A Simple and Rapid Cryopreservation Technique for Ciliates: A Long-Term Storage Procedure Used for Marine Scuticociliates.

Pseudocohnilembus persalinus is a free-living marine scuticociliate that, as a new model organism, has been used in a wide variety of studies. However, long-term laboratory maintenance for this species is mainly achieved by subculture that requires rigorous culture environments and, too often, cultures of the organism die out for a variety of reasons. Successful transport of viable cultures also poses problems for researchers. This study describes a simple and rapid protocol for long-term cryopreservation of P. persalinus. The effects of physiological states of individuals before freezing, the type and concentration of cryoprotectant, and optimal temperatures for freezing and thawing were assessed. A cryopreservation protocol, using a mixture of 30% glycerol and 70% concentrated P. persalinus cell culture, incorporating rate-controlled freezing at -80 °C before liquid nitrogen storage, maintained a high recovery efficiency after 8 wk of storage. These results suggest that broader application of this protocol to build a cryopreserved marine protozoa culture bank for biological studies may be possible.

Accumulation and Dissolution of Magnetite Crystals in a Magnetically Responsive Ciliate.

Magnetotactic bacteria (MTB) represent a group of microorganisms that are widespread in aquatic habitats and thrive at oxic-anoxic interfaces. They are able to scavenge high concentrations of iron thanks to the biomineralization of magnetic crystals in their unique organelles, the so-called magnetosome chains. Although their biodiversity has been intensively studied, their ecology and impact on iron cycling remain largely unexplored. Predation by protozoa was suggested as one of the ecological processes that could be involved in the release of iron back into the ecosystem. Magnetic protozoa were previously observed in aquatic environments, but their diversity and the fate of particulate iron during grazing are poorly documented. In this study, we report the morphological and molecular characterizations of a magnetically responsive MTB-grazing protozoan able to ingest high quantities of MTB. This protozoan is tentatively identified as Uronema marinum, a ciliate known to be a predator of bacteria. Using light and electron microscopy, we investigated in detail the vacuoles in which the lysis of phagocytized prokaryotes occurs. We carried out high-resolution observations of aligned magnetosome chains and ongoing dissolution of crystals. Particulate iron in the ciliate represented approximately 0.01% of its total volume. We show the ubiquity of this interaction in other types of environments and describe different grazing strategies. These data contribute to the mounting evidence that the interactions between MTB and protozoa might play a significant role in iron turnover in microaerophilic habitats.IMPORTANCE Identifying participants of each biogeochemical cycle is a prerequisite to our understanding of ecosystem functioning. Magnetotactic bacteria (MTB) participate in iron cycling by concentrating large amounts of biomineralized iron minerals in their cells, which impacts their chemical environment at, or below, the oxic-anoxic transition zone in aquatic habitats. It was shown that some protozoa inhabiting this niche could become magnetic by the ingestion of magnetic crystals biomineralized by grazed MTB. In this study, we show that magnetic MTB grazers are commonly observed in marine and freshwater sediments and can sometimes accumulate very large amounts of particulate iron. We describe here different phagocytosis strategies, determined using magnetic particles from MTB as tracers after their ingestion by the protozoa. This study paves the way for potential scientific or medical applications using MTB grazers as magnetosome hyperaccumulators.

A New Molecular Approach Based on the Secondary Structure of Ribosomal RNA for Phylogenetic Analysis of Mobilid Ciliates.

We analyzed the secondary structure of the small subunit (SSU) rRNA genes of Mobilida (Ciliophora, Peritrichia) and found that the secondary structures of some regions within the SSU-rRNA gene are distinct between the families Trichodinidae and Urceolariidae. Therefore, some of these important regions including H10, H11, H17, H47, H29, H30, H37, E10-1, H45-H46, and V4 (E23-4, E23-7) could be used as the barcodes for classification of these two families. In contrast, V4 (E23-1, E23-2) belongs to a hypervariable region and is not a good barcode at the genus level because of its great inter-specific variation. Our results indicated that the comprehensive analysis of the secondary structure of SSU-rRNA genes is a reliable auxiliary approach for phylogenic study of mobilid ciliates. It was further found that the coevolution between hosts or habitats and the Mobilida ciliates was existent, because the host types and their habitats were critical ecological factors that influenced the evolution of Mobilida ciliates.

Development of a fatty acid and RNA stable isotope probing-based method for tracking protist grazing on bacteria in wastewater.

Removal of potential pathogenic bacteria, for example, during wastewater treatment, is effected by sorption, filtration, natural die-off, lysis by viruses, and grazing by protists, but the actual contribution of grazing has never been assessed quantitatively. A methodical approach for analyzing the grazing of protists on (13)C-labeled prey bacteria was developed which enables mass balances of the carbon turnover to be drawn, including yield estimation. Model experiments for validating the approach were performed in closed microcosms with the ciliate Uronema sp. and (13)C-labeled Escherichia coli as model prey. The transfer of bacterial (13)C into grazing protist biomass was investigated by fatty acid (FA) and RNA stable isotope probing (SIP). Uronema sp. showed ingestion rates of ∼390 bacteria protist(-1) h(-1), and the temporal patterns of (13)C assimilation from the prey bacteria to the protist FA were identified. Nine fatty acids specific for Uronema sp. were found (20:0, i20:0, 22:0, 24:0, 20:1ω9c, 20:1ω9t, 22:1ω9c, 22:1ω9t, and 24:1). Four of these fatty acids (22:0, 20:1ω9t, 22:1ω9c, and 22:1ω9t) were enriched very rapidly after 3 h, indicating grazing on bacteria without concomitant cell division. Other fatty acids (20:0, i20:0, and 20:1ω9c) were found to be indicative of growth with cell division. The fatty acids were found to be labeled with a percentage of labeled carbon (atoms percent [atom%]) up to 50. Eighteen percent of the E. coli-derived (13)C was incorporated into Uronema biomass, whereas 11% was mineralized. Around 5 mol bacterial carbon was necessary in order to produce 1 mol protist carbon (y(x)(/)(s) ≈ 0.2), and the temporal pattern of (13)C labeling of protist rRNA was also shown. A consumption of around 1,000 prey bacteria (∼98 atom% (13)C) per protist cell appears to be sufficient to provide detectable amounts of label in the protist RNA. The large shift in the buoyant density fraction of (13)C-labeled protist RNA demonstrated a high incorporation of (13)C, and reverse transcription-real-time PCR (RT-qPCR) confirmed that protist rRNA increasingly dominated in the heavy RNA fraction.

Ultrastructure, encystment and cyst wall composition of the resting cyst of the peritrich ciliate Opisthonecta henneguyi.

The cyst wall of Opisthonecta henneguyi has been studied ultrastructurally and cytochemically by light and electron microscopy, as well as by chemical and electrophoretic analyses, to examine the structure of the cyst wall and its composition. The cyst wall consists of four morphologically distinct layers. The ectocyst is a thin dense layer. The mesocyst is the thickest layer and is composed of a compact material. The endocyst is a thin layer like the ectocyst, but less dense. The granular layer varies in thickness and is composed of a granular material. In the resting cyst, kinetosomes of both oral apparatus and trochal band as well as the myoneme system are maintained, and only cilia are resorbed. The sugars present in the cyst wall are predominantly N-acetylglucosamine (90%) and glucose (10%). The mesocyst is composed of chitin, and the endocyst includes glycoproteins and acid mucopolysaccharides. During secretion of the cyst wall, the endocyst and granular layer are secreted from precursors synthesized "de novo". No cytoplasmic precursors of ectocyst and mesocyst have been detected.