Global perspective of environmental distribution and diversity of Perkinsea (Alveolata) explored by a meta-analysis of eDNA surveys.

Perkinsea constitutes a lineage within the Alveolata eukaryotic superphylum, mainly composed of parasitic organisms. Some described species represent significant ecological and economic threats due to their invasive ability and pathogenicity, which can lead to mortality events. However, the genetic diversity of these described species is just the tip of the iceberg. Environmental surveys targeting this lineage are still scarce and mainly limited to the Northern Hemisphere. Here, we aim to conduct an in depth exploration of the Perkinsea group, uncovering the diversity across a variety of environments, including those beyond freshwater and marine ecosystems. We seek to identify and describe putative novel organisms based on their genetic signatures. In this study, we conducted an extensive analysis of a metabarcoding dataset, focusing on the V4 region of the 18S rRNA gene (the EukBank dataset), to investigate the diversity, distribution and environmental preferences of the Perkinsea. Our results reveal a remarkable diversity within the Perkinsea, with 1568 Amplicon Sequence Variants (ASVs) identified across thousands of environmental samples. Surprisingly, we showed a substantial diversity of Perkinsea within soil samples (269 ASVs), challenging the previous assumption that this group is confined to marine and freshwater environments. In addition, we revealed that a notable proportion of Perkinsea ASVs (428 ASVs) could correspond to putative new organisms, encompassing the well-established taxonomic group Perkinsidae. Finally, our study shed light on previously unveiled taxonomic groups, including the Xcellidae, and revealed their environmental distribution. These findings demonstrate that Perkinsea exhibits far greater diversity than previously detected and surprisingly extends beyond marine and freshwater environments. The meta-analysis conducted in this study has unveiled the existence of previously unknown clusters within the Perkinsea lineage, solely identified based on their genetic signatures. Considering the ecological and economic importance of described Perkinsea species, these results suggest that Perkinsea may play a significant, yet previously unrecognized, role across a wide range of environments, spanning from soil environments to the abyssal zone of the open ocean with important implications for ecosystem functioning.

Co-infection of two eukaryotic pathogens within clam populations in Arcachon Bay.

The parasitic species Perkinsus olseni (= atlanticus) (Perkinsea, Alveolata) infects a wide range of mollusc species and is responsible for mortality events and economic losses in the aquaculture industry and fisheries worldwide. Thus far, most studies conducted in this field have approached the problem from a "one parasite-one disease" perspective, notably with regards to commercially relevant clam species, while the impact of other Perkinsus species should also be considered as it could play a key role in the disease phenotype and dynamics. Co-infection of P. olseni and P. chesapeaki has already been sporadically described in Manila clam populations in Europe. Here, we describe for the first time the parasitic distribution of two Perkinsus species, P. olseni and P. chesapeaki, in individual clam organs and in five different locations across Arcachon Bay (France), using simultaneous in situ detection by quantitative PCR (qPCR) duplex methodology. We show that P. olseni single-infection largely dominated prevalence (46-84%) with high intensities of infection (7.2 to 8.5 log-nb of copies. g-1of wet tissue of Manila clam) depending on location, suggesting that infection is driven by the abiotic characteristics of stations and physiological states of the host. Conversely, single P. chesapeaki infections were observed in only two sampling stations, Ile aux Oiseaux and Gujan, with low prevalences 2 and 14%, respectively. Interestingly, the co-infection by both Perkinsus spp., ranging in prevalence from 12 to 34%, was distributed across four stations of Arcachon Bay, and was detected in one or two organs maximum. Within these co-infected organs, P. olseni largely dominated the global parasitic load. Hence, the co-infection dynamics between P. olseni and P. chesapeaki may rely on a facilitating role of P. olseni in developing a primary infection which in turn may help P. chesapeaki infect R. philippinarum as a reservoir for a preferred host. This ecological study demonstrates that the detection and quantification of both parasitic species, P. olseni and P. chesapeaki, is essential and timely in resolving cryptic infections and their consequences on individual hosts and clam populations.

Freshwater protists: unveiling the unexplored in a large floodplain system.

Protists play a fundamental role in all ecosystems, but we are still far from estimating the total diversity of many lineages, in particular in highly diverse environments, such as freshwater. Here, we survey the protist diversity of the Paraná River using metabarcoding, and we applied an approach that includes sequence similarity and phylogeny to evaluate the degree of genetic novelty of the protists' communities against the sequences described in the reference database PR2 . We observed that ~28% of the amplicon sequence variants were classified as novel according to their similarity with sequences from the reference database; most of them were related to heterotrophic groups traditionally overlooked in freshwater systems. This lack of knowledge extended to those groups within the green algae (Archaeplastida) that are well documented such as Mamiellophyceae, and also to the less studied Pedinophyceae, for which we found sequences representing novel deep-branching clusters. Among the groups with potential novel protists, Bicosoecida (Stramenopiles) were the best represented, followed by Codosiga (Opisthokonta), and the Perkinsea (Alveolata). This illustrates the lack of knowledge on freshwater planktonic protists and also the need for isolation and/or cultivation of new organisms to better understand their role in ecosystem functioning.

New Perkinsea Parasitoids of Dinoflagellates Distantly Related to Parviluciferaceae Members.

Perkinsea is a phylogenetic group of protists that includes parasites of distantly related hosts. However, its diversity is still mainly composed of environmental sequences, mostly obtained from freshwater environments. Efforts to isolate and culture parasitoids of dinoflagellates have led to the description of several phylogenetically closely related species constituting the Parviluciferaceae family. In this study, two new parasitoid species infecting dinoflagellates during recurrent coastal blooms are reported. Using the ribosomal RNA (rRNA) gene phylogenies, we show that both cluster within Perkinsea, one of them at the base of Parviluciferaceae and the other in a distinct branch unrelated to other described species. The establishment of host-parasite lab cultures of the latter allowed its morphological characterization, resulting in the formal description of Maranthos nigrum gen. nov., sp. nov. The life-cycle development of the two parasitoids is generally the same as that of other members of the Parviluciferaceae family but they differ in the features of the trophont and sporont stages, including the arrangement of zoospores during the mature sporangium stage and the lack of specialized structures that release the zoospores into the environment. Laboratory cross-infection experiments showed that the parasitoid host range is restricted to dinoflagellates, although it extends across several different genera. The maximum prevalence reached in the tested host populations was lower than in other Parviluciferaceae members. The findings from this study suggest that Perkinsea representatives infecting dinoflagellates are more widespread than previously thought.

Expanded host and geographic range of tadpole associations with the Severe Perkinsea Infection group.

Severe Perkinsea infection is an emerging disease of amphibians, specifically tadpoles. Disease presentation correlates with liver infections of a subclade of Perkinsea (Alveolata) protists, named Pathogenic Perkinsea Clade (PPC). Tadpole mortality events associated with PPC infections have been reported across North America, from Alaska to Florida. Here, we investigate the geographic and host range of PPC associations in seemingly healthy tadpoles sampled from Panama, a biogeographic provenance critically affected by amphibian decline. To complement this work, we also investigate a mortality event among Hyla arborea tadpoles in captive-bred UK specimens. PPC SSU rDNA was detected in 10 of 81 Panama tadpoles tested, and H. arborea tadpoles from the UK. Phylogenies of the Perkinsea SSU rDNA sequences demonstrate they are highly similar to PPC sequences sampled from mortality events in the USA, and phylogenetic analysis of tadpole mitochondrial SSU rDNA demonstrates, for the first time, PPC associations in diverse hylids. These data provide further understanding of the biogeography and host range of this putative pathogenic group, factors likely to be important for conservation planning.

Development of duplex TaqMan-based real-time PCR assay for the simultaneous detection of Perkinsus olseni and P. chesapeaki in host Manila clam tissue samples.

The aetiological agent Perkinsus olseni is globally recognised as a major threat for shellfish production considering its wide geographical distribution across Asia, Europe, Australia and South America. Another species, Perkinsus chesapeaki, which has never been known to be associated with significant mortality events, was recently detected along French coasts infecting clam populations sporadically in association with P. olseni. Identifying potential cryptic infections affecting Ruditapes philippinarum is essential to develop appropriate host resource management strategies. Here, we developed a molecular method based on duplex real-time quantitative PCR for the simultaneous detection of these two parasites, P. olseni and P. chesapeaki, in the different clam tissues: gills, digestive gland, foot, mantle, adductor muscle and the rest of the soft body. We firstly checked the presence of possible PCR inhibitors in host tissue samples. The qPCR reactions were inhibited depending on the nature of the host organ. The mantle and the rest of the soft body have a high inhibitory effect from threshold of host gDNA concentration of 2 ng.µL-1, the adductor muscle and the foot have an intermediate inhibition of 5 ng.µL-1, and the gills and digestive gland do not show any inhibition of the qPCR reaction even at the highest host gDNA concentration of 20 ng.µL-1. Then, using the gills as a template, the suitability of the molecular technique was checked in comparison with the Ray's Fluid Thioglycolate Medium methodology recommended by the World Organisation for Animal Health. The duplex qPCR method brought new insights and unveiled cryptic infections as the co-occurrence of P. olseni and P. chesapeaki from in situ tissue samples in contrast to the RFTM diagnosis. The development of this duplex qPCR method is a fundamental work to monitor in situ co-infections that will lead to optimised resource management and conservation strategies to deal with emerging diseases.

A novel duplex qPCR assay for stepwise detection of multiple Perkinsea protistan infections of amphibian tissues.

Alveolate protists within the phylum Perkinsea have been found to infect amphibians across a broad taxonomic and geographic range. Phylogenetic analysis has suggested the existence of two clades of amphibian Perkinsea: a putatively non-pathogenic clade linked to asymptomatic infections of tadpoles in Africa, Europe and South America, and a putatively pathogenic clade linked to disease and mass mortality events of tadpoles in North America. Here, we describe the development of a duplex TaqMan qPCR assay to detect and discriminate between rDNA sequences from both clades of Perkinsea in amphibian tissues. The assay uses a single primer pair to target an 18S small subunit (SSU) ribosomal RNA (rRNA) gene region shared between the two clades, and two dual-labelled probes to target a region within this fragment that is diagnostic for each clade. This assay enables rapid screening for each of the two Perkinsea groups, allowing for detection, primarily of the phylogenetic group associated with disease outbreaks, and secondarily for the phylogenetic group with no current disease relationship identified. Incorporation of our novel qPCR assay into the routine surveillance of amphibian populations will allow for the assessment of the incidence of each protist clade, thereby providing an improved understanding of Perkinsea infection pervasiveness and a method to underpin future conservation planning.

Emerging Parasitic Protists: The Case of Perkinsea.

The last century has witnessed an increasing rate of new disease emergence across the world leading to permanent loss of biodiversity. Perkinsea is a microeukaryotic parasitic phylum composed of four main lineages of parasitic protists with broad host ranges. Some of them represent major ecological and economical threats because of their geographically invasive ability and pathogenicity (leading to mortality events). In marine environments, three lineages are currently described, the Parviluciferaceae, the Perkinsidae, and the Xcellidae, infecting, respectively, dinoflagellates, mollusks, and fish. In contrast, only one lineage is officially described in freshwater environments: the severe Perkinsea infectious agent infecting frog tadpoles. The advent of high-throughput sequencing methods, mainly based on 18S rRNA assays, showed that Perkinsea is far more diverse than the previously four described lineages especially in freshwater environments. Indeed, some lineages could be parasites of green microalgae, but a formal nature of the interaction needs to be explored. Hence, to date, most of the newly described aquatic clusters are only defined by their environmental sequences and are still not (yet) associated with any host. The unveiling of this microbial black box presents a multitude of research challenges to understand their ecological roles and ultimately to prevent their most negative impacts. This review summarizes the biological and ecological traits of Perkinsea-their diversity, life cycle, host preferences, pathogenicity, and highlights their diversity and ubiquity in association with a wide range of hosts.

Intracellular Infection of Diverse Diatoms by an Evolutionary Distinct Relative of the Fungi.

The Fungi are a diverse kingdom, dominating terrestrial environments and driving important ecologies. Although fungi, and the related Opisthosporidia, interact with photosynthetic organisms on land and in freshwater as parasites, symbionts, and/or saprotrophic degraders [1, 2], such interactions in the marine environment are poorly understood [3-8]. One newly identified uncultured marine lineage has been named novel chytrid-like-clade-1 (NCLC1) [4] or basal-clone-group-I [5, 6]. We use ribosomal RNA (rRNA) encoding gene phylogenies to demonstrate that NCLC1 is a distinct branch within the Opisthosporidia (Holomycota) [7]. Opisthosporidia are a diverse and largely uncultured group that form a sister branch to the Fungi or, alternatively, the deepest branch within the Fungi, depending on how the boundary to this kingdom is inferred [9]. Using culture-free lineage-specific rRNA-targeted fluorescent in situ hybridization (FISH) microscopy, we demonstrate that NCLC1 cells form intracellular infection of key diatom species, establishing that intracellular colonization of a eukaryotic host is a consistent lifestyle across the Opisthosporidia [8-11]. NCLC1 infection-associated loss and/or envelopment of the diatom nuclei infers a necrotrophic-pathogenic interaction. Diatoms are one of the most diverse and ecologically important phytoplankton groups, acting as dominant primary producers and driving carbon fixation and storage in many aquatic environments [12-14]. Our results provide insight into the diversity of microbial eukaryotes that interact with diatoms. We suggest that such interactions can play a key role in diatom associated ecosystem functions, such as the marine carbon pump through necrotrophic-parasitism, facilitating the export of diatoms to the sediment [15, 16].

Pathology and Case Definition of Severe Perkinsea Infection of Frogs.

Severe Perkinsea infection (SPI) is an emerging disease of frogs responsible for mass mortalities of tadpoles across the United States. It is caused by protozoa belonging to the phylum Perkinsozoa that form a distinct group referred to as the Pathogenic Perkinsea Clade of frogs. In this work, we provide detailed description of gross and histologic lesions from 178 naturally infected tadpoles, including 10 species from 22 mortality events and 6 amphibian health monitoring studies from diverse geographic areas. On external examination, we observed abdominal distension (10, 5.6%), cutaneous erythema and petechia (3, 1.7%), subcutaneous edema (3, 1.7%), and areas of white skin discoloration (3, 1.7%). On macroscopic examination of internal organs, we found hepatomegaly (68, 38.2%), splenomegaly (51, 28.7%), nephromegaly (47, 26.4%), ascites (15, 8.4%), segmental irregular thickening and white discoloration of the intestine (8, 4.5%), pancreatomegaly (4, 2.2%), and pancreatic petechia (1, 0.6%). Histologically, over 60% of the liver (148/165, 89.7%), kidney (113/147, 76.9%), spleen (96/97, 99%), and pancreas (46/68, 67.6%) were invaded by myriad intracellular and extracellular Perkinsea hypnospore-like and trophozoite-like organisms. Numerous other tissues were affected to a lesser extent. Mild histiocytic inflammation with fewer lymphocytes or eosinophils was commonly observed in areas of infection that were not obscured by lympho-granulocytic hematopoietic tissue. In light of these observations, we suggest a logical pathogenesis sequence. Finally, we propose a "case definition" for SPI to promote standardized communication of results and prevent misdiagnosis with epidemiological and pathologically overlapping diseases such as ranavirosis.

Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton.

Phytoplankton community structure is shaped by both bottom-up factors, such as nutrient availability, and top-down processes, such as predation. Here we show that marine viruses can blur these distinctions, being able to amend how host cells acquire nutrients from their environment while also predating and lysing their algal hosts. Viral genomes often encode genes derived from their host. These genes may allow the virus to manipulate host metabolism to improve viral fitness. We identify in the genome of a phytoplankton virus, which infects the small green alga Ostreococcus tauri, a host-derived ammonium transporter. This gene is transcribed during infection and when expressed in yeast mutants the viral protein is located to the plasma membrane and rescues growth when cultured with ammonium as the sole nitrogen source. We also show that viral infection alters the nature of nitrogen compound uptake of host cells, by both increasing substrate affinity and allowing the host to access diverse nitrogen sources. This is important because the availability of nitrogen often limits phytoplankton growth. Collectively, these data show that a virus can acquire genes encoding nutrient transporters from a host genome and that expression of the viral gene can alter the nutrient uptake behavior of host cells. These results have implications for understanding how viruses manipulate the physiology and ecology of phytoplankton, influence marine nutrient cycles, and act as vectors for horizontal gene transfer.

Ultrastructure of Selenidium pendula, the Type Species of Archigregarines, and Phylogenetic Relations to Other Marine Apicomplexa.

Archigregarines, an early branching lineage within Apicomplexa, are a poorly-known group of invertebrate parasites. By their phylogenetic position, archigregarines are an important lineage to understand the functional transition that occurred between free-living flagellated predators to obligatory parasites in Apicomplexa. In this study, we provide new ultrastructural data and phylogenies based on SSU rDNA sequences using the type species of archigregarines, the Selenidiidae Selenidium pendulaGiard, 1884. We describe for the first time the syzygy and early gamogony at the ultrastructural level, revealing a characteristic nuclear multiplication with centrocones, cryptomitosis, filamentous network of chromatin, a cyst wall secretion and a 9+0 flagellar axoneme of the male gamete. S. pendula belongs to a monophyletic lineage that includes several other related species, all infecting Sedentaria Polychaeta (Spionidae, Sabellaridae, Sabellidae and Cirratulidae). All of these Selenidium species exhibit similar biological characters: a cell cortex with the plasma membrane - inner membrane complex - subpellicular microtubule sets, an apical complex with the conoid, numerous rhoptries and micronemes, a myzocytosis with large food vacuoles, a nuclear multiplication during syzygy and young gamonts. Two other distantly related Selenidium-like lineages infect Terebellidae and Sipunculida, underlying the ability of archigregarines to parasite a wide range of marine hosts.

Nematopsis temporariae (Gregarinasina, Apicomplexa, Alveolata) is an intracellular infectious agent of tadpole livers.

Amphibians are in decline as a result of habitat destruction, climate change and infectious diseases. Tadpoles are thought susceptible to infections because they are dependent on only an innate immune system (e.g. macrophages). This is because the frog adaptive immune system does not function until later stages of their life cycle. In 1920, Nöller described a putative infectious agent of tadpoles named Nematopsis temporariae, which he putatively assigned to gregarine protists (Apicomplexa). Here, we identify a gregarine infection of tadpoles using both microscopy and ribosomal DNA sequencing of three different frog species (Rana temporaria, R. dalmatina, and Hyla arborea). We show that this protist lineage belongs to the subclass Gregarinasina Dufour 1828 and is regularly present in macrophages located in liver sinusoids of tadpoles, confirming the only known case of a gregarine infection of a vertebrate.

A role for fungi as parasites in the black box of marine trophic interactions.

The role of microbial parasites in aquatic environments remains under-investigated and likely under-estimated limiting our understanding of trophic interactions and biogeochemical cycles (Lefèvre et al., ; Worden et al., ). All natural interactions between microbes are difficult to capture and study but those occurring in the open ocean more so, because the scale and the complexity of the system makes repeat sampling a challenge. Lepère and colleagues, by combining a range of cell recovery and detection techniques, have identified parasitic interactions between putative fungi and eukaryotic algae (Lepère et al., ) allowing us to peer into the black box of microbial parasitism in the marine water column.

Molecular diversity and distribution of marine fungi across 130 European environmental samples.

Environmental DNA and culture-based analyses have suggested that fungi are present in low diversity and in low abundance in many marine environments, especially in the upper water column. Here, we use a dual approach involving high-throughput diversity tag sequencing from both DNA and RNA templates and fluorescent cell counts to evaluate the diversity and relative abundance of fungi across marine samples taken from six European near-shore sites. We removed very rare fungal operational taxonomic units (OTUs) selecting only OTUs recovered from multiple samples for a detailed analysis. This approach identified a set of 71 fungal 'OTU clusters' that account for 66% of all the sequences assigned to the Fungi. Phylogenetic analyses demonstrated that this diversity includes a significant number of chytrid-like lineages that had not been previously described, indicating that the marine environment encompasses a number of zoosporic fungi that are new to taxonomic inventories. Using the sequence datasets, we identified cases where fungal OTUs were sampled across multiple geographical sites and between different sampling depths. This was especially clear in one relatively abundant and diverse phylogroup tentatively named Novel Chytrid-Like-Clade 1 (NCLC1). For comparison, a subset of the water column samples was also investigated using fluorescent microscopy to examine the abundance of eukaryotes with chitin cell walls. Comparisons of relative abundance of RNA-derived fungal tag sequences and chitin cell-wall counts demonstrate that fungi constitute a low fraction of the eukaryotic community in these water column samples. Taken together, these results demonstrate the phylogenetic position and environmental distribution of 71 lineages, improving our understanding of the diversity and abundance of fungi in marine environments.

Cryptic infection of a broad taxonomic and geographic diversity of tadpoles by Perkinsea protists.

The decline of amphibian populations, particularly frogs, is often cited as an example in support of the claim that Earth is undergoing its sixth mass extinction event. Amphibians seem to be particularly sensitive to emerging diseases (e.g., fungal and viral pathogens), yet the diversity and geographic distribution of infectious agents are only starting to be investigated. Recent work has linked a previously undescribed protist with mass-mortality events in the United States, in which infected frog tadpoles have an abnormally enlarged yellowish liver filled with protist cells of a presumed parasite. Phylogenetic analyses revealed that this infectious agent was affiliated with the Perkinsea: a parasitic group within the alveolates exemplified by Perkinsus sp., a "marine" protist responsible for mass-mortality events in commercial shellfish populations. Using small subunit (SSU) ribosomal DNA (rDNA) sequencing, we developed a targeted PCR protocol for preferentially sampling a clade of the Perkinsea. We tested this protocol on freshwater environmental DNA, revealing a wide diversity of Perkinsea lineages in these environments. Then, we used the same protocol to test for Perkinsea-like lineages in livers of 182 tadpoles from multiple families of frogs. We identified a distinct Perkinsea clade, encompassing a low level of SSU rDNA variation different from the lineage previously associated with tadpole mass-mortality events. Members of this clade were present in 38 tadpoles sampled from 14 distinct genera/phylogroups, from five countries across three continents. These data provide, to our knowledge, the first evidence that Perkinsea-like protists infect tadpoles across a wide taxonomic range of frogs in tropical and temperate environments, including oceanic islands.

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.

Diverse molecular signatures for ribosomally 'active' Perkinsea in marine sediments.

BACKGROUND: Perkinsea are a parasitic lineage within the eukaryotic superphylum Alveolata. Recent studies making use of environmental small sub-unit ribosomal RNA gene (SSU rDNA) sequencing methodologies have detected a significant diversity and abundance of Perkinsea-like phylotypes in freshwater environments. In contrast only a few Perkinsea environmental sequences have been retrieved from marine samples and only two groups of Perkinsea have been cultured and morphologically described and these are parasites of marine molluscs or marine protists. These two marine groups form separate and distantly related phylogenetic clusters, composed of closely related lineages on SSU rDNA trees. Here, we test the hypothesis that Perkinsea are a hitherto under-sampled group in marine environments. Using 454 diversity 'tag' sequencing we investigate the diversity and distribution of these protists in marine sediments and water column samples taken from the Deep Chlorophyll Maximum (DCM) and sub-surface using both DNA and RNA as the source template and sampling four European offshore locations. RESULTS: We detected the presence of 265 sequences branching with known Perkinsea, the majority of them recovered from marine sediments. Moreover, 27% of these sequences were sampled from RNA derived cDNA libraries. Phylogenetic analyses classify a large proportion of these sequences into 38 cluster groups (including 30 novel marine cluster groups), which share less than 97% sequence similarity suggesting this diversity encompasses a range of biologically and ecologically distinct organisms. CONCLUSIONS: These results demonstrate that the Perkinsea lineage is considerably more diverse than previously detected in marine environments. This wide diversity of Perkinsea-like protists is largely retrieved in marine sediment with a significant proportion detected in RNA derived libraries suggesting this diversity represents ribosomally 'active' and intact cells. Given the phylogenetic range of hosts infected by known Perkinsea parasites, these data suggest that Perkinsea either play a significant but hitherto unrecognized role as parasites in marine sediments and/or members of this group are present in the marine sediment possibly as part of the 'seed bank' microbial community.

Genetic diversity of Amoebophryidae (Syndiniales) during Alexandrium catenella/tamarense (Dinophyceae) blooms in the Thau lagoon (Mediterranean Sea, France).

During toxic spring and fall blooms produced by the dinoflagellate Alexandrium in the Thau lagoon (Mediterranean Sea), we monitored the presence of Amoebophryidae (Syndiniales), a group of parasites virulent toward a wide range of dinoflagellate hosts. A PCR-biased approach unveiled the presence of at least 10 different parasitic groups during Alexandrium proliferation. However, fluorescent in situ hybridization failed to reveal parasitic infection inside Alexandrium cells in field populations. In contrast, several co-occurring, less abundant thecate dinoflagellate species were infected by Amoebophryidae, showing up to 10% of infected cells. We concluded that Alexandrium populations were not infected by these local parasites, at least during our survey. In order to check this resistance capacity on a more global scale, we cross-infected several Alexandrium strains isolated from the Thau lagoon with one strain of the parasite Amoebophrya sp. originating from Salt Pond, MA, USA. All of these hosts were strongly infected by the North American parasite, leading to the conclusion that blooming Alexandrium in the Thau lagoon were not particularly resistant to this kind of parasite. These results provide additional evidence that dinoflagellates may become invasive when they successfully escaped their natural enemies in time and/or space (the "enemy release" hypothesis).