Nematopsis temporariae (Gregarinasina, Apicomplexa, Alveolata) 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 the 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. This article is protected by copyright. All rights reserved.Marie Curie Intra-European and EMBO Long-Term Fellowships . Grant Number: FP7-PEOPLE-2011-IEF-299815-PARAFROGS and ATL-1069-2011. Czech Science Foundation . Grant Number: GBP505/12/G112-ECI

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

This is the final published PDF. Available from BMC via the DOI in this record.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.Marie Curie Intra-European Fellowship grantEMBO Long-Term fellowshipGordon and Betty Moore Foundatio

Virus, bactéries et protistes pathogènes du phytoplancton, le rôle insoupçonné des parasites dans le fonctionnement des écosystèmes aquatiques

International audienceMicroscopiques, les parasites sont la plupart du temps invisibles, mais présents partout. Ils infectent tous les organismes du monde vivant. La dernière décennie a révélé une incroyable diversité chez les parasites viraux, bactériens et eucaryotes. Ceux infectant le phytoplancton pourraient avoir une importance capitale dans la dynamique des populations algales et dans le fonctionnement des écosystèmes aquatiques, mais leur rôle est encore très largement méconnu à ce jour (Brussaard, 2004). Sur ces questions, la recherche ne fait que commencer

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

This is the final version. Available on open access from Elsevier via the DOI in this recordData and Code Availability: All data and code are available with DOI’s given in the methods section. Specifically, physical and chemical parameters of the water column obtained using CTD ocean water sampling are available at http://biomarks.eu/ctd007 (and replicated here at figshare DOI: 10.6084/m9.figshare.9821936). The phylogenetic tree file, masked and unmasked SSU rDNA alignments are available at Zenodo repository: DOI 10.5281/zenodo.2788876. All sequence data used were derived from the NCBI ‘GenBank’ database and accession numbers are provided in Figure. 1B. The R code used to test statistical association in the FISH data are available at Zenodo repository: DOI 10.5281/zenodo.2788876.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, such interactions in the marine environment are poorly understood. One newly identified uncultured marine lineage has been named novel chytrid-like-clade-1 (NCLC1) or basal-clone-group-I. We use ribosomal RNA (rRNA) encoding gene phylogenies to demonstrate that NCLC1 is a distinct branch within the Opisthosporidia (Holomycota). 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. 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. 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. 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.Agence Nationale de la Recherche (ANR)Genome CanadaDonald Hill Family FellowshipRoyal SocietyBiodivERsAGordon and Betty Moore Foundatio

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

Journal ArticleEnvironmental 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.Gordon and Betty Moore FoundationDeutsche ForschungsgemeinschaftMarie Curie International Outgoing FellowshipMarie Curie Intra-European FellowshipEMBO Long-Term fellowshi

Groups without cultured representatives dominate eukaryotic picophytoplankton in the oligotrophic South East Pacific Ocean

Background: Photosynthetic picoeukaryotes (PPE) with a cell size less than 3 µm play a critical role in oceanic primary production. In recent years, the composition of marine picoeukaryote communities has been intensively investigated by molecular approaches, but their photosynthetic fraction remains poorly characterized. This is largely because the classical approach that relies on constructing 18S rRNA gene clone libraries from filtered seawater samples using universal eukaryotic primers is heavily biased toward heterotrophs, especially alveolates and stramenopiles, despite the fact that autotrophic cells in general outnumber heterotrophic ones in the euphotic zone. Methodology/Principal Findings: In order to better assess the composition of the eukaryotic picophytoplankton in the South East Pacific Ocean, encompassing the most oligotrophic oceanic regions on earth, we used a novel approach based on flow cytometry sorting followed by construction of 18S rRNA gene clone libraries. This strategy dramatically increased the recovery of sequences from putative autotrophic groups. The composition of the PPE community appeared highly variable both vertically down the water column and horizontally across the South East Pacific Ocean. In the central gyre, uncultivated lineages dominated: a recently discovered clade of Prasinophyceae (IX), clades of marine Chrysophyceae and Haptophyta, the latter division containing a potentially new class besides Prymnesiophyceae and Pavlophyceae. In contrast, on the edge of the gyre and in the coastal Chilean upwelling, groups with cultivated representatives (Prasinophyceae clade VII and Mamiellales) dominated. Conclusions/Significance: Our data demonstrate that a very large fraction of the eukaryotic picophytoplankton still escapes cultivation. The use of flow cytometry sorting should prove very useful to better characterize specific plankton populations by molecular approaches such as gene cloning or metagenomics, and also to obtain into culture strains representative of these novel groups

Extensive genetic diversity and rapid population differentiation during blooms of Alexandrium fundyense (Dinophyceae) in an isolated salt pond on Cape Cod, MA, USA

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Ecology and Evolution 2 (2012): 2588–2599, doi:10.1002/ece3.373.In Massachusetts, paralytic shellfish poisoning (PSP) is annually recurrent along the coastline, including within several small embayments on Cape Cod. One such system, the Nauset Marsh System (NMS), supports extensive marshes and a thriving shellfishing industry. Over the last decade, PSP in the NMS has grown significantly worse; however, the origins and dynamics of the toxic Alexandrium fundyense (Balech) populations that bloom within the NMS are not well known. This study examined a collection of 412 strains isolated from the NMS and the Gulf of Maine (GOM) in 2006–2007 to investigate the genetic characteristics of localized blooms and assess connectivity with coastal populations. Comparisons of genetic differentiation showed that A. fundyense blooms in the NMS exhibited extensive clonal diversity and were genetically distinct from populations in the GOM. In both project years, genetic differentiation was observed among temporal samples collected from the NMS, sometimes occurring on the order of approximately 7 days. The underlying reasons for temporal differentiation are unknown, but may be due, in part, to life-cycle characteristics unique to the populations in shallow embayments, or possibly driven by selection from parasitism and zooplankton grazing; these results highlight the need to investigate the role of selective forces in the genetic dynamics of bloom populations. The small geographic scale and limited connectivity of NMS salt ponds provide a novel system for investigating regulators of blooms, as well as the influence of selective forces on population structure, all of which are otherwise difficult or impossible to study in the adjacent open-coastal waters or within larger estuaries.This study was funded through the Woods Hole Center for Oceans and Human Health, National Science Foundation OCE-0430724 and National Institutes of Health 1 P50 ES012742-01, and National Science Foundation OCE-0911031. Funding was also provided by NOAA Grant NA06NOS4780245

Dynamics of Alexandrium fundyense blooms and shellfish toxicity in the Nauset Marsh System of Cape Cod (Massachusetts, USA)

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Harmful Algae 12 (2011): 26–38, doi:10.1016/j.hal.2011.08.009.Paralytic Shellfish Poisoning (PSP) toxins are annually recurrent along the Massachusetts coastline (USA), which includes many small embayments and salt ponds. Among these is the Nauset Marsh System (NMS), which has a long history of PSP toxicity. Little is known, however, about the bloom dynamics of the causative organism Alexandrium fundyense within that economically and socially important system. The overall goal of this work was to characterize the distribution and dynamics of A. fundyense blooms within the NMS and adjacent coastal waters by documenting the distribution and abundance of resting cysts and vegetative cells. Cysts were found predominantly in three drowned kettle holes or salt ponds at the distal ends of the NMS - Salt Pond, Mill Pond, and Town Cove. The central region of the NMS had a much lower concentration of cysts. Two types of A. fundyense blooms were observed. One originated entirely within the estuary, seeded by cysts in the three seedbeds. These blooms developed independently of each other and of the A. fundyense population observed in adjacent coastal waters outside the NMS. The temporal development of the blooms was different in the three salt ponds, with initiation differing by as much as 30 days. These differences do not appear to reflect the initial cyst abundances in these locations, and may simply result from higher cell retention and higher nutrient concentrations in Mill Pond, the first site to bloom. Germination of cysts accounted for a small percentage of the peak cell densities in the ponds, so population size was influenced more by the factors affecting growth than by cyst abundance. Subsurface cell aggregation (surface avoidance) limited advection of the vegetative A. fundyense cells out of the salt ponds through the shallow inlet channels. Thus, the upper reaches of the NMS are at the greatest risk for PSP since the highest cyst abundances and cell concentrations were found there. After these localized blooms in the salt ponds peaked and declined, a second, late season bloom occurred within the central portions of the NMS. The timing of this second bloom relative to those within the salt ponds and the coastal circulation patterns at that time strongly suggest that those cells originated from a regional A. fundyense bloom in the Gulf of Maine, delivered to the central marsh from coastal waters outside the NMS through Nauset Inlet. These results will guide policy decisions about water quality as well as shellfish monitoring and utilization within the NMS and highlight the potential for “surgical” closures of shellfish during PSP events, leaving some areas open for harvesting while others are closed.This work was supported by NOAA Grant NA06OAR4170021, NPS Grant H238015504 and by the Woods Hole Center for Oceans and Human Health through NSF Grants OCE-0911031 and OCE-0430724 and NIEHS Grant 1P50-ES01274201. B.G.C. was supported by a Xunta de Galicia Ángeles Alvariño fellowship and the Stanley W. Watson Chair for Excellence in Oceanography under a Postdoctoral program at the Woods Hole Oceanographic Institution

Reproductive plasticity and local adaptation in the host-parasite system formed by the toxic Alexandrium minutum and teh dinoflagellate parasite Parvilucifera sinerae

A parasite threat stimulates adaptive shifts in the life-history strategy (sexual recombination rate) of the toxic bloom-forming dinoflagellate Alexandrium minutum Halim. This microalgae divides asexually when clonal but can also form mobile zygotes (planozygotes) when compatible clones are crossed. Planozygotes usually form resistant dormant stages (resting cysts) although they can also divide. In this study, asexual and sexual cultures were infected with the parasite Parvilucifera sinerae (Perkinsozoa) and the resulting clones classified as susceptible (S), low susceptible (LS), or resistant (R) to the infection. R and LS clones were never of Mediterranean origin, pointing to local adaptation of the parasite. (S × S) crosses were infected faster than either of the parental clones growing asexually. By contrast, (S × R) crosses were resistant to the parasite and produced no resting cysts, even when planozygotes were formed. Therefore, in infected cultures, the planozygotes mainly divided instead of encysting, thus increasing the rate at which recombinant progeny formed. This strategy against infection seems to combine the benefits of quickly producing asexual offspring and increasing recombination. As the susceptibility of the crosses was dependent on parental sexual compatibility, and cultures established by the division of (R × S) planozygotes (F1 offspring) also formed R or LS cultures, resistance may be regulated by several genes or through maternal effects.Preprin

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

International audienceAlthough 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