Hemolysis Phaeocystis

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).


Aanesen RT, Eilertsen HC, Stabell OB (1998) Light-induced toxic properties of the marine alga Phaeocys-tis pouchetii towards cod larvae. Aquat Toxicol 40:109-121

Admiraal W, Werner D (1983) Utilisation of limiting concentrations of ortho-phosphate and production of extracellular organic phosphate in culture of marine diatoms. J Plankton Res 5:495-513 Arrigo KR, Robinson DH, Worthen DL, Dunbar RB, Di-Tullio GR, VanWoert M and, Lizotte MP (1999) Phy-toplankton community structure and the drawdown of nutrients and CO2 in the Southern Ocean. Science 283:365-367

Arzul G, Gentien P, Bodennec G (1998) Potential toxicity of microalgal polyunsaturated fatty acids (PUFAs). In: Baudimant G, Guezennec JH, Roy P, Samain JF (eds) Marine lipids. IFREMIER, Nantes, pp 53-62 Brussaard CPD, Kuipers B, Veldhuis MJW (2005) A mesocosm study of Phaeocystis globosa population dynamics - 1. Regulatory role of viruses in bloom. Harmful Algae 4:859-874 Brussaard CPD (2006) Phaeocystis and its interaction with viruses. Biogeochemistry (this issue) Cadée GC, Hegeman J (2002) Phytoplankton in the Marsdiep at the end of the 20th century; 30 years monitoring biomass, primary production and Phaeo-cystis blooms. J Sea Res 48:97-110 de Boer MK, Tyl MR, Vrieling EG, van Rijssel M (2004) Effects of salinity and nutrient conditions on growth and haemolytic activity of Fibrocapsa japonica (Raph-idophyceae). Aquat Microb Ecol 37:171-181 DiTullio GR, Grebmeier JM, Arrigo KR, Lizotte MP, Robinson DH, Leventer A, Barry JB, VanWoert ML, Dunbar RB (2000) Rapid and early export of Phaeo-cystis antarctica blooms in the Ross Sea, Antarctica. Nature 404:595-598 Eilertsen HC, Raa J (1995) Toxins in seawater produced by a common phytoplankter: Phaeocystis pouchetii. J Mar Biotechnol 3:115-119 Eschbach E, Scharsack JP, John U, Medlin LK (2001) Improved erythrocyte lysis assay in microtitre plates for sensitive detection and efficient measurement of hae-molytic compounds from ichtyotoxic algae. J Appl Toxicol 21:513-519 Estep KW, Nejstgaard JC, Skjoldal HR, Rey F (1990) Predation by copepods upon natural populations of Phaeocystis pouchetii as a function of the physiological state of the prey. Mar Ecol Prog Ser 67:235-249 Fistarol GO, Legrand C, Graneli E (2003) Allelopathic effect of Prymnesium parvum on a natural plankton community. Mar Ecol Prog Ser 255:115-125 Fu M, Koulman A, Van Rijssel M, Lützen A, De Boer MK, Tyl MR, Liebezeit G (2004) Chemical characterisation of three haemolytic compounds from the microalgal species Fibrocapsa japonica (Raphidophyceae). Tox-icon 43:355-363 Guillard RRL, Helleburst JA (1971) Growth and production of extracellular substances by two strain of Phaeocystis pouchetii. J Phycol 7:330-338 Hansen E, Eilertsen HC, Ernstsen A, Geneviere AM (2003) Anti-mitotic activity towards sea urchin embryos in extracts from the marine haptophycean Phaeo-cystis pouchetii (Hariot) Lagerheim collected along the coast of northern Norway. Toxicon 41:803-812 Hansen E, Ernstsen A, Eilertsen HC (2004) Isolation and characterisation of a cytotoxic polyunsaturated aldehyde from the marine phytoplankter Phaeocystis pouchetii (Hariot) Lagerheim. Toxicology 199:207217

Hamm CE, Rousseau V (2003) Composition, assimilation and degradation of Phaeocystis globosa-derived fatty acids in the North Sea. J Sea Res 50:271-283 Hay ME, Kubanek J (2002) Community and ecosystem level consequences of chemical cues in the phytoplank-ton. J Chem Ecol 28:2001-2016 He J, Shi Z, Zhang Y, Liu Y, Jiang T, Yin Y, Qi Y (1999) Morphological characteristics and toxins of Phaeocys-tis cf pouchetii (Prymnesiophyceae). Oceanol Limnol Sin 30:76-83

Huang CJ, Dong QX, Zheng L (1999) Taxonomic and ecological studies on a large scale Phaeocystis pouchetii bloom in the Southeast Coast of China during late 1997. Oceanol Limnol Sin 30:581-590 Huntley M, Tande K, Eilertsen HC (1987) On the trophic fate of Phaeocystis pouchetii (Hariot). II. Grazing rates of Calanus hyperboreus (Kroyer) on diatoms and different size categories of Phaeocystis pouchetii. J Exp Mar Biol Ecol 110:197-212 Kuroda A, Nakashima T, Yamaguchi K, Oda T (2005) Isolation and characterization of light-dependent haemo-lytic cytotoxin from harmful red tide phytoplankton Chattonella marina. Comp Biochem Physiol C 141:297-305

Jacobsen A (2000) New aspects of bloom dynamics of Phaeocystis pouchetii (Haptophyta) in Norwegian waters. Ph. D. thesis, University of Bergen Jakobsen HH, Tang KW (2002) Effects of protozoan grazing on colony formation in Phaeocystis globosa (Prymnesiophyceae) and the potential costs and benefits. Aquat Microb Ecol 27:261-273 Jebram D (1980) Prospection for a sufficient nutrition for the cosmopolitic marine bryozoan Electra pilosa (Linnaeus). Zoologische Jahrbücher Abteilung für Systematik, Ökologie und Geographie der Tiere 107:368-390

Johansson N, Granéli E (1999) Influence of different nutrient conditions on cell density, chemical composition and toxicity of Prymnesium parvum (haptophyta) in semi-continuous cultures. J Exp Mar Biol Ecol 239:243-258

Jüttner F (2001) Liberation of 5,8,11,14,17-eicosapantae-noic acid and other polyunsaturated fatty acids from lipids as a grazer defence reaction in epilithic diatom biofilms. J Phycol 37:744-755 Lancelot C, Billen G, Sournia A, Weisse T, Colijn F, Vel-dhuis MJW, Davies A, Wassmann P (1987) Phaeocys-tis blooms and nutrient enrichment in the continental coastal zones of the North Sea. Ambio 16:38-46 Legrand C, Rengefors K, Fistarol GO, Granéli E (2003) Allelopathy in phytoplankton - biochemical, ecological and evolutionary aspects. Phycologia 42:406-419 Marshall JA, Ross T, Pyecroft S, Hallegraeff G (2005) Superoxide production by marine microalgae. II. Towards understanding ecological consequences and possible functions. Mar Biol 147:541-549 Menden-Deuer S, Lessard EJ (2000) Carbon to volume relationships for dinoflagellates, diatoms, and other protist plankton. Limnol Oceanogr 45:569-579 Nejstgaard JC, Frischer ME, Verity PG, Anderson JT, Jac-obsen A, Zirbel MJ, Larsen A, Martínez-Martínez J, Sazhin AF, Walters T, Bronk DA, Whipple SJ, Borrett SR, Patten BC, Long JD (2006) Plankton development and trophic transfer in sea water enclosures with nutrients and Phaeocystis pouchetii added. Mar Ecol Prog Ser 321:99-121 Nejstgaard JC, Tang KW, Steinke M, Dutz J, Koski M, Antajan E, Long JD (2006) Zooplankton grazing on Phaeocystis: a critical review and future challenges. Biogeochemistry. This volume Noordkamp DJB, Schotten M, Gieskes WWC, Forney LJ, Gottschal JC, van Rijssel M (1998) High acrylate concentrations in the mucus of Phaeocystis globosa colonies. Aquat Microb Ecol.16:45-52 Noordkamp DJB, Gieskes WWC, Gottschal JC, Forney LJ, van Rijssel M (2000) Acrylate in Phaeocystis colonies does not affect the surrounding bacteria. J Sea Res 43:287-296

Paffenhöfer GA, Ianora A, Miralto A, Turner JT, Kleppel GS, d'Alcala MR, Casotti R, Caldwell GS, Pohnert G, Fontana A, Muller-Navarra D, Jonasdottir S, Armbrust V, Bamstedt U, Ban S, Bentley MG, Boersma M, Bundy M, Buttino I, Calbet A, Carlotti F, Carotenuto Y, d'Ippolito G, Frost B, Guisande C, Lampert W, Lee RF, Mazza S, Mazzocchi MG, Nejstgaard JC, Poulet SA, Romano G, Smetacek V, Uye S, Wakeham S, Watson S, Wichard T (2005) Colloquium on diatom-copepod interactions. Mar Ecol Prog Ser 286:293-305 Parsons T, Maita Y, Lalli C (1984) A manual of chemical and biological methods for seawater analysis. Perg-amon Press, Oxford, UK, 173 pp Peng XC, Yang WD, Liu JS, Peng ZY, Lu SH, Ding WZ (2005) Characterization of the hemolytic properties of an extract from Phaeocystis globosa Scherffel. J Integrative plant biol Y47:165-171 Pohnert G, Boland W (2002) The oxylipin chemistry of attraction and defence in brown algae and diatoms. Nat Prod Rep 19:108-122 Savage RE (1930) The influence of Phaeocystis on the migration of the herring. Fish Invest Lond (Ser II) 12:1-14

Schoemann V, Becquevort S, Stefels J, Rousseau V, Lancelot C (2005) Phaeocystis blooms in the global ocean and their controlling mechanisms: a review. J Sea Res 53:43-66

Sieburth JM (1960) Acrylic acid an "antibiotic" principle in Phaeocystis blooms in Antarctic waters. Science 132:676-677

Sieburth JM (1961) Antibiotic properties of acrylic acid, a factor in gastrointestinal antibiosis of polar marine animals. J Bacteriol 82:72-79 Simonsen S, Moestrup 0 (1997) Toxicity tests in eight species of Chrysochromulina (Haptophyta). Can J Bot 75:129-136

Stabell OB, Aanesen RT, Eilertsen HC (1999) Toxic peculiarities of the marine alga Phaeocystis pouchetii detected by in vivo and in vitro bioassay methods. Aquat Toxicol 44:279-288 Stefels J, Dijkhuizen L (1996) Characteristics of DMSP-lyase in Phaeocystis sp. (Prymnesiophyceae). Mar Ecol Prog Ser 131:307-313 Stolte W, Panosso R, Gisselson LA, Graneli E (2002) Utilization efficiency of nitrogen associated with riverine dissolved organic carbon (>1 kDa) by two toxin-producing phytoplankton species. Aquat Microb Ecol 29:97-105

Svensen C, Egge JK, Stiansen JE (2001) Can silicate and turbulence regulate the vertical flux of biogenic matter? A mesocosm study. Mar Ecol Prog Ser 217:67-80 Sverdrup LE, Källqvist T, Kelley AE, Fürst CS, Hagen SB (2001) Comparative toxicity of acrylic acid to marine and freshwater microalgae and the significance for environmental effects assessments. Chemosphere 45:653-658

Tang KW (2003) Grazing and colony size development in Phaeocystis globosa (Prymnesiophyceae): the role of a chemical signal. J of Plankton Res 25:831-842 Wolfe GV, Steinke M, Kirst GO (1997) Grazing-activated chemical defence in a unicellular marine alga. Nature 387:894-897

Yasumoto T, Underdal B, Aune T, Hormazabal V, Skul-berg OM, Oshima Y (1990) Screening for hemolytic and ichthyotoxic components of Chrysochromulina polylepis and Gyrodinium aurolum from Norwegian coastal waters. In: Graneli E., Sundström B, Edler L, Anderson DM (eds) Toxic marine phytoplankton. Elsevier, New York, pp 436-440

Biogeochemistry (2007) 83:201-215 DOI 10.1007/s10533-007-9096-0

Was this article helpful?

0 0

Post a comment