The colonization of two Phaeocystis species Prymnesiophyceae by pennate diatoms and other protists a significant contribution to colony biomass

Andrey F. Sazhin • L. Felipe Artigas • Jens C. Nejstgaard • Marc E. Frischer

Received: 2 October 2005 / Accepted: 12 June 2006 / Published online: 14 March 2007 © Springer Science+Business Media B.V. 2007

Abstract The association of Phaeocystis spp. with small pennate diatoms during three Phaeo-cystis-dominated spring blooms were investigated in the Eastern English Channel (2003 and 2004) and in coastal waters of Western Norway during a mesocosm experiment (2005). In each of these studies, colonization of the surface of large Phaeocystis spp. colonies by small needle-shaped diatoms (Pseudo-nitzschia spp.) were observed. In the English Channel the diatom Pseudo-nitzs-chia delicatissima colonized the surface of large (>100 ^m) Phaeocystis globosa colonies. The abundance of Pseudo-nitzschia delicatissima reached 130 cells per colony and formed up to 70% of the total carbon associated with Phaeo-cystis cells during late bloom stages. In Norwegian

P.P. Shirshov Institute of Oceanology RAS, Nakhimovsky Prospect, Moscow, 117851, Russia e-mail: [email protected]

UMR 8013 ELICO (FORTEC), MREN-Universite du Littoral Cote d'Opale, Wimereux, France

J. C. Nejstgaard

Department of Biology, University of Bergen, Bergen, Norway

M. E. Frischer

Skidaway Institute of Oceanography, Savannah, GA, 31411, USA

waters, the surface of large (>250 mm) Phaeocystis pouchetii colonies were colonized by Pseudo-nitzschia cf. granii var. curvata and to a lesser degree by other phytoplankton and protist species, although the abundance of these diatoms was never greater than 40 cells per colony. Based on these observations we suggest that diatoms utilize Phaeocystis colonies not only as habitat, but that they are able to utilize the colonial matrix as a growth substrate. Furthermore, these observations indicate that a considerable fraction of biomass (chlorophyll) associated with Phaeocystis colonies, especially large colonies concerned with intense and prolonged blooms, are due to co-occurring plankton species and not exclusively Phaeocystis cells.

Keywords Biomass estimate, colonies • Colonization • Phaeocystis bloom • Pseudo-nitzschia species

Introduction

The coexistence of Phaeocystis species with pen-nate diatoms and other protists, although not universally observed, is a well-known and common phenomenon (Hasle 1964; Rousseau et al. 1994; Hasle and Syvertsen 1997; Peperzak etal. 1998; Wassmann et al. 1999; Throndsen et al. 2003; Hamm and Rousseau 2003). In general, the colonization of the microphytobenthos is well known in shelf waters and in the near-shore regions of seas and oceans, pennate diatoms are also known to dominate in the community of nano- and microalgae on the surface of macrophytes and zoobenthos (e.g. Proshkina-Lavrenko 1963; Sapozhnikov 2003 and many others).

Since the genus Phaeocystis was first described over 100 years ago (Pouchet 1892), a large number of observations and studies have reported the conspicuous bloom-forming Phaeocystis spp. (see, e.g., review of Schoemann et al. 2005). However, for a long time the presence of the small needle-shaped Nitzschia species on and/or in Phaeocystis colonies was reported only by Hasle and co-workers (Hasle 1964; Hasle and Syvertsen 1997). These investigators described two diatoms species in association with the surface of Phaeocystis pouchetii colonies: Pseudo-nitzschia delicatissima and Pseudo-nitzschia granii var. curvata (Thrond-sen et al. 2003). Wassmann et al. (1999) reported abundant populations of Pseudo-nitzschia cf. pseudodelicatissima and the cryptophyte flagellate Plagioselmis sp., associated with colonies of P. pouchetii in the Barents Sea. In other studies microscopic examination of senescent Phaeocystis colonies and foam revealed the presence of large numbers of the pennate diatoms (Nitzschia species) on the surface of Phaeocystis globosa (Peperzak et al. 1998; Hamm and Rousseau 2003).

During our studies, we observed an abundance of the small needle-shaped Pseudo-nitzschia species on Phaeocystis colonies provoking interest in both qualitative and quantitative analysis of this phenomenon. If Pseudo-nitzschia species comprise a significant fraction of total Phaeocystis colony biomass, it is essential to take this fact into consideration in the studies of food webs, vertical fluxes, biogeochemical element fluxes, etc. since Phaeocystis is a widely distributed phytoplankter and it often develops massive blooms (Schoe-mann et al. 2005).

Material and methods

During a bloom of P. globosa in March-May 2003 and in February-April 2004 water samples were collected at several stations in the Eastern English Channel in the coastal waters off Boulogne—Wimereux, France (Fig. 1). Water samples were collected from three depth ranges including surface waters (0.5-2 m), mid-depth waters (10-12 m), and water from just above the bottom (20-22 m) using Niskin bottles during several cruises of the RVs "Sepia II" and "Cotes de la Manche". Coastal-offshore transects and 24 h drifting experiments were carried out at two sites, one located off the Wimereux-Slack estuaries and another located southward in the Bay of Somme.

Samples of P. pouchetii were also collected from blooms in a mesocosm experiment conducted at the marine biological field station, University of Bergen, Western Norway (60°16' N, 05°14' E), on 01-27 April 2005 (Fig. 2). The experiment was conducted essentially as described by Nejstgaard et al. (2006) in floating 11 m3 polyethylene enclosures (4.5 m deep, 2 m diameter). The mesocosms were transparent with 90% penetration of photosynthetically active radiation (PAR). Mesocosms were filled on 31 March by pumping fjord water from a depth of 5 m. The water column was well mixed with an airlift-system, pumping 401 water min"1. In order to allow the introduction of new species, to avoid substantial pH changes due to primary production, and to replace the water sampled during the mesocosm experiment, 10% of the mesocosm water was renewed daily starting from April 1 by pumping fjord water from outside the mesocosm from a depth of 2.5 m. An intense bloom of P. pouchetii was stimulated after fertilization with NO3 (16 mm) and PO4 (1 mm).

Whole colonies and cells within colonies (non-motile stage) of Phaeocystis were identified and enumerated by light microscopy. The samples were either live or preserved with 1 % glutaralde-hyde-lugol solution (Rousseau et al. 1990). In addition, we used epifluorescence microscopy to enumerate and identify flagellate forms (motile stage) of Phaeocystis and microplankton (Sherr et al. 2000). In our modified procedure, the samples were stained with primulin, fixed with 3.6% glutaraldehyde solution with 10% glycerol added for better preservation, and gently filtered onto black-stained Nucleopore filters (0.4 mm). Identical microscopy procedures were applied for samples

Fig. 1 Map of the sampling area in the Eastern English Channel

from the Eastern English Channel and Western Norway. Our microscopic estimation of the number of nonmotile cells of Phaeocystis inside colonies of different volumes corresponded to the results obtained by Rousseau et al. (1994), i.e., according to the following regression equation: log Ncell = 0.51 log V + 3.67 where Ncell is the colony cell number and V is the colonial volume expressed in mm3. For the identification of diatom species, we used a taxonomic key based on light microscope observations (Hasle et al. 1996; Hasle and Syvertsen 1997; Throndsen et al. 2003); and electron microscope observations (Hasle et al. 1996; Priisholm et al. 2002). For Pseudo-nitzschia delicatissima from the English Channel, scanning electron microscopy (SEM) electron micrographs were obtained using a LEO 438 VP scanning electron microscope. Cell volume was calculated by approximation to the closest sample 3D shapes and converted into C according to Menden-Deuer and Lessard (2000). The volume of diatoms was generally less than 3000 pm3, so we applied the following volume-to-carbon conversion formula for protist plankton: pg C cell-1 = 0.216 x volume0939.

From the English Channel, 84 samples were counted. Most of the samples were preserved and colonies of Phaeocystis were enumerated in the total sample volume collected (100-250 ml). The samples were settled for minimum 24 h, then gently concentrated by removing surface water with a plastic syringes (ca. 10 mm diameter), passed through a 5 pm nylon mesh. All volume of concentrated subsamples (5-10 ml) was observed in 1 ml-Naumann counting chamber (Naumann 1922).

Because samples were collected from a large variety of hydrographical conditions, it was not possible to statistically analyze the samples with respect to environmental conditions (i.e., time, location, and depth). For this reason, the samples from the English Channel were divided into different groups depending on the stage of the P. globosa bloom. These stages were determined on the basis of various characteristics: the presence or absence of colonies, their size and shape and the number of cells within the colonies. An additional indication of one or another stage of the P. globosa bloom was provided by other algae, mainly diatoms which were also counted in the samples. Succession changes in phytoplankton community allowed us to make more precise grouping of the data. Three groups were distinguished. The first group included the samples collected in March and several samples collected at the end of February (start of the P. globosa bloom), the second group included the samples collected in April (middle of the bloom), and the third included the samples collected in May (end of the bloom). Within each group, independently of collection date, location and depth, the data were counted as one set.

In the Western Norway mesocosm experiment only live samples were analyzed. The development of the P. pouchetii bloom in the fertilized meso-cosm was followed daily from 1 April-27 April,

2005. During this period about 30 samples were analyzed. In each sample at least 500 colonies were counted and at least 20 colonies were measured.

Results

Pseudo-nitzschia delicatissima1 (usually as single or formed pairs) colonized the surface of the P. globosa colonies at all depths from the Eastern English Channel (Fig. 3a-c). Diatom growth usually started when colony size was over 100 pm (typical biomass values for P. globosa cells colony-1 are about 380 pg C) and the bloom was rather intense. At this stage, diatoms were observed on 5-14% of the P. globosa colonies, with about 5-7 Pseudo-nitzschia delicatissima cells per colony (88-124 pg C colony-1) (Fig. 4). Over the course of bloom development, the frequency of colonization increased to about 30%. After one month, when colony size reached 300-600 pm (typical biomass values for P. globosa cells colony-1 about 1140-1795 pg C), the number of Pseudo-nitzschia delicatissima increased

1 In a recent article (a combination of the morphological and molecular findings) Pseudo-nitzschia delicatissima was shown to be a complex of three different species (Pseudo-nitzschia delicatissima, P. decipiens sp. nov. and P. dolorosa sp. nov. (Lundholm et al. 2006).

to 25-50 cells per colony (442-883 pg C colony-1). Two months after the beginning of the bloom, nearly 100% of P. globosa colonies were colonized by diatoms, the average colony size was above 1000 mm (biomass values for P. globosa cells colony-1 were above 3560 pg C), and the number of Pseudo-nitzschia delicatissima varied from a few to 120-130 cells per colony (2119-2296 pg C colony-1). Similar observations were made both in 2003 and 2004. In these studies, over the duration of the bloom, the average contribution of Pseudo-nitzschia delicatissima to the total biomass associated with P. globosa colonies was 46% of total carbon (non-motile P. globosa cells and diatoms). However, during late bloom stages, the biomass of Pseudo-nitzschia delicatissima accounted for up to 70% of the total carbon of the Phaeocystis cells (nonmotile stage) within the colonies. In these estimates carbon from the colony matrix was not included since non-cellular material associated with the colonies contains very little carbon (Rijssel et al. 1997).

During the bloom of P. pouchetii in the meso-cosm experiment (Norway) the first diatoms appeared when the colonies reached a size of 250 x 180 mm containing about 50 nonmotile cells (544 pg C colony-1). At this stage in colony development the colonies of P. pouchetii were transi-tioning from a spherical to ellipsoid shape and we began to observe their colonization by the diatom Pseudo-nitzschia cf. granii var. curvata (Fig. 5a, b). During the following two weeks, the mean size of the P. pouchetii colonies continued to increase to about 370 x 350 mm containing about 200 cells colony-1 (2176 pg C of cells/colony) and the number of Pseudo-nitzschia cf. granii var. curvata varied from 4 to 59 cells per colony (136-2011 pg C of cells colony-1), with a mean of 18 cells per colony (613 pg C of cells colony-1). However, fewer than 1% (0.22-0.92%) of Phaeocystis colonies was colonized by diatoms (Fig. 6). In addition to Pseudo-nitzschia cf. granii var. curvata, P. pouchetii colonies were occasionally colonized by other diatom species. For example, we observed a P. pouchetii colony with 12 cells of Nitzschia frigida on the surface and other colonies with 1-3 cells of Cylindrotheca closterium (Fig. 7). In two samples we observed P. pouchetii colonies with attached suctorian ciliates (Acineta tuberosa) (Fig. 8). Cili-ates of the genus Acineta have been reported to be

Fig. 3 (a-c) Diatom Pseudo-nitzschia delicatissima inhabiting the surface of the Phaeocystis globosa colonies from the Eastern English Channel (a light microscopy, b epiflu-orescence microscopy, c scanning electron microscopy)

epibionts of decapods, cladocerans, copepods, ostracods isopods and amphipods (Fernandez-Leborans and Tato-Porto 2000; Arndt et al. 2005).

Discussion

In this report we present observations of Phaeocystis colonies being colonized by different small

Fig. 4 Bloom and evolution of Phaeocystis globosa colonies and diatom Pseudo-nitzschia delica-tissima from the Eastern English Channel in 2003 and 2004

Nitzschia Colony
Fig. 5 (a, b) Diatom Pseudo-nitzschia cf. granii var. curv-ata inhabiting the surface of Phaeocystis pouchetii colonies from Western Norway (light microscopy)

needle-shaped Pseudo-nitzschia species to such an extent that the estimated diatom carbon at times was similar to or even exceeded the contribution

April

2003 + 2004

of the nonmotile Phaeocystis cells within colonies. The colonization of P. globosa by Pseudo-nitzchia delicatissima during two successive years in the English Channel, of P. pouchetii by other Pseudo-nitzschia species in the Norwegian mesocosm experiment, and earlier reports by other authors, confirm that colonization of Phaeocystis colonies by diatoms and other protists appears to be widespread and common phenomenon. For example, the presence of Calliacantha natans (Choanoflag-ellida) in high abundance on senescent colonies of P. pouchetii was reported from the Eastern Bering Sea shelf (Sukhanova and Flint 2001), ovoid heterotrophic gymnodinoid dinoflagellates were found on senescent Phaeocystis colonies (Peper-zak etal. 1998), and preliminary observations of Phaeocystis antarctica colonies collected in the Ross Sea show that small (less than 10 pm) het-erotrophic protists were associated with the colonial matrix (Shields and Smith 2005). Since the colonies of Phaeocystis species may be intensely colonized by pigment-containing algae such as diatoms, care should be taken when estimating Phaeocystis colony biomass using pigment-based methods. For example, quantification by Chl a, or even high-performance liquid chromatography (HPLC) and chemical taxonomy (CHEMTAX) without direct inspection of the algal material (cf. Irigoien et al. 2004) may lead to significant over-estimation of the Phaeocystis biomass. As seen in

Fig. 6 Bloom and evolution of Phaeocystis pouch-etii colonies (a) and diatom Pseudo-nitzschia cf. granii var. curvata (b) from Western Norway. The squares refer to the number of colonies and the circles to the average size of the colonies

Fig. 6 Bloom and evolution of Phaeocystis pouch-etii colonies (a) and diatom Pseudo-nitzschia cf. granii var. curvata (b) from Western Norway. The squares refer to the number of colonies and the circles to the average size of the colonies

Fig. 3b, the majority of total chlorophyll associated with a Phaeocystis colony can be derived from diatoms on the colony rather than Phaeocys-tis cells within the colony. Therefore to accurately quantify Phaeocystis biomass, particularly during intense and prolonged blooms, it is necessary to consider the accompanying algae on the colonies and unambiguous methods such as microscopy should be routinely utilized.

The underlying processes influencing the association between Phaeocystis colonies and diatoms remains incompletely understood. The relationship

Fig. 7 Diatom Cylindrotheca closterium inhabiting the surface of a Phaeocystis pouchetii colony from Western Norway (light microscopy)

between colonized Phaeocystis and diatoms may represent either an active or passive association. Recent studies in the Southern Ocean have demonstrated that P. antarctica and diatoms have similar photosynthetic capabilities and that growth of both can co-occur under similar light conditions (Hilst and Smith 2002). If this is also the case for P. globosa, P. pouchetii, and associated Pseudo-nitzschia species, the colonization of Phaeocystis colonies by diatoms might be expected to occur without advantage or disadvantage to either species under similar favorable growth conditions. Alternatively, it is well known that diatoms are able to use various organic substances, especially when photosynthesis is limited (Lewin and Helle-bust 1976; Molloy and Syrett 1988; Petterson and

Sahlsten 1990; Graham and Wilcox 2000). It is therefore possible that diatoms might use Phaeo-cystis spp. colonies not only as habitat, but may be able to utilize organic and inorganic substances derived from or associated with the colonial matrix. Such a commensalism could be increased as the Phaeocystis colony becomes older and less defended due to deterioration of the colony membrane (Hamm et al., 1999; Hamm, 2000) or becomes less allelopathic (Long et al. submitted). Still another possibility is that Phaeocystis might benefit from an association with Pseudo-nitzschia in which case their association would represent a true symbiosis. It has been reported that Pseudo-nitzschia species can produce specific aldehydes that reduce copepod fecundity (Miralto et al. 1999) and thus, the presence of Pseudo-nitzschia spp. on Phaeocystis colonies might provide a level of grazing protection to aging colonies. However, in our opinion the interactions of Pseudo-nitzs-chia species and Phaeocystis colonies are most likely to be of the facultative commensalism type, although additional studies are needed to investigate this hypothesis. Regardless, conceptual models of the life cycle of Phaeocystis spp. (Whipple et al. 2005) especially ecological significance of the colonial life form should now take into account Pseudo-nitzschia/Phaeocystis association.

Acknowledgments We are indebted to Dr. Lucie Courcot from Universite du Littoral Cote d'Opale, Wimereux, France for SEM photographs that were useful for identification of the Pseudo-nitzschia delicatissima. We thank the crews of R.V. Sepia II and Côtes de la Manche for their help during cruises in the Eastern English Channel, as well as staff of Université of Littoral and University of Bergen for their technical support and help. Anna Boyette (Skid-away Institute of Oceanography) prepared the figures. This work was part of CPER "Bloom of Phaeocystis" and "PNEC Manche Orientale" French regional and national programs for Eastern Channel Monitoring. Jens C. Nejstg-aard was supported by the Norwegian Research Council (152714/120). This study was also funded by the US National Science Foundation Office of Polar Programs grant (OPP—00-83381) and the US Department of Energy Biotechnology Investigations-Ocean Margins Program (FG02-98EF 62531).

References

Arndt CE, Fernandez-Leborans G, Seuthe L, Berge J, Gulliksen B (2005) Ciliated epibionts on the Arctic

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