Hydrobiologia (2011) 659:37–48 DOI 10.1007/s10750-010-0268-x DISREGARDED DIVERSITY AND ECOLOGICAL POTENTIALS Review Article Perkinsozoa, a well-known marine protozoan flagellate parasite group, newly identified in lacustrine systems: a review Jean-François Mangot • Didier Debroas Isabelle Domaizon • Received: 19 August 2009 / Accepted: 12 April 2010 / Published online: 16 May 2010 Ó Springer Science+Business Media B.V. 2010 Abstract The recurrent detection of parasitic zoospores among aquatic heterotrophic flagellates (HFs) has recently modified our view of how the microbial loop is organized, and called into question the role of eukaryotic parasites in the aquatic trophic food web. The Perkinsozoa group, already known to play a significant role as parasite in marine systems, is of special interest here, since it has recently been detected in several lakes by constructing clone libraries. In marine systems, this group is known to consist solely of intracellular parasites of molluscs or phytoplanktonic species, but their hosts in freshwater environments are still unknown, and little is yet known about their functional importance in planktonic systems. This review summarizes the main Guest editors: T. Sime-Ngando & N. Niquil / Disregarded Microbial Diversity and Ecological Potentials in Aquatic Systems J.-F. Mangot (&)
I. Domaizon INRA – UMR 42 CARRTEL, Centre Alpin de Recherche sur les Réseaux Trophiques des Ecosystèmes Limniques, Thonon-les-bains Cedex 74203, France e-mail: jean-francois.mangot@wanadoo.fr J.-F. Mangot
D. Debroas LMGE, Laboratoire ‘‘Microorganismes: Génome & Environnement’’, UMR CNRS 6023, Campus des Cézeaux, 24, av. des Landais, Aubière Cedex 63177, France information currently available about Perkinsozoa through a description of their phylogenetic position, their life cycles, and regulatory factors, and the consideration of the specificities of their hosts in marine systems, and the few data recently acquired in lakes. Keywords Perkinsozoa
Eukaryotic parasites
Marine and lacustrine systems Introduction Numerous molecular studies have recently characterized picoplanktonic assemblages by amplifying and sequencing the gene coding for 18S rRNA gene, both in marine and freshwater systems (Dı́ez et al., 2001; Moreira & López-Garcı́a, 2002; Lefranc et al., 2005; Lepère et al., 2006, 2008; Lefèvre et al., 2008). These data have called into question the role of eukaryotic parasites in aquatic systems. More precisely, the importance of putative parasites within small eukaryotes has made it clear that the concept of a microbial loop within which heterotrophic flagellates (HFs) being thought to be mainly bacterivorous (Azam et al., 1983; Sanders et al., 1989) does not adequately describe the various interactions within the microbial food web. The presence of parasitic zoospores among HFs has led us to envisage greater taxonomic and functional heterogeneity among these heterotrophs, 123 38 particularly if parasitism is taken into account. Parasitism is indeed a model of interaction, which increases the complexity of the trophic network by extending food chains, increasing connectance, and the efficiency of transfers; particularly as these parasites have relatively complex life cycles, and are known to infect organisms of various trophic levels (Arias-Gonzáles & Morand, 2006). Most aquatic organisms are known to contain intracellular symbionts (viruses, archaea, bacteria, fungi, and other eukaryotes parasites) (Canter & Lund, 1969; Cole, 1982; Embley et al., 1992; Holfeld, 1998; Erard-Le Denn et al., 2000; Brussaard, 2004; Park et al., 2004). Also, while viral parasitism on bacterioplankton in aquatic systems has been well documented for the last 10 years (Fuhrman, 1999; Wommack & Colwell, 2000; Bettarel et al., 2003, 2004; Weinbauer, 2004), studies focusing on eukaryotic parasites have generally been overlooked. Probably because little is yet known about the identity and diversity of these parasites in aquatic systems, eukaryotic parasitism is often poorly taken into account in functional models (Arias-Gonzáles & Morand, 2006; Lafferty et al., 2006). However, some significant regulating effects have been reported in the literature, and have highlighted the significant impact of parasitism on algal community control in marine systems (Johansson & Coats, 2002; Park et al., 2002; Ibelings et al., 2004; Kagami et al., 2004, 2010). For instance, Syndiniales parasites are able to infect marine dinoflagellates, which are responsible for toxic red tides, also known as harmful algal blooms (HAB), which can cause illness and even human death (Zingone & Enevoldsen, 2000). Chambouvet et al. (2008) recently revealed that the dinoflagellate parasitoid successions in a natural estuary are correlated to the rapid development of four major species of phytoplanktonic dinoflagellate populations (Heterocapsa rotundata, Scrippsiella trochoidea, Alexandrium minutum, and Heterocapsa triquetra). On the basis of such examples, it is obvious that planktonic food web models must now take the role of eukaryotic parasites into account (Arias-Gonzáles & Morand, 2006; Lafferty et al., 2006, 2008). Parasitic interactions increase the complexity of the trophic network both through direct effects (host–cell mortality, parasitic zoospores used as a resource by grazers) and indirect impacts (cascade effects on the competition between phytoplanktonic species, and on 123 Hydrobiologia (2011) 659:37–48 the redistribution and recycling of organic matter after cell lysis) that modify connectance and the efficiency of transfers (Kagami et al., 2004, 2010; Arias-Gonzáles & Morand, 2006). In aquatic ecosystems, three major eukaryotic lineages are known to include potentially parasitic organisms. Organisms belonging to the fungal group of Chytridiomycota are known mainly as eukaryotic parasites of phytoplankton (Goldstein, 1960; Canter & Lund, 1969; Ibelings et al., 2004). Some parasites have also been reported within the Cercozoa (Rhizaria phyla), in which a handful of taxa are parasites of diatoms (Cavalier-Smith & Chao, 2003). However, alveolates exhibit the most considerable diversity of known eukaryotic parasites, including various morphologically distinctive parasites among the three traditional phyla of Ciliophora, Dinoflagellata, and Apicomplexa (Cavalier-Smith, 1993) and the newly included Chromerida (Moore et al., 2008; Obornı́k et al., 2009). Parasitic activities of alveolates have been reported in a wide range of ecosystems (soil, aquatic, etc.). In order to begin with, apicomplexans (ex-sporozoan) and ciliates are major parasites of marine and terrestrial populations (crustaceans, fish, or mammals), and can cause extensive health and economic damage to human populations, particularly those in the developing world (Hakimi & Deitsch, 2007). Leaving aside these two alveolates phyla, Dinoflagellata constitute the main source of aquatic parasites (Blastodiniales, Syndiniales). Among them, Syndiniales constitute an atypical order of dinoflagellates, consisting of wellknown and exclusively marine parasites (Hematodinium spp., Amoebophrya spp.), which display a hostspecific infection strategy (Groisillier et al., 2006; Chambouvet et al., 2008; Guillou et al., 2008). Finally, Chromera velia, a newly cultured photosynthetic symbiont of the stony coral Plesiastrea purpurea, constitutes the latest symbiotic alveolates defined until now (Moore et al., 2008). Based on the available sequences, chromerids may form a sister group to the parasitic apicomplexans and predatory colpodellids rather than the photoautotrophic dinoflagellates (Moore et al., 2008; Obornı́k et al., 2009), and its discovery allows to speculate on the origin of symbiosis and parasitism among the Alveolata. The Perkinsozoa group, a sister-group of the dinoflagellates (also known as perkinsids or perkinseans), is structured around two well-known marine Hydrobiologia (2011) 659:37–48 39 parasites, Perkinsus marinus (Mackin et al., 1950) and Parvilucifera infectans (Norén et al., 1999) (Fig. 1). Perkinsids are intracellular parasites that form a relatively close sister lineage to generally freeliving dinoflagellates (Norén et al., 1999; Leander & Keeling, 2003). Little is known about Perkinsozoa diversity, especially in freshwater, and this constitutes a real handicap for attempts to understand the role of parasites in aquatic systems more precisely. Since the middle of the 2000s, based on recent descriptive studies (Lefranc et al., 2005; Richards et al., 2005; Lefèvre et al., 2007; Lepère et al., 2008) attempts have been made to characterize the composition of the picoeukaryotic population (\5 lm) in several lakes using cloning–sequencing approaches. The recurrent detection of sequences of putative parasites in these studies explains the new interest in this group of eukaryotic parasites in lacustrine systems where their presence had hitherto been unknown. We propose to review the main information available about this phylogenetic group in both marine and freshwater systems. Fig. 1 Phylogenetic tree showing the position of each clades of Perkinsozoa among the alveolates based on their small subunit ribosomal RNA (SSU rRNA) sequences. The names of the lakes in which sequences were identified are indicated with their access number. Numbers at branch nodes indicate significant bootstrap confidence values in percent (BV [90%). The tree was constructed (maximum parsimony criteria), and the BVs for terminal nodes were calculated both using the ARB software package (Ludwig et al., 2004) The phylogenetic position of Perkinsozoa phyla among the lineage of alveolates Perkinsids have mainly been studied in marine environments (Mackin et al., 1950; Norén et al., 1999; Park et al., 2002, 2004, 2006; Casas et al., 123 40 2008). Although there is now strong evidence that perkinsozoans constitute a sister group of dinoflagellates (Moore et al., 2008), their systematic position has long been controversial. They have been alternatively linked to Apicomplexa or to Dinoflagellata (Coss et al., 2001; Brugerolle, 2002). Initially, the classification of organisms within this genus was based on a variety of morphological criteria, especially at the ultrastructural scale. The debate about their phylogenetic position is illustrated by the example of Perkinsus marinus, the best-known parasite in this group. Initially described as a fungus named Dermocystidium marinus by Mackin et al. (1950), on the basis of the observation of the position of the nucleus close to the cell wall and the presence of a centrally located endosome, a subsequent reexamination of its morphological features led to its being transferred to the protozoan phylum Labyrinthomorpha, and designated as Labyrinthomixa marina (Mackin & Ray, 1966). The evaluation of zoospore ultrastructure by Levine (1978) led to a further reconsideration of the phylogenetic position of Perkinsus marinus and the establishment of a new Perkinsean taxon in the protozoan phylum Apicomplexa. Indeed, zoospores of this species differ from those of all other apicomplexans in having a mastigoneme-bearing anterior flagellum, and having an atypical conoid apparatus on the apical complex structure. More recently, studies based on morphological observations and phylogenetic analyses of alveolate DNA sequences, and especially actin gene sequences (Reece et al., 1997; Siddall et al., 1997), led to their exclusion from the phylum Apicomplexa, and the conclusion that Perkinsus was related to the dinoflagellates. However, on the basis of ultrastructural features and phylogenetic studies of the 18S rRNA sequence of a new parasitic species, Parvilucifera infectans, Norén et al. (1999) established a new phylum, Perkinsozoa, within the Alveolata. However, the apparent widespread serological affinity between Perkinsus marinus and diverse parasitic dinoflagellates suggested a closer phylogenetic link to the syndinean dinoflagellate lineage (Bushek et al., 2002). This newly established phylum is assumed to be entirely parasitic (Moreira & López-Garcı́a, 2002), and it consists of two freshwater clades as defined by Lepère et al. (2008) by applying the criteria of Zwart et al. (2002). According to these authors, an environmental clade comprises at least two sequences that 123 Hydrobiologia (2011) 659:37–48 are at least 95% identical and originate from at least two different aquatic sites. Thus, we have defined two clades inside the phylum of Perkinsozoa (named clade 1 and clade 2), each structured around two marine species of parasites, Perkinsus marinus (parasite of molluscs) and Parvilucifera infectans (parasite of dinoflagellates) (Fig. 1). Indeed, in order to determine the phylogenetic affiliation of perkinsozoan species reported here with other congeneric species, a phylogenetic analysis thanks to ARB software was performed (Ludwig et al., 2004). The rooted tree identified two clades highly supported by bootstrap value (BV) of 99% (Fig. 1). Among the second clade of Perkinsozoa named clade 2, Parvilucifera infectans with other environmental sequences reported here constituted a higher-order clade with a strong BV (98%). Perkinsus marinus, often considered as part of the Perkinsozoa clade 1, seems to form only an additional branch apart of this clade which is constituted by environmental sequences supported by a strong BV (100%). Perkinsozoa in marine systems: a natural cause of diseases in oyster cultures and of toxic dinoflagellate proliferations Perkinsus spp. are known to be parasites of molluscs. These hosts are easily sampled in situ, moreover, problems involving infected molluscs have an economic impact as many of these species are commercialized; consequently Perkinsus spp. are the most studied perkinsozoan species. Recently, an assay tool based on a polymerase chain reaction coupled with restriction fragment length polymorphism (PCR– RFLP) of the rRNA internal-transcribed spacers (ITS region) has made it possible to target these parasites. This methodology was validated to discriminate between the various Perkinsus species (P. marinus, P. olseni, P. mediterraneus, and P. chesapeaki), to provide rapid identification, and the final goal was to prevent recurrent diseases (Abollo et al., 2006). Perkinsus marinus is a well-known agent of the disease known as ‘‘Dermo’’, which is the main cause of mortality of bivalve molluscs in American coastal waters. The eastern oyster, Crassostrea virginica, and the Pacific oyster, Crassostrea cortenziensis, are both parasited by Perkinsus marinus, along the Gulf of Mexico and Atlantic coast of North America Hydrobiologia (2011) 659:37–48 (Burreson et al., 1994; Soniat, 1996), and along the Pacific coast of Mexico, respectively (Cáceres-Martı́nez et al., 2008). In European coastal waters, some species of the Perkinsus genus are also able to infect other bivalve hosts. This is the case of Perkinsus mediterraneus, a parasite of the European flat oyster Ostrea edulis (Casas et al., 2008), and P. atlanticus, a parasite of the Portuguese clam Ruditapes decussatus (Azevedo, 1989; Ordas & Figueras, 1998). As illustrated by these examples, Perkinsus species are very widely geographically distributed, which partly reflects their host distribution (Fig. 2). It seems that a variety of levels of host specificities can be observed. Perkinsus marinus seems to solely infect the oyster Crassostrea, while, the clam Ruditapes decussatus known to be infected by P. atlanticus in European waters can also be parasited by P. qugwadi on the west coast of Canada (Blackbourn et al., 1998). Most of the Perkinsus spp. seem to be specific to one type of bivalve (clams or oysters); however, some of them are able to infect various types of host, such as Perkinsus olseni which is a parasite of both clams and abalones. Depending on its geographic location, P. olseni, which is known to infect the Spanish Ruditapes decussatus in Mediterranean waters (Elandaloussi et al., 2009), elsewhere also infects the Korean Venus clam Protothaca jedoensis (Park et al., 2006) and the Australian abalone species, Haliotis rubra and Haliotis laevigata (Lester et al., 1990). Perkinsid infection of species other than 41 molluscs has been reported with the case of P. chabelardi, a parasite of sardine eggs (Gestal et al., 2006). The prevalence of P. marinus, P. atlanticus, and P. olseni on their respective hosts are highly variable, and can sometimes reach very high values (up to 100%) (Burreson et al., 1994; Ngo & Choi, 2004). Seasonal variations have been reported by several authors (Ngo & Choi, 2004; GullianKlanian et al., 2008); these changes in prevalence are linked to various environmental factors (temperature, salinity, phosphorous, silica variations, etc.). The Perkinsozoa group also includes various parasites of protistan species. Parvilucifera infectans is known to infect 26 different microalgal species, 17 of which belong to 10 different dinoflagellate genera (Park et al., 2004). The distribution of this parasite extends from Australian to Norwegian seas, where they commonly infect photosynthetic and heterotrophic dinoflagellates (Park et al., 2004) (Fig. 2). Although the rare mortality rates estimated until now were low (\0.2% of the host population per day, Park et al. 2004), several authors suggest that Parvilucifera infectans could have a significant regulatory effect on its host, contributing to bloom dissipation and controlling dinoflagellate populations (Gisselson et al., 2002; Park et al., 2004). Other species phylogenetically close to P infectans have recently been isolated and studied. They include Parvilucifera sinerae sp. nov., a perkinsid isolated from a bloom of the toxic dinoflagellate Alexandrium minutum in Mediterranean Sea (Figueroa et al., Fig. 2 Global distribution of Perkinsus spp. (white ellipsoidal symbol) and of Parvilucifera spp. (gray ellipsoidal symbol). Each symbol notes locations where infections have been observed in molluscs or phytoplanktonic organisms 123 42 Hydrobiologia (2011) 659:37–48 2008), and Parvilucifera prorocentri sp. nov., a novel parasite found to infect the marine benthic dinoflagellate Prorocentrum fukuyoi (Leander & Hoppenrath, 2008). The pathogenicity of Perkinsus spp. has been relatively thoroughly investigated and quantified, but this is not the case for Parvilucifera spp. which constitute a newly described group of parasites for which no quantitative prevalence data are available. Life cycle and regulatory factors of Perkinsozoa phyla in aquatic systems The life cycles of the three genera of perkinsids, Perkinsus spp., Parvilucifera spp., and Cryptophagus spp., display some common characteristics. For instance, like chytrids, perkinsids have a free living stage (a zoosporic flagellate) during their life cycle (Figs. 3, 4). This infectious stage is characterized by the presence of two flagella on the apical complex, reminiscent of Apicomplexa (Fig. 5). The first steps in the infection process have been studied in detail in the case of Perkinsus spp., a parasite of molluscs, but are less known for its two sister genera Parvilucifera spp. and Cryptophagus spp. After the phagocyting of Perkinsus trophozoites by host hemocytes (Fernández-Robledo et al., 2008), a pale appearance of the digestive gland was observed, the mantle was retracted, gonad development inhibited, growth retarded, and eventually the hosts died (Mackin, Fig. 3 Negative staining of the zoospore of Perkinsus sp. isolated from Macoma balthica. Scale bar = 2 lm (from Coss et al., 2001, p. 49) (Ó by the Society of Protozoologists, 2001. With permission from Wiley-Blackwell) 123 Fig. 4 Free-living stage of Perkinsozoa clade 1 hybridized by TSA–FISH (probe: PERKIN_01) observed by epifluorescence microscopy. Scale bar = 10 lm Fig. 5 Ultrastructure section of Perkinsus atlanticus zoospore, showing the nucleus (N), mitochondrion (M), the plastid (arrow), and the apical end (*) containing such apical complex elements as rhoptries (R). Near the zoospores, some transverse section of the flagella (F) Scale bar = 0.5 lm (from TelesGrilo et al., 2007, p. 164) (Ó Elsevier GmbH, 2007. With permission from Elsevier Science) Hydrobiologia (2011) 659:37–48 1951). The rest of the life cycle of Perkinsozoa shows some similarities among the three genera, such as the presence of a similar intracellular stage, the trophont. Perkinsus spp. and Parvilucifera spp. trophonts secrete a ‘‘sporangium’’ inside the host cell, within which the sporozoites differentiate (Norén et al., 1999; Coss et al., 2001), whereas Cryptophagus spp. zoospores grow and directly divide within the host cytoplasm and are not surrounded by a sporangium or a cyst envelope (Brugerolle, 2002). Thanks to these studies, a general life cycle scenario of perkinsids parasites can now be proposed: After the adhesion of the sporozoite to the host cell surface, the zoospore penetrates into the host cytoplasm. The trophont then grows within the host cytoplasm, and undergoes multiple divisions either in sporangia (Perkinsus spp. and Parvilucifera spp.) or within the host cytoplasm 43 (Cryptophagus spp.). After 4–5 successive nuclear mitoses by schizogony, trophozoite/sporozoite differentiation occurs, and buds onto the host cell surface (Fig. 6). Sporozoites acquire flagella, micronemes and other apical structures, and are then release outside the host structure. Sometimes, a resting cystlike stage can occur during the trophozoite stage. It seems likely that the development of populations of Perkinsozoa parasites could be controlled by various abiotic factors, notably temperature. Indeed, the proliferation of all the Perkinsus species is correlated with warm summer water temperatures (above 20°C), which is when pathogenicity and the associated mortality are the highest (Soudant et al., 2005). The same authors report a slight, but significant, decrease in the percentage of viable cells (PVC) after cold or heat treatments (-80, -20, and 40°C). Furthermore, temperature may have also an impact on the development of the hosts of Perkinsus. The gonadal development of Ruditapes philippinarum appeared to be more efficient at higher temperature (18°C, Delgado & Camacho, 2007). As a result, temperature may have direct and indirect effects, on the viability of Perkinsozoa species and the metabolism of their hosts, respectively. However, some of them are more tolerant to variations in various environmental factors. P. atlanticus is able to adapt to very different culture media, salinity, and temperature conditions, and its development seems to be independent of density (Ordas & Figueras, 1998). Detection and recent interest in the Perkinsozoa phylum in lacustrine systems Fig. 6 Light micrograph of the different live stages of cultured Perkinsus mediterraneus cells in JL-ODRP-2F medium. Trophozoites (T) showing a granular cytoplasm with a large vacuole (v) and a prominent vacuoplast (vp). Perkinsus mediterraneus divides by schizogony, and schizonts (S) vary in size. Scale bar: 20 lm (from Casas et al., 2008, p. 37) (Ó by the International Society of Protistologists, 2008. With permission from Wiley-Blackwell) The characterization of small-eukaryote diversity (\5 lm) conducted in various lakes with differing trophic status (Lefranc et al., 2005) has recently revealed the presence of Perkinsozoa in freshwater systems. This study showed that sequences affiliated to Perkinsozoa can account for up to 60% of the clone library (18S rRNA gene) obtained from the eutrophic lake Aydat (summer sampling in the euphotic zone). The sequences obtained for this lake were affiliated with low similarities (86–90%) to Perkinsus marinus. The unexpected qualitative importance of perkinsids in lacustrine systems led to the emergence of numerous questions about their functional roles in lacustrine food webs. Several recent studies also dealing with the 123 44 characterization of lacustrine picoeukaryotic diversity have confirmed the recurrent presence and importance of this group. Perkinsozoa have been detected in two geographically distinct, oligotrophic lakes, the American Lake George (euphotic zone) (Richards et al., 2005) and the French Lake Pavin, respectively (oxic and oxycline zones) (Lefèvre et al., 2007, 2008). More recently, Lepère et al. (2008) have reported their presence in the 18S rRNA gene library of perkinsids sequences in the epilimnion of Lake Bourget in spring and summer, providing semi-quantitative information about this group. In this mesotrophic system, sequences of Perkinsozoa account for 15.2% of the different OTUs obtained during the summer (August) (29.2% of the clones), whereas in May, the proportion was only about 5% of both sequences and clones. All these perkinsid sequences obtained from sequencing in various lacustrine systems can be divided to form two clades of Perkinsozoa (named clade 1 and clade 2). Recently, on the basis of these molecular data, specific oligonucleotide probes have been designed to target each clade of Perkinsozoa to quantify and estimate the in situ dynamics of this group in Lake Bourget (Mangot et al., 2009). The temporal dynamics of the free-living stage (zoospore) of clade 1 and clade 2 of Perkinsozoa (size fraction \5 lm), as analyzed by epifluorescence microscopy and TSA-FISH (Fig. 4), have all revealed an increase in abundance in summer in the epilimnion of the Lake Bourget (up to 30% of all eukaryotes targeted by the EUK1209R probe in July and August), possibly corresponding to an infectious peak. As already reported in marine systems (Soudant et al., 2005), the dynamics of freshwater perkinsozoans are strongly correlated to temperature (Lefranc et al., 2005, Lefèvre et al., 2007, 2008; Lepère et al., 2008; Mangot et al., 2009). In Lake Bourget, the dynamics of Perkinsozoa clade 1 coincide significantly (RDA analysis, P \ 0.05) with the abundance patterns of Peridinium and Ceratium (Dinoflagellates), whereas the dynamics of Perkinsozoa clade 2 are correlated to the presence of Dinobryon (Chrysophyceae). Although some putative phytoplanktonic host– Perkinsozoa links could be suggested on the basis of this study, no definite association (host/parasite) was demonstrated. Only one study has reported the presence of an algal-parasitic Perkinsozoa, Rastrimonas subtilis gen. et sp. nov (Brugerolle, 2003) in freshwater environments. This taxon was previously known as Cryptophagus subtilis (Brugerolle, 2002), a parasite of 123 Hydrobiologia (2011) 659:37–48 cryptophytes. The ultrastructure and life cycle of this freshwater perkinsid have been studied using cultures of the cryptophyte Chilomonas paramaecium, originally collected from a river. Because no genetic characterization of this taxon was obtained by sequencing, its affiliation to one of the two clades of Perkinsozoa defined here is not possible. However, the description of its ultrastructure given by Brugerolle strongly supports the affiliation of R. subtilis to the phylum of Perkinsozoa (Brugerolle, 2002). Conclusion It is now obvious that eukaryotic parasitism, hitherto generally overlooked in aquatic studies, could in fact play a critical role in the functioning of these ecosystems. For instance, the Perkinsozoa, a sistergroup of dinoflagellates, are a good example of organisms that have generally been classified as unidentified HFs, but which could affect planktonic populations. These parasites have mainly been studied in marine environments, where they constitute one of the main causes of mortality in some molluscs (oysters, clams, abalones, etc.) and phytoplanktonic species (especially, dinoflagellates) worldwide. Valuable knowledge has been acquired about the role of some Perkinsozoa in marine systems, but very little information is known about this parasite group in freshwater systems. It is only recently, thanks to the development of molecular techniques, that Perkinsozoa has been detected and quantified in lakes. Some tools are now available that can target these groups (notably specific oligonucleotide probes), but further research is now needed to connect the potential quantitative importance of these parasites to their precise functional roles in lacustrine systems, notably through cell–scale approaches. Isolating infected hosts, coupled with molecular analysis (genome amplification) of these single cells could reveal the association between lacustrine perkinsids and their hosts. The use of double staining (host/parasite) using TSA–FISH would, in a second step, provide some essential information about prevalence and host/ parasite in situ dynamics. 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Typical freshwater bacteria: an analysis of available 16S rNRA gene sequences from plankton of lakes and rivers. Aquatic Microbial Ecology 28: 141–155. Author Biographies Jean-François Mangot is a PhD student in lacustrine microbial ecology at Blaise Pascal (Clermont-Ferrand) and Savoie Universities, France. His PhD thesis deals with the study of the dynamics and the diversity of potential eukaryotic parasite microorganisms in lacustrine systems, especially the group of Perkinsozoa recently discovered in freshwater ecosystems. Didier Debroas is Professor at Blaise Pascal University in the fields of microbial ecology and bioinformatics. He began his scientific career by studying the microbial ecology of the rumen ecosystem and he has been working since 1993 on the aquatic ecosystem. At present, he is the leader of the group ‘‘Environmental Microbiology and Bioinformatics’’ at the LMGE lab (CNRS, Aubière – http:// www.lmge.univ-bpclermont.fr). Currently, his main research concerns (i) the taxonomic and functional diversities of lacustrine picoplankton and (ii) the community genetic potential of these microorganisms. His research group is developing (i) some methodologies for analysing diversity and functions at the cell level (cell-sorting, metagenomics, etc.) and (ii) bioinformatic tools for dealing with data sequences originated from massive parallel sequencing. 123 48 Isabelle Domaizon is researcher at the hydrobiological INRA center (CARRTEL, Thonon, France). She is currently the leader of the group ‘Biodiversity Functioning Evolution of Lacustrine Ecosystem’. Her major research interest deals with the structure and functioning of microbial food webs in lakes; the general objective is to identify the links between the structure of planktonic communities and the functioning of lacustrine pelagic systems. The specific research 123 Hydrobiologia (2011) 659:37–48 questions addressed concern (i) the diversity of microbial eukaryotes, (ii) the trophic interactions and pathways within microbial trophic networks and up to higher trophic levels, and (iii) the regulatory factors involved in the dynamics, structure, diversity and activity of planktonic micro-organisms.