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

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

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

New Insights into the Diversity of Marine Picoeukaryotes

Over the last decade, culture-independent surveys of marine picoeukaryotic diversity based on 18S ribosomal DNA clone libraries have unveiled numerous sequences of novel high-rank taxa. This newfound diversity has significantly altered our understanding of marine microbial food webs and the evolution of eukaryotes. However, the current picture of marine eukaryotic biodiversity may be significantly skewed by PCR amplification biases, occurrence of rDNA genes in multiple copies within a single cell, and the capacity of DNA to persist as extracellular material. In this study we performed an analysis of the metagenomic dataset from the Global Ocean Survey (GOS) expedition, seeking eukaryotic ribosomal signatures. This PCR-free approach revealed similar phylogenetic patterns to clone library surveys, suggesting that PCR steps do not impose major biases in the exploration of environmental DNA. The different cell size fractions within the GOS dataset, however, displayed a distinct picture. High protistan diversity in the <0.8 µm size fraction, in particular sequences from radiolarians and ciliates (and their absence in the 0.8–3 µm fraction), suggest that most of the DNA in this fraction comes from extracellular material from larger cells. In addition, we compared the phylogenetic patterns from rDNA and reverse transcribed rRNA 18S clone libraries from the same sample harvested in the Mediterranean Sea. The libraries revealed major differences, with taxa such as pelagophytes or picobiliphytes only detected in the 18S rRNA library. MAST (Marine Stramenopiles) appeared as potentially prominent grazers and we observed a significant decrease in the contribution of alveolate and radiolarian sequences, which overwhelmingly dominated rDNA libraries. The rRNA approach appears to be less affected by taxon-specific rDNA copy number and likely better depicts the biogeochemical significance of marine protists

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 appear 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 USA, 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 rDNA sequencing, we developed a targeted PCR protocol for preferentially sampling a clade of the Perkinsea. We test 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. This provides the first evidence that Perkinsea-like protists infect tadpoles across a wide taxonomic range of frogs in tropical and temperate environments, including oceanic islands.Linnean Society of LondonSystematics Association UKMarie Curie Intra-European Fellowship GrantEuropean Molecular Biology OrganizationGordon and Betty Moore FoundationNERCLeverhume TrustBBSRCCanadian Institute for Advanced ResearchCzech Science Foundation GrantInstitute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republi