledgments We would like to thank T. S0rlie, A. Aadnesen, and H. Gjertsen for their service at the Espeg-rend Weld station, S.R. Borrett and S.J. Whipple for light measurements, and M. Hordnes, E. F.Skjoldal, and S. Tor-kildsen for technical support. J.C. Nejstgaard was supported by the Norwegian Research Council (152714/120). The logistics and costs of the mesocosm study, and the contributions of P.G. Verity were supported by US National Science Foundation grant OPP-00-83381. M. van Rijssel and A.C. Alderkamp received funding for the Weldwork by the Dutch Schure-Beijerink-Popping Fund (SBP/JK/2003-14).
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Biogeochemistry (2007) 83:201-215 DOI 10.1007/s10533-007-9096-0
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