John R. Dolan

Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir

ABSTRACT Bacterioplankton from a meso-eutrophic dam reservoir was size fractionated to reduce (<0.8-μm treatment) or enhance (<5-μm treatment) protistan grazing and then incubated in situ for 96 h in dialysis bags. Time course samples were taken from the bags and the reservoir to estimate bacterial abundance, mean cell volume, production, protistan grazing, viral abundance, and frequency of visibly infected cells. Shifts in bacterial community composition (BCC) were examined by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rDNA genes from the different treatments, and fluorescence in situ hybridization (FISH) with previously employed and newly designed oligonucleotide probes. Changes in bacterioplankton characteristics were clearly linked to changes in mortality rates. In the reservoir, where bacterial production about equaled protist grazing and viral mortality, community characteristics were nearly invariant. In the “grazer-free” (0.8-μm-filtered) treatment, subject only to a relatively low mortality rate (∼17% day−1) from viral lysis, bacteria increased markedly in concentration. While the mean bacterial cell volume was invariant, DGGE indicated a shift in BCC and FISH revealed an increase in the proportion of one lineage within the beta proteobacteria. In the grazing-enhanced treatment (5-μm filtrate), grazing mortality was ∼200% and viral lysis resulted in mortality of 30% of daily production. Cell concentrations declined, and grazing-resistant flocs and filaments eventually dominated the biomass, together accounting for >80% of the total bacteria by the end of the experiment. Once again, BCC changed strongly and a significant fraction of the large filaments was detected using a FISH probe targeted to members of the Flectobacillus lineage. Shifts of BCC were also reflected in DGGE patterns and in the increases in the relative importance of both beta proteobacteria and members of the Cytophaga-Flavobacterium cluster, which consistently formed different parts of the bacterial flocs. Viral concentrations and frequencies of infected cells were highly significantly correlated with grazing rates, suggesting that protistan grazing may stimulate viral activity.

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote Small Sub-Unit rRNA sequences with curated taxonomy

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR2, http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.

Patterns of Rare and Abundant Marine Microbial Eukaryotes

BACKGROUND Biological communities are normally composed of a few abundant and many rare species. This pattern is particularly prominent in microbial communities, in which most constituent taxa are usually extremely rare. Although abundant and rare subcommunities may present intrinsic characteristics that could be crucial for understanding community dynamics and ecosystem functioning, microbiologists normally do not differentiate between them. Here, we investigate abundant and rare subcommunities of marine microbial eukaryotes, a crucial group of organisms that remains among the least-explored biodiversity components of the biosphere. We surveyed surface waters of six separate coastal locations in Europe, independently considering the picoplankton, nanoplankton, and microplankton/mesoplankton organismal size fractions. RESULTS Deep Illumina sequencing of the 18S rRNA indicated that the abundant regional community was mostly structured by organismal size fraction, whereas the rare regional community was mainly structured by geographic origin. However, some abundant and rare taxa presented similar biogeography, pointing to spatiotemporal structure in the rare microeukaryote biosphere. Abundant and rare subcommunities presented regular proportions across samples, indicating similar species-abundance distributions despite taxonomic compositional variation. Several taxa were abundant in one location and rare in other locations, suggesting large oscillations in abundance. The substantial amount of metabolically active lineages found in the rare biosphere suggests that this subcommunity constitutes a diversity reservoir that can respond rapidly to environmental change. CONCLUSIONS We propose that marine planktonic microeukaryote assemblages incorporate dynamic and metabolically active abundant and rare subcommunities, with contrasting structuring patterns but fairly regular proportions, across space and time.

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.

Viral activity in two contrasting lake ecosystems

ABSTRACT For aquatic systems, especially freshwaters, there is little data on the long-term (i.e., >6-month period) and depth-related variability of viruses. In this study, we examined virus-induced mortality of heterotrophic bacteria over a 10-month period and throughout the water column in two lakes of the French Massif Central, the oligomesotrophic Lake Pavin and the eutrophic Lake Aydat. Concurrently, we estimated nonviral mortality through heterotrophic nanoflagellate and ciliate bacterivory. Overall, viral infection parameters were much less variable than bacterial production. We found that the frequency of visibly infected cells (FVIC), estimated using transmission electron microscopy, peaked in both lakes at the end of spring (May to June) and in early autumn (September to October). FVIC values were significantly higher in Lake Pavin (mean [M] = 1.6%) than in Lake Aydat (M = 1.1%), whereas the opposite trend was observed for burst sizes, which averaged 25.7 and 30.2 virus particles bacterium−1, respectively. We detected no significant depth-related differences in FVIC or burst size. We found that in both lakes the removal of bacterial production by flagellate grazing (MPavin = 37.7%, MAydat = 18.5%) was nearly always more than the production removed by viral lysis (MPavin = 16.2%, MAydat = 19%) or ciliate grazing (MPavin = 2.7%, MAydat = 8.8%). However, at specific times and locations, viral lysis prevailed over protistan grazing, for example, in the anoxic hypolimnion of Lake Aydat. In addition, viral mortality represented a relatively constant mortality source in a bacterial community showing large variations in growth rate and subject to large variations in loss rates from grazers. Finally, although viruses did not represent the main agent of bacterial mortality, our data seem to show that their relative importance was higher in the less productive system.

Accuracy of protist diversity assessments: morphology compared with cloning and direct pyrosequencing of 18S rRNA genes and ITS regions using the conspicuous tintinnid ciliates as a case study

Deep-sequencing technologies are becoming nearly routine to describe microbial community composition in environmental samples. The 18S ribosomal DNA (rDNA) pyrosequencing has revealed a vast diversity of infrequent sequences, leading to the proposition of the existence of an extremely diverse microbial ‘rare biosphere’. Although rare microbes no doubt exist, critical views suggest that many rare sequences may actually be artifacts. However, information about how diversity revealed by molecular methods relates to that revealed by classical morphology approaches is practically nonexistent. To address this issue, we used different approaches to assess the diversity of tintinnid ciliates, a species-rich group in which species can be easily distinguished morphologically. We studied two Mediterranean marine samples with different patterns of tintinnid diversity. We estimated tintinnid diversity in these samples employing morphological observations and both classical cloning and sequencing and pyrosequencing of two different markers, the 18S rDNA and the internal transcribed spacer (ITS) regions, applying a variety of computational approaches currently used to analyze pyrosequence reads. We found that both molecular approaches were efficient in detecting the tintinnid species observed by microscopy and revealed similar phylogenetic structures of the tintinnid community at the species level. However, depending on the method used to analyze the pyrosequencing results, we observed discrepancies with the morphology-based assessments up to several orders of magnitude. In several cases, the inferred number of operational taxonomic units (OTUs) largely exceeded the total number of tintinnid cells in the samples. Such inflation of the OTU numbers corresponded to ‘rare biosphere’ taxa, composed largely of artifacts. Our results suggest that a careful and rigorous analysis of pyrosequencing data sets, including data denoising and sequence clustering with well-adjusted parameters, is necessary to accurately describe microbial biodiversity using this molecular approach.

Microzooplankton diversity: relationships of tintinnid ciliates with resources, competitors and predators from the Atlantic Coast of Morocco to the Eastern Mediterranean

We examined tintinnid (loricate ciliate microzooplankton) diversity using data from 11 stations between the Moroccan upwelling system and the oligotrophic Eastern Mediterranean. Taxonomic and morphological diversity of tintinnids was compared to phytoplankton distribution and size-structure, to the abundance of competitors in the form of oligotrich ciliates, and predators as copepods. Tintinnid taxonomic diversity was estimated as numbers of species and the Shannon Index, H 0 ; morphological diversity was quantified by substituting size classes of lorica dimensions for species. Total chlorophyll was partitioned into micro-, nano- and pico-fractions using pigment data and a size-diversity was estimated by considering the 3 size classes as 3 species. Along a west-to-east gradient, average water column concentrations of most organism groups declined approximately an order of magnitude yielding tight correlations. However, tintinnid diversity, both taxonomic and morphological, increased from the Atlantic upwelling station into the western basin of the Mediterranean, and declined slightly towards the Eastern Mediterranean, paralleling shifts in the chlorophyll size-diversity estimate. Diversity varied with absolute or relative abundance of oligotrich or copepods, but different diversity metrics were significantly correlated only with phytoplankton size-diversity. We conclude that tintinnid diversity more closely reflects resource diversity than competitive interactions or predation. r 2002 Elsevier Science Ltd. All rights reserved.

Planktonic ciliate distribution relative to a deep chlorophyll maximum: Catalan Sea, N.W. Mediterranean, June 1993

Vertical distributions and relative contributions of distinct trophic guilds of ciliates were investigated in an oligotrophic system with a deep chlorophyll maximum (DCM) in early summer. Ciliates were classified as heterotrophic: micro and nano ciliates, tintinnids and predacious forms or photosynthetic: large mixotrophic oligotrichs (Laboea strobilia, Tontonia spp.), and the autotrophic Mesodinium rubrum. Variability between vertical profiles (0–200 m) was relatively low with station to station differences (C.V.s of ∼30%) generally larger than temporal (1–4 day) differences (C.V.s of ∼15%), for integrated concentrations. Total ciliate biomass, based on volume estimates integrated from 0–80 m, averaged ∼ 125 mg C m−2, compared to ∼35 mg m−2 for chlorophyll a (chl a), yielding a ciliate to chi ratio of 3.6, well within the range of 1 to 6 reported for the euphotic zones of most oceanic systems. Heterotrophic ciliate concentrations were correlated with chl a concentration (r = 0.83and0.82, biomass and cells l−1, respectively) and averaged ∼230 cells 1−1 in near surface samples (chl a = 0.1 μg 1−1) to ∼850 cells 1−1 at 50 m depth, coinciding with the DCM (chl a = 1–2 μg 1−1). Tintinnid ciliates were diverse (36 species in 19 genera) but a minor part of heterotrophic ciliates. Nanociliates represented <1% of heterotrophic or total ciliate biomass, in contrast to reports on near-shore ciliate communities. Predacious ciliates were very rare. Large mixotrophic oligotrichs, while a minor portion of ciliate cells l−1, were an important part of total ciliate biomass, representing 63% at 5 m and 21 % integrated over 0–80 m. Mesodinium rubrum was found throughout the water column, usually with a sub-surface peak (∼100 cells 1−1). Concentrations of neither large mixotrophic oligotrichs, nor the autotrophic M. rubrum, were correlated with chl a. Estimates of the contribution of photosynthetic ciliate chi (mixotrophic and autotrophic) to total chl a (based on literature values of chla cell−1) ranged from ∼20% in some surface samples to <0.5% in the DCM.

Seasonal abundances of planktonic ciliates and microflagellates in mesohaline Chesapeake Bay waters

Ciliate, heterotrophic microflagellate (hflag) and autotrophic microflagellate (aflag) abundances are reported for mesohaline Chesapeake Bay waters based on samples gathered from April through October 1985–1987. Total water column averages for ciliate and microflagellate abundances were typical of eutrophic marine systems. Ciliate density ranged from 17·2 cells ml−1 in April to 1·8 cells ml−1 in September; hflag ranged from 3·7 × 103 cells ml−1 in June to 1·1 × 103 cells ml−1 in October. In spring the majority of ciliate and hflag standing stocks (70% and 64%, respectively) were located in bottom and transition waters; during summer months the majority (approximately 85% of both groups) were in surface and transition waters. During fall, ciliate stock was concentrated (72%) in surface waters and hflag were relatively evenly distributed in the three water column zones. Ciliate and microflagellate numbers were not directly related to chlorphyll α concentration except in the bottom layer, where simultaneous declines accompanied anoxia. Ciliate concentrations correlated with total numbers of microflagellates and hflag abundance, but not aflag density. We discuss the relative importance of predation and food availability in regulating ciliate and hflag concentrations in mesohaline Chesapeake Bay waters.

Microphagous ciliates in mesohaline Chesapeake Bay waters: Estimates of growth rates and consumption by copepods

Growth rates of microphagous ciliates (forms which feed primarily on picoplankton-sized prey) were estimated, along with rates of their consumption by copepods, in shipboard experiments conducted in the mesohaline portion of Chesapeake Bay, USA, under contrasting water column conditions in April, June, and August 1987. Estimates were based on temporal changes in cell densities in size-fractionated water samples incubated under in situ conditions. In April, at low temperatures (7 to 10°C) and with oxygen present throughout the water column, similar generation times of ca. 1 to 1.5 d were estimated for surface and deep water (24 m) ciliate populations. In June, water was anoxic below 12 m and a distinct anoxic microphage community grew at about twice the rate of the surface community with generation times of ca. 7 and 14 h, respectively. In August, bottom water was again anoxic, but the sameStrobilidium sp. dominated both surface and deep waters with low or no growth apparent in anoxic waters and a generation time of ca. 8 h in surface waters. Copepod (primarilyAcartia tonsa Dana nauplii) clearance rates for microphagous ciliates in surface waters were 0.11, 0.56, and 0.53 ml h−1 copepod−1 for April, June and August, respectively. Calculation of removal rates, based on average densities, indicated that from 34 to 200% of surface waters were cleared d−1 of microphagous ciliates by copepods.