In situ prey selection of mixotrophic and heterotrophic flagellates in Antarctic oligotrophic lakes: An analysis of the digestive vacuole content

We investigated the selective predation of mixotrophic and heterotrophic flagellates (MF and HF) on different heterotrophic prokaryote phylotypes (HPP; Bacteria + Archaea) living in natural assemblages from oligotrophic Antarctic lakes. In situ prey preference was analyzed for the first time on different mixotrophic taxa (Pseudopedinella sp., Ochromonas-like cells, Chrysophyceae >5 μm). The relative abundances of seven different HPP hybridized by CARD-FISH (catalyzed reporter deposition-fluorescent in situ hybridization) in natural community were compared with the proportions of hybridized cells inside digestive vacuoles. Our results showed some general trends to selectivity over some HPP. Alphaproteobacteria and Betaproteobacteria were the most abundant groups, and strikingly, a negative selection trend was detected in most samples by all bacterivorous protists. In contrast, for Actinobacteria a positive selection trend was observed in most samples, whereas Bacteroidetes seemed to be randomly preyed upon. Interestingly, similar prey preferences were observed in all bacterivorous flagellates. Our results suggest that phylogenetic affiliation determines part of the process of prey selection by protists in these lakes. Nevertheless, other features, such as cell size, morphology and the presence of the S-layer, might also significantly contribute to prey selectivity on the HPP. © 2012 The Author 2012. Published by Oxford University Press. All rights reserved.Fil:Queimaliños, C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Schiaffino, M.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Izaguirre, I. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Unrein, F. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

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

Evaluation of Alternative High-Throughput Sequencing Methodologies for the Monitoring of Marine Picoplanktonic Biodiversity Based on rRNA Gene Amplicons

16 pages, 6 figures, 2 tablesSequencing of rRNA gene polymerase chain reaction amplicons (rRNA tags) is the most common approach for investigating microbial diversity. The recent development of high-throughput sequencing (HTS) technologies has enabled the exploration of microbial biodiversity at an unprecedented scale, greatly expanding our knowledge on the microbiomes of marine ecosystems. These approaches provide accurate, fast, and cost efficient observations of the marine communities, and thus, may be suitable tools in biodiversity monitoring programs. To reach this goal, consistent and comparable methodologies must be used over time and within sites. Here, we have performed a cross-platform study of the two most common HTS methodologies, i.e., 454-pyrosequencing and Illumina tags to evaluate their usefulness in biodiversity monitoring and assessment of environmental status. Picoplankton biodiversity has been compared through both methodologies by sequencing the 16 and 18 S rRNA genes of a set of samples collected in the coast of Barcelona (NW Mediterranean). The results show that, despite differences observed in the rare OTUs retrieved, both platforms provide a comparable view of the marine picoplankton communities. On a taxonomic level, there was an accurate overlap in the detected phyla between the two methods and the overall estimates of alpha- and betadiversity were comparable. In addition, we explored the concept of “indicator species” and found that certain taxa (i.e., members of the Gammaproteobacteria among others) as well as the ratio between some phylogenetic groups (i.e., the ratio of Alphaproteobacteria/Gammaproteobacteria, Alteromonas/SAR11, and Alteromonas + Oceanospirillales/SAR11) have potential for being useful indicators of environmental status. The data show that implementing new protocols and identifying indicators of environmental status based on rRNA amplicon sequencing is feasible, and that is worth exploring whether the identified indices are universally applicableThis manuscript is a result of DEVOTES (DEVelopment Of innovative Tools for understanding marine biodiversity and assessing GEnS) project, funded by the European Union (grant agreement no. 308392), and a MINECO Grant GRADIENTS Fine-scale structure of cross-shore GRADIENTS along the Mediterranean coast (CTM2012-39476-C02)Peer Reviewe

Marked changes in diversity and relative activity of picoeukaryotes with depth in the world ocean

Microbial eukaryotes are key components of the ocean plankton. Yet, our understanding of their community composition and activity in different water layers of the ocean is limited, particularly for picoeukaryotes (0.2–3 µm cell size). Here, we examined the picoeukaryotic communities inhabiting different vertical zones of the tropical and subtropical global ocean: surface, deep chlorophyll maximum, mesopelagic (including the deep scattering layer and oxygen minimum zones), and bathypelagic. Communities were analysed by high-tthroughput sequencing of the 18S rRNA gene (V4 region) as represented by DNA (community structure) and RNA (metabolism), followed by delineation of Operational Taxonomic Units (OTUs) at 99% similarity. We found a stratification of the picoeukaryotic communities along the water column, with assemblages corresponding to the sunlit and dark ocean. Specific taxonomic groups either increased (e.g., Chrysophyceae or Bicosoecida) or decreased (e.g., Dinoflagellata or MAST-3) in abundance with depth. We used the rRNA:rDNA ratio of each OTU as a proxy of metabolic activity. The highest relative activity was found in the mesopelagic layer for most taxonomic groups, and the lowest in the bathypelagic. Altogether, we characterize the change in community structure and metabolic activity of picoeukaryotes with depth in the global ocean, suggesting a hotspot of activity in the mesopelagic

Effect of venipuncture site on hematologic and serum biochemical parameters in marginated tortoise (Testudo marginata)

Blood samples were obtained from the dorsal coccygeal vein and the brachial vein of five adult (four females and one male) and two subadult males of marginated tortoise (Testudo marginata) and hematologic and biochemical parameters were compared. Significant differences were found for red blood cell count, hematocrit, hemoglobin concentration, total proteins, uric acid, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, calcium, and phosphorus, which were greater in the brachial vein samples. Hemodilution due to lymph was observed when collecting blood from the dorsal coccygeal vein, and it is thought to be the cause of the differences found. This research documented that the brachial vein is a more reliable and consistent venipuncture site than dorsal coccygeal vein in marginated tortoise

Assessment of microbial plankton diversity as an ecological indicator in the NW Mediterranean coast

High-throughput sequencing of microbial assemblages has been proposed as an alternative methodology to the traditional ones used in marine monitoring and environmental assessment. Here, we evaluated pico- and nanoplankton diversity as ecological indicators in NW Mediterranean coastal waters by comparing their diversity in samples subjected to varying degrees of continental pressures. Using metabarcoding of the 16S and 18S rRNA genes, we explored whether alphadiversity indices, abundance of Operational Taxonomic Units and taxonomic groups (and their ratios) provide information on the ecological quality of coastal waters. Our results revealed that only eukaryotic diversity metrics and a limited number of prokaryotic and eukaryotic taxa displayed potential in assessing continental influences in our surveyed area, resulting thus in a restrained potential of microbial plankton diversity as an ecological indicator. Therefore, incorporating microbial plankton diversity in environmental assessment could not always result in a significant improvement of current marine monitoring strategies.Preprint2,35

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

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