Haemolytic activity of live Phaeocystis pouchetii during mesocosm blooms

Marion van Rijssel • Anne-Carlijn Alderkamp • Jens C. Nejstgaard • Andrey F. Sazhin • Peter G. Verity

Received: 28 November 2005 / Accepted: 12 June 2006 / Published online: 10 March 2007 © Springer Science+Business Media B.V. 2007

Abstract Chemical defence is a potential mechanism contributing to the success of Phaeocystis species that repeatedly dominate the phytoplank-ton in coastal areas. Species within the genus Phaeocystis have long been suspected of imposing negative effects on co-occurring organisms. Recently a number of toxins have been extracted and identified from Phaeocystis samples, but it is not clear if they do enhance the competitive advantage of Phaeocystis species.

In the present study the cytotoxic impact of live Phaeocystis pouchetii to human blood cells in close proximity, regardless of the nature of the responsible mechanism, was quantified using a

M. van Rijssel (&) • A.-C. Alderkamp Department of Marine Biology, Centre for Ecological and Evolutionary Studies, University of Groningen, P.O. Box 14, 9750AA Haren, The Netherlands e-mail: [email protected]

J. C. Nejstgaard

Department of Fisheries and Marine Biology, University of Bergen, P.O. Box 7800, 5020 Bergen, Norway

P. P. Shirshov Institute of Oceanology,

Russian Academy of Science,

36 Nakhimovski Prospekt, 117997 Moscow, Russia

Skidaway Institute of Oceanography,

10 Oceans Science Circle, Savannah, GA, 31411, USA

bioassay. Haemolytic activity was measured during blooms of P. pouchetii in mesocosms. These environments were chosen to mimic natural conditions including chemically mediated interactions that could trigger defensive and/or allelopathic responses of Phaeocystis.

Haemolytic activity correlated with P. pouch-etii numbers and was absent during the preceding diatom bloom. Samples containing live P. pouchetii cells showed the highest activity, while filtered sea water and cell extracts were less haemolytic or without effect. Dose-response curves were linear up to 70% lysis, and haemolysis in samples containing live P. pouchetii cells reached EC50 values comparable to known toxic prymnesiophytes (1.9 * 107 cells l_1). Haemolytic activity was enhanced by increased temperature and light. The results indicate that unprotected and thus presumably vulnerable cells present in a P. pouchetii bloom may lyse within days.

Keywords Allelopathy • Chemical defence • Prymnesiophyte • PUFA • PUA

Introduction

Phaeocystis is an algal genus which regularly dominates vernal blooms in coastal regions all over the world, especially in temperate and higher-latitude waters. These almost monospecific blooms (Lancelot et al. 1987) have a great impact on the local marine food web because they produce the bulk of the primary production in springtime (Arrigo etal. 1999; DiTullio et al. 2000; Schoemann et al. 2005, introduction of this issue). There is likely a combination of mechanisms behind the bloom forming capacity of this genus. For example, Phaeocystis is able to take advantage of eutrophication (Lancelot et al. 1987; Cadee and Hegeman 2002), resulting in high biomass production. Blooms are typically dominated by the colonial form of these species and adjustment of colony size could be a response to escape grazing pressure, thereby reducing population losses [Jacobsen and Tang 2002; Tang 2003, see however Nejstgaard et al. (this issue) for a discussion on this mechanism]. Envelopment of the cells by a colonial mucous layer could be another mechanism to reduce losses because only the motile cells seem to be susceptible to viral lysis (Brussaard et al. 2005, this issue).

Also, Phaeocystis has been suspected for a long time of having a negative effect on co-occurring organisms. Penguins died after consumption of krill that fed on P. antarctica (Sieburth 1960, 1961). Schools of herring seem to avoid Phaeocystis blooms (Savage 1930), and mass mortality of caged fish occurred during a P. globosa bloom in the China Sea (Huang et al. 1999). Cod larvae died in the presence of natural densities of P. pouchetii (Eilertsen and Raa 1995; Aanesen et al. 1998) and negative effects of Phaeocystis were recorded on the bryozoan Electra pilosa (Jebram 1980). Bacterial consumption rates of acrylate in field samples increased when the > 20 pm fraction containing P. globosa colonies was removed (Noordkamp et al. 2000). And then there is the so-called legend of Phaeocystis unpalatability (Huntley et al. 1987) that says that healthy colonies are not consumed due to some sort of chemical deterrence (Estep et al. 1990). The observed negative effects of Phaeocystis presence on other organisms may be a key to its bloom forming capacity; chemical deterrence could be a way to reduce grazing (Nejstgaard et al., this issue) as well as inhibiting competitors (allelopathy), thereby increasing fitness.

Up to now, three toxic components that could be involved in chemical deterrence have been identified in Phaeocystis species: acrylate, a poly-unsaturated aldehyde, and a haemolytic glyco-lipid. (1) Acrylate is produced by Phaeocystis (Guillard and Hellebust 1971; Sieburth 1960) upon enzymatic cleavage of dimethylsulphonio-propionate (DMSP, Stefels and Dijkhuizen 1996) and accumulates in mM concentrations in the colonial mucous layer (Noordkamp et al. 1998). During growth, however, acrylate is unlikely to cause harmful effects on nearby living cells because it is not excreted from the colonies (Noordkamp et al. 2000). Additionally, the concentration of acrylate present in the water column is not expected to exceed the 4 pM observed in senescent cultures (Noordkamp et al. 2000). This is much lower than the mM range of L(E)C50 values reported for marine organisms (Sverdrup et al. 2001). Therefore, acrylate is not a likely component to be involved in allelopathy. Acry-late produced by Phaeocystis could, however, have a negative impact on grazers (and their consumers) when Phaeocystis cells accumulate in their guts. In these acidic environments the high concentrations of acrylate will be in the proton-ated toxic form (below pH 4.25). A grazing-acti-vated chemical defence system based on the conversion of DMSP into DMS and acrylate upon cell damage was already described for another prymnesiophyte, Emiliania huxleyi (Wolfe et al. 1997).

(2) The isolation and identification of an unsat-urated aldehyde from P. pouchetii (Hansen et al. 2004) may indicate a line of defence that was recently revealed for diatoms (Paffenhofer et al. 2005, and references therein). Membrane lipids are converted into mildly toxic polyunsaturated fatty acids (PUFAs) by a grazing-activated enzymatic conversion. In the presence of reactive oxygen species (ROS) PUFAs may be converted into highly toxic polyunsaturated aldehydes (PUAs). In laboratory tests these PUAs negatively affect copepod fecundity and egg-hatching, and induce apoptosis in sea urchin embryos and cytotoxicity in human cell lines (Pohnert and Boland 2002). Precursors for PUAs, such as the PUFA eicosa-pentaenoic acid (EPA), are abundantly present in P. globosa (Hamm and Rousseau 2003). Although these haemolytic PUFAs (Arzul et al. 1998; Fu et al. 2004) provide essential nutrition for copepods high concentrations may be harmful (Juttner 2001). Extracted culture fluid of P. pouchetii containing PUAs and corresponding with densities of 106 cells per ml completely blocked DNA replication in sea urchin embryos (Hansen et al. 2003; Hansen et al. 2004). Extraction of cells or sea water yielded fractions that were mildly haemolytic as well as anaesthetic upon injection in flies (Stabell et al. 1999).

(3) The massive P. globosa bloom of 1997 in the coastal waters of southeast China induced fish mortality and the economic losses were substantial (Huang et al. 1999). A haemolysin that was isolated and characterised as a glycolipid with a digalactose and a PUFA (heptadecadienoyl) group was found to be responsible for the fish mortality (He et al. 1999) by induction of pores in the cell membrane of target cells (Peng et al. 2005). Both the isolated toxin and supernatant of the P. globosa cultures inhibited cultures of other microalgae (J.-S. Liu pers. comm.).

Now that evidence is accumulating about toxic substances produced by Phaeocystis species, field studies are needed to assess if these toxins are used in chemical warfare against predators and/or competitors (allelopathy). In this study we tested the hypothesis that P. pouchetii excretes a lytic agent. The potential impact of live P. pouchetii to cells in close proximity was quantified in a bioas-say during a P. pouchetii bloom in a mesocosm experiment. Red blood cells were selected as model targets representing unprotected cells, and lysis was quantified by monitoring dissolved haemoglobin. Phaeocystis blooms in mesocosms, with natural plankton communities containing all trophic levels up to macrozooplankton, were used to simulate in situ conditions including the chemically mediated interactions (Hay and Kubanek 2002, and references therein) that could trigger defensive responses of P. pouchetii.

Material and methods

Mesocosm set up

Haemolytic activity was studied in P. pouchetii blooms in experiments conducted from 27 February (experimental day 1) to 3 April 2003

at the marine biological field station in Raunefjorden, outside Bergen, Norway (60°16'N, 05°14'E). Three transparent floating polyethylene enclosures were used [4.5 m deep, 2 m diameter, ca 11 m3, 0.12 mm thick walls with 90 % penetration of photosynthetically active radiation (PAR)]. Details of the location and general mesocosm design can be found in Svensen et al. (2001) and at the website of the University of Bergen (http:// www.ifm.uib.no/LSF/inst2.html).

The mesocosms were filled on 27 February by pumping nutrient-poor fjord water from 5 m depth. The water column was well mixed with an airlift system, pumping 401 min-1 (Jacobsen, 2000). In order to allow the introduction of new species, to avoid substantial pH changes due to primary production, and to replace water sampled during the mesocosm experiment, 10% of the water was renewed daily in each mesocosm from 3 March by pumping (peristaltic) fjord water from about 2.5 m depth outside the mesocosms. On 3 March, mesocosms M2 and M3 were enriched with nitrate (NaNO3) and phosphate (NaH2PO4) to final concentrations of 16 |jM and 1 |jM, according to the Redfield ratio, to stimulate the development of a bloom of P. pouchetii. Meso-cosm M1 received no nutrients and served as a control. Nutrient outflow with renewal of water was compensated for by daily additions of nutrients (1.6 ^M nitrate and 0.1 |jM phosphate per mesocosm in M2 and M3). On 20 March daily nutrient replacement in M2 was stopped to induce a fast decline of the P. pouchetii bloom.

Sampling

Sampling was performed at least every third day, in the morning, 4 h after sunrise, using 15 l carboys that were filled at the surface of the meso-cosms. Samples were taken gently to avoid disruption of the colonies. Carboys were stored in the dark at ambient temperature (ca. 4°C). Cells were collected within 1 h from 250 ml subsamples filtered over GF/F filters (47 mm, Whatman) using gravity only. The GF/F filtrate was collected and stored in the dark at 4°C until analysis on the same day. The filters were transferred into test tubes containing 5 ml artificial seawater at 31.5 PSU (ambient salt concentration) which contained 23.4 g NaCl, 9.35 g MgCl2-6H2O, 0.50 g CaCl2.2H2O, 3.05 g Na2SO4, and 0.81 g K2SO4 per litre (Admiraal and Werner 1983). To release water soluble cell content, cells on the filter were disrupted by three sonic bursts (5 s, amplitude 90, 28 Watts) with a probe (Sonics VibratellTM), followed by centrifugation (30 min, 8000 x g). The supernatant (hereafter referred to as extract) was ready to be tested for haemolysis. After experimental day 24 it was no longer possible to filter the 250 ml using gravity and sampling on filters was stopped.

Phytoplankton analysis

Phytoplankton abundance and species composition were determined in 60 ml samples that were fixed with glutaraldehyde (0.5% final concentration) and stored at 4°C. Samples were settled onto black-stained Nuclepore filters with 0.4 mm pore size and then frozen. Cells were counted at 200x, 400x and/or 600-1250x magnification on a light and epifluorescence Olympus or LUMAM-P8 microscope. Cellular carbon of the various phytoplankton species was determined using the conversion described by Menden-Deuer and Lessard (2000). Samples for chlorophyll a (Chl a) were filtered onto 0.45 mm cellulose-acetate filters (Sartorius AG, Germany), immediately extracted in 90% acetone overnight, and analyzed according to Parsons et al. (1984) on a Turner Designs 10-AU fluorometer.

Erythrocyte lysis analysis (ELA)

Whole mesocosm samples, GF/F filtrate and extracts (up to 23 March 2003) were diluted 1:1 with blood cell suspension. For whole-sample transfer, every pipette tip used was clipped with a pair of scissors to have a>3 mm diameter inlet, making sure to include colonies while sampling. The blood cell suspension was prepared by adding five drops of fresh human blood to 30 ml buffer (Eschbach et al. 2001), centrifugation (5 min, 4500 x g) and addition of the same volume of buffer to the pellet. Standard incubations were done for 24 h in triplicate in test vials (2.5 ml, Eppendorf) at 15°C, 7 mmol photons m~2 s_1. After incubation, intact blood cells and phytoplankton were removed by centrifugation (5 min, 4500 x g) and the supernatant was transferred into a cuvette and measured at 414 nm to quantify haemoglobin. Artificial seawater diluted 1:1 with ELA buffer was the 0% control, a sonified blood suspension diluted with artificial sea-water was used as 100% lysis control. For comparison with other haemolytic studies a dose response curve with saponin (Sigma) was made to estimate the EC50 value for this control substance.

In addition to standard incubations, tests were performed. Dose-response curves were made with dilution series of whole mesocosm samples in artificial sea water. The effect of the incubation temperature (15°C) was also compared with a series at ambient temperature (4°C, all sample handling and incubation in cold room). Effects of light conditions were tested by incubations in the dark (wrapped in aluminium foil), 7 and at 40 mmol photons m~2 s_1.

Data analysis

Results of regression analysis (Excel 2003, Microsoft Office) are presented as significant with the symbol P (<0.05); P* (<0.01), P" (<0.001), P*" (<0.0001), or by the actual P value.

Results

Bloom description

In the beginning of the experiment a diatom bloom dominated by Chaetoceros socialis developed in all mesocosms. Maximal densities of 4.5, 5.9 and 5.2x106 cells per litre were measured on day 12 of the experiment for M1, M2, and M3, respectively (ca. 4.5 ug r1 Chl a in all mesocosms, Fig. 1, Table 1). After day 15, P. pouchetii blooms developed in all mesocosms, but were more pronounced in the two nutrient-enriched mesocosms (M2 and M3) compared to the control bag (M1). In M1 maximal P. pouchetii numbers were 9-106 cells per litre representing 55% of total carbon biomass at 2-3 mg total Chl a l_1. In M2, that was fertilized until experimental day 21, maximal P. pouchetii numbers were 4.3 x107 cells per litre (92% of total biomass at 23 mg Chl a l_1), and in M3 5x107 cells o

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