A multigene family encoding R-SNAREs in the ciliate Paramecium tetraurelia

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane interactions and are conventionally divided into Q-SNAREs and R-SNAREs according to the possession of a glutamine or arginine residue at the core of their SNARE domain. Here, we describe a set of R-SNAREs from the ciliate Paramecium tetraurelia consisting of seven families encoded by 12 genes that are expressed simultaneously. The complexity of the endomembrane system in Paramecium can explain this high number of genes. All P. tetraurelia synaptobrevins (PtSybs) possess a SNARE domain and show homology to the Longin family of R-SNAREs such as Ykt6, Sec22 and tetanus toxin-insensitive VAMP (TI-VAMP). We localized four exemplary PtSyb subfamilies with GFP constructs and antibodies on the light and electron microscopic level. PtSyb1-1, PtSyb1-2 and PtSyb3-1 were found in the endoplasmic reticulum, whereas PtSyb2 is localized exclusively in the contractile vacuole complex. PtSyb6 was found cytosolic but also resides in regularly arranged structures at the cell cortex (parasomal sacs), the cytoproct and oral apparatus, probably representing endocytotic compartments. With gene silencing, we showed that the R-SNARE of the contractile vacuole complex, PtSyb2, functions to maintain structural integrity as well as functionality of the osmoregulatory system but also affects cell division.

Viral, bacterial and ciliate abundance and viral-bacterial diversity in mesocosm experiments, 2007-2008, Kongsfjorden

Two mesocosm experiments, PAME-I and PAME-II were conducted in 2007 and 2008 to investigate fate of organic carbon in the arctic microbial food web. Mesocosms were nutrient fertilized initially to induce phytoplankton bloom development. In PAME-I eight units (each 700 L) formed two four point gradients of additional DOC in form of glucose (0, 0.5, 1 and 3 times Redfield ratio in terms of carbon relative to the nitrogen and phosphorus additions) (Fig. 1). All the eight units also got a daily dose of NH4+ and PO4**3- in Redfield ratio. Two gradients were set up, one with silicate addition, performed in the Arctic location Ny Ålesund, Svalbard, have previously been reported to give different food-web level responses to similar nutrient perturbations. In PAME-II all ten units (each 900 L) formed two four point gradients of additional DOC in form of glucose (0, 0.5, 1, 2 and 3 times Redfield ratio in terms of carbon relative to nitrogen and phosphorus additions). The two gradients in glucose were kept silicate replete. NH4+ was used as the DIN source in one gradient (units 1 to 5) and NO3- in the other (units 6-9). All units got a daily dose of PO4**3- in Redfield ratio. Prokaryotes and viruses were measured by flow cytometry, while ciliate abundances were counted using a Flow Cam. Viral and bacterial diversity was measured by PFGE and DGGE, respectively. In PAME-II the abundance of ciliates was lower than in PAME-I, presumably caused by higher copepod grazing. The abundances of prokaryotes and viruses were also lower in PAME-II compared to PAME-I. Further, less diversity was detected in the viral community (FCM and PFGE) in PAME-II, and no response was observed in the bacterial community structure due to addition of organic carbon.

Ciliate abundance in waters of the Argentine shelf and the Drake Passage, the south-western Atlantic

Ciliates from sub-surface waters of the Argentine shelf and the Drake Passage under austral summer and autumn conditions were examined and compared for the first time. In both environments, the taxonomic structure of ciliates was related to temperature and salinity, and aloricate oligotrichs dominated in density (80%) over loricate oligotrichs, litostomatids and prostomatids, while the microplanktonic fraction prevailed in terms of biomass (90%) over the nanociliates. Myrionecta rubra was found all along the Argentine shelf only in autumn, but showed isolated peaks of abundance (10**3 ind./L) during summer. Mean values of density and biomass of total ciliates decreased ca. 2-fold from the shelf-slope to oceanic waters, while potential maximum production of aloricate oligotrichs decreased 9-fold, in relation with the drop in chlorophyll a concentration and the latitudinal decline of temperature, also reflected in maximum growth rates. Fifty percent of total ciliate abundance was represented by local increases (maximum: 20 000 ind./L and 25 µg C/L), which were spatially superimposed with ranges of seawater temperature and chlorophyll a concentrations of 10-15°C and 0.6-6 µg/L, respectively and were found in the nearby of fronts located on the shelf and the slope.

Ingestion of a bacterivorous ciliate by the oyster Crassostrea gigas: Protozoa as a trophic link between picoplankton and benthic suspension-feeders

The linked concepts of 'microbial loop' and 'protozoan trophic link' have been very well documented in filter-feeding microzooplankton such as copepods, but have not been applied to energy transfer to benthic suspension-feeding macrofauna, with the exception of the recent demonstration of heterotrophic flagellate assimilation by mussels. The oyster Crassostrea gigas obtains energy resources by filtering microalgae (similar to 5 to 100 mu m). However, in turbid estuaries, light-limited phytoplanktonic production cannot entirely account for oyster energy requirements. Conversely, picoplankters (<2 mu m), which are main effecters of coastal energy flow and matter cycling, are not efficiently retained by oyster filtration. Ciliate protozoal as both micro-sized cells (similar to 5 to 100 run) and bacteria grazers, may represent a major intermediary in trophic transfer between picoplankton and metazoa. The ciliate Uronema was intensely cultured and labelled, using the cyanobacteria Synechococcus as an auto-fluorescent biomarker. The labelled ciliates were offered as potential prey to oysters. We report here the first experimental evidence of a significant retention and ingestion of ciliates by oysters, supporting the role of protozoa as a realistic trophic link between picoplankters and filter-feeding bivalves and thus enhancing their potential importance in estuarine microbial food webs.

Novel ciliate genetic code variants including the reassignment of all three stop codons to sense codons in Condylostoma magnum

mRNA translation in many ciliates utilizes variant genetic codes where stop codons are reassigned to specify amino acids. To characterize the repertoire of ciliate genetic codes, we analyzed ciliate transcriptomes from marine environments. Using codon substitution frequencies in ciliate protein-coding genes and their orthologs, we inferred the genetic codes of 24 ciliate species. Nine did not match genetic code tables currently assigned by NCBI. Surprisingly, we identified a novel genetic code where all three standard stop codons (TAA, TAG, and TGA) specify amino acids in Condylostoma magnum. We provide evidence suggesting that the functions of these codons in C. magnum depend on their location within mRNA. They are decoded as amino acids at internal positions, but specify translation termination when in close proximity to an mRNA 3' end. The frequency of stop codons in protein coding sequences of closely related Climacostomum virens suggests that it may represent a transitory state.mRNA translation in many ciliates utilizes variant genetic codes where stop codons are reassigned to specify amino acids. To characterize the repertoire of ciliate genetic codes, we analyzed ciliate transcriptomes from marine environments. Using codon substitution frequencies in ciliate protein-coding genes and their orthologs, we inferred the genetic codes of 24 ciliate species. Nine did not match genetic code tables currently assigned by NCBI. Surprisingly, we identified a novel genetic code where all three standard stop codons (TAA, TAG, and TGA) specify amino acids in Condylostoma magnum. We provide evidence suggesting that the functions of these codons in C. magnum depend on their location within mRNA. They are decoded as amino acids at internal positions, but specify translation termination when in close proximity to an mRNA 3' end. The frequency of stop codons in protein coding sequences of closely related Climacostomum virens suggests that it may represent a transitory state.

Chemosensory response of marine flagellate towards L- and D- dissolved free amino acids generated during heavy grazing on bacteria

This study investigated the generation of dissolved free amino acids (DFAA) by the bacterivorous flagellate Rhynchomonas nasuta when feeding on abundant prey. Specifically, it examined whether this flagellate protist exhibits a chemosensory response towards those amino acids. The concentrations of glycine and the and D-enantiomers of glutamate, serine, threonine, alanine, and leucine were determined in co-cultures of the flagellate and bacteria. Glycine, L- and D-alanine, and L-serine were found to accumulate under these conditions in amounts that correlated positively with flagellate abundance, suggesting that protists are involved in their generation. Investigations of the chemotactic response of young and old foraging protists to the same amino acids, offered in concentrations similar to those previously generated, showed that glycine elicited the strongest attraction in both age groups. Young protists were strongly attracted to all the assayed amino acids, whereas older protists maintained a high level of attraction only for glycine. These results suggest that glycine generated by protists actively grazing in bacterially enriched patches functions as an infochemical, signaling to foraging protists the presence of available prey in the aquatic environment.

Plankton associated with medusae of the freshwater jellyfish Craspedacusta sowerbyi (Lankester) in a Thames backwater

Samples of plankton were taken from Broom Water in August 1997 after a sighting of medusae on 11th August. Broom Water is about 8 m wide, and extends 250 m from the main channel of the Thames, above the weir at Teddington. On 11th August medusae were so abundant that it was possible to collect 20 in ten minutes. They were rising to the surface in bright sunlight, then sinking slowly down through the water. Examination of a medusa's tentacles under a microscope revealed the presence of a commensal protozoan, a ciliate Trichodina pediculus. Over 20 species of phytoplankton were found in Broom Water. Most of the species are common and widespread, but it was a surprise to find Errerella bornhemiensis with its characteristic pyramidal colonies, which is a relatively rare species. Zooplankters in Broom Water consisted of Rotifera and Crustacea. Zooplankton is the main food of Craspedacusta and it was found that the crustaceans but not the rotifers did undergo significant changes during the period 11-19th August. The major changes were a big increase in the percentage of cyclopoids, and a marked decrease in Bosmina. This could be because the delicate cuticle of Bosmina is much more susceptible to the stinging cells of the medusae compared with the tougher exoskeleton of the cyclopoid.

The diversity and ecological role of protozoa in fresh waters

Protozoa feed on and regulate the abundance of most types of aquatic microorganisms, and they are an integral part of all aquatic microbial food webs. Being so small, aerobic protozoa thrive at low oxygen tensions, where they feed (largely unaffected by metazoan grazing) on the abundance of other microorganisms. In anaerobic environments, they are the only phagotrophic organisms, and they live in unique symbiotic consortia with methanogens, sulphate reducers and non-sulphur purple bacteria. The number of extant species of protozoa may be quite modest (the global number of ciliate species is estimated at 3000), and most of them probably have cosmopolitan distributions. This will undoubtedly make it easier to carry out further tasks, e.g. understanding the role of protozoan species diversity in the natural environment.

稀有植物太行花的保护生物学研究

悬崖代表着一个残遗的、较少受到干扰的古生境，悬崖也是生物多样性的避难所。这个特殊生境中分布种类丰富的稀有植物和特有植物，而这些稀有和特有植物又往往以悬崖为唯一的生境，在长期的演化历史进程中形成彼此相互适应的关系。这种分布在特殊生境中的稀有植物为保护研究提出了新的研究方向和问题。本文以蔷薇科的中国特有植物太行花（Taihangia rupestris Yü et Li）为对象，对这个仅分布在有限的地理范围内悬崖上的稀有物种进行了保护生物学的研究。 利用RAPD分子标记技术对太行花8个居群的遗传多样性和遗传结构进行了检测分析。结果表明太行花居群内维持着较高的遗传多样性水平，未显示遭受过严重遗传瓶颈的迹象。同时，居群间遗传分化显著，与居群间的地理距离相关，聚类分析得到的树系图与太行花种下两个变种（太行花原变种Taihangia rupestris var. rupestris和缘毛太行花Taihangia rupestris var. ciliate Yü et Li）的划分相符。 通过栽培实验，研究了太行花对光照和养分的反应，从生态学角度检测植物对环境因子变化的适应能力。结果表明光照处理显著影响太行花的气体交换参数，且显示出随季节不同而变化的趋势。在8月第一次测量时，光合速率随着光照水平的增加而增加;而在9月底第二次测量时，高光下植株的光合速率反低于中光条件下的植株。光合光反应曲线表明太行花对不同光照水平（高光、中光和低光）均显示出一定的光合适应性。 光照对太行花的生长反应、形态、解剖等指标均具有显著的作用。在收获时，高光下植物形成小而厚的叶片，更快的叶片形成速率足以弥补单叶面积上光合速率的下降，使植株维持最大的总生物量。中光条件下的植株具有更长的叶柄，更大的叶面积，大而薄的叶片，和更大的叶面积比，被认为是对较弱光照环境适应的表现。而光照对叶片的气孔指标（气孔密度、气孔指数和大小）没有作用，养分施加对太行花的影响也甚微。总体来说，太行花显示出对光照变化的生理适应性和形态可塑性，光照和养分这两个环境条件均非影响太行花局限分布的关键因素。 同时，我们采用扫描电镜手段跟踪观察了太行花的花的早期发育过程。太行花早期发育过程中苞片内残留的退化花痕迹表明，太行花的顶生单花其实是有限聚伞花序中其它花芽败育的结果，显示出从花序向单花演化的趋势。同时，雌蕊的发育在早期是正常的，未显示出退化痕迹。 根据以上结果表明，局限分布在特殊生境上的太行花并未显示出生理适应幅度的狭窄性和遗传多样性水平的降低，因此，太行花在长期的进化过程中形成了对特殊生境——悬崖的适应性，遗传衰退和缺乏适应性不是稀有植物太行花局限分布的原因。在对太行花的保护中应进一步加强对其生境的保护，并在进行迁地保护和回归引种时应兼顾不同变种和地理分布的居群，以实现对其遗传完整性的有效保护。

Reconsideration of Phylogenetic Relationships of the Subclass Peritrichia (Ciliophora, Oligohymenophorea) Based on Small Subunit Ribosomal RNA Gene Sequences, with the Establishment of a New Subclass Mobilia Kahl, 1933

Based on its characteristic oral apparatus, the ciliate subclass Peritrichia has long been recognized as a monophyletic assemblage composed of the orders Mobilida and Sessilida. Following the application of molecular methods, the monophyly of Peritrichia has recently been questioned. We investigated the phylogenetic relationships of the peritrichous ciliates based on four further complete small subunit ribosomal RNA sequences of mobilids, namely Urceolaria urechi, Trichodina meretricis, Trichodina sinonovaculae, and Trichodina ruditapicis. In all phylogenetic trees, the mobilids never clustered with the sessilids, but instead formed a monophyletic assemblage related to the peniculines. By contrast, the sessilids formed a sister clade with the hymenostomes at a terminal position within the Oligohymenophorea. We therefore formally separate the mobilids from the sessilids (Peritrichia sensu stricto) and establish a new subclass, Mobilia Kahl, 1933, which contains the order Mobilida Kahl, 1933. We argue that the oral apparatus in the mobilians and sessilid peritrichs is a homoplasy, probably due to convergent evolution driven by their similar life-styles and feeding strategies. Morphologically, the mobilians are distinguished from all other oligohymenophoreans by the presence of the adhesive disc, this character being a synapomorphy for the Mobilia.

Cloning, characterization, and gene expression analysis of a novel cadmium metallothionein gene in Tetrahymena pigmentosa

A novel cadmium-inducible metallothionein (MT) gene (Tpig-MT1) was cloned and sequenced from the ciliate Tetrahymena pigmentosa. The number of deduced amino acids is 118. The polypeptide possesses CCC and CC clusters characteristic of typical Tetrahymena Cd-inducible MTs. The structure of Tpig-MT1 is different from the reported Cd-MT in T. pyriformis, T. thermophila and T. pigmentosa. Tpig-MT1 contains two intragenic tandem repeats with 72.9% identity described as Tpig-MT1 (repeat A1) and Tpig-MT1 (repeat A2). The transcriptional response of Tpig-MT1 gene to different heavy metals (Cd, Cu, Zn, Hg, Pb) and oxidative stress (H2O2) was measured using real-time quantitative PCR. The results showed that the gene was quickly induced (1 h) by the five heavy metals and the order of expression level was Hg>Pb>Cd>Cu>Zn. The induction effect of H2O2 was 5-fold after about 15 min, but soon decreased to a non-significant level (30 min). The genetic diversity of Tetrahymena MT genes is discussed in relation to the unique structure of the Tpig-MT1 gene and other reported Cd-MT isoforms. (C) 2008 Elsevier B.V. All rights reserved.

Light and scanning electron microscopic study of Balantidium ctenopharyngodoni Chen, 1955 (Class : Litostomatea) from China

Redescription of Balantidium ctenopharyngodoni "Chen (Acta Hydrobiol Sin 1:123-164, 1955)", collected from the hindgut of grass carp (Ctenopharyngodon idella), especially the segment of 6-10 cm upstream from the anus, from Honghu Lake, Hubei Province, central China in November 2005, is presented in this paper to complete Chen's description at both light and scanning electron microscopic levels. Some revisions were done: the vestibulum is fairly symmetrical, with compactly arranged cilia rather than assembled membrane bordering on the left vestibular side; four contractile vacuoles actually exist in the latter body, three of which surround the posterior portion of the macronucleus, whereas the fourth lies antero-left to it. Somatic monokinetids were compared among the species of genus Balantidium. The cysts were described, and possible infection routes of B. ctenopharyngodoni were also discussed.

Population genetic structure of Carchesium polypinum (Ciliophora : Peritrichia) in four chinese lakes inferred from ISSR fingerprinting: High diversity but low differentiation

Although the peritrichous ciliate Carchesium polypinum is common in freshwater, its population genetic structure is largely unknown. We used inter-simple sequence repeat (ISSR) fingerprinting to analyze the genetic structure of 48 different isolates of the species from four lakes in Wuhan, central China. Using eight polymorphic primers, 81 discernible DNA fragments were detected, among which 76 (93.83%) were polymorphic, indicating high genetic diversity at the isolate level. Further, Nei's gene diversity (h) and Shannon's Information index (I) between the different isolates both revealed a remarkable genetic diversity, higher than previously indicated by their morphology. At the same time, substantial gene flow was found. So the main factors responsible for the high level of diversity within populations are probably due to conjugation (sexual reproduction) and wide distribution of swarmers. Analysis of molecular variance (AMOVA) showed that there was low genetic differentiation among the four populations probably due to common ancestry and flooding events. The cluster analysis and principal component analysis (PCA) suggested that genotypes isolated from the same lake displayed a higher genetic similarity than those from different lakes. Both analyses separated C. polypinum isolates into subgroups according to the geographical locations. However, there is only a weak positive correlation between the genetic distance and geographical distance, suggesting a minor effect of geographical distance on the distribution of genetic diversity between populations of C. polypinum at the local level. In conclusion, our studies clearly demonstrated that a single morphospecies may harbor high levels of genetic diversity, and that the degree of resolution offered by morphology as a marker for measuring distribution patterns of genetically distinct entities is too low.

Cloning and characterization of a new multi-stress inducible metallothionein gene in Tetrahymena pyriformis

A new multi-stress-inducible metallothionein (MT) gene isoform has been cloned and characterized from the ciliate Tetrahymena pyriformis. Both the 5'- and 3'-UT regions of the Tp-MT2 gene are very different from the previously reported Tp-MT1 isoform in this organism and from other described MT genes in Tetrahymena pigmentosa and Tetrahymena thermophila. The putative protein sequence of Tp-MT2 contains cysteine clusters with characteristics of the typical Tetrahymena Cd-inducible MT genes. However, the sequence has a special feature of four intragenic tandem repeats within its first half, with a conserved structural pattern x(5/8)CCCx(6)CCx(6)CxCxNCxCCK. To investigate the transcriptional activities of both Tp-MT2 and Tp-MT1 genes toward heavy metals (Cd, Hg, Cu, Zn) and H2O2, the mRNA levels of these two isoforms were evaluated by means of real-time quantitative PCR. Results showed that Tp-MT2 had a higher basal expression level than Tp-MT1 and both genes were induced by Cd, Hg, Cu, and Zn ions after short exposure (I h), although to different extents. Cd was the most effective metal inducer of both two isoforms, but the relative expression level of Tp-MT2 was much lower than that of Tp-MT1. Different expression patterns were also shown between the two genes when treated with Cd over a period of 24 h. We suggest that TpMT-1 plays the role of a multi-inducible stress gene, while TpMT-2 may have a more specific function in basal metal homeostasis although it may have undergone a functional differentiation process. The putative functional significance and evolutionary mode of the TpMT-2 isoform are discussed. (c) 2006 Elsevier GmbH. All rights reserved.