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on non-nutrient agar medium with inactivated E. coli and subjected to PCR with various primer pairs (amplify mainly the 18S-small ribosomal RNA). Free-living amoebae were intensively detected by PCR in two collection regions (Yeoju and Yangpeong city around Southern Han-Liver). PCR products obtained from water samples of Yeoju and Yangpeong city were subjected to gene sequencing. The similarity of 18S-rRNA sequences were compared with various reference amoebae in GeneBank, and they showed 86-99% homology with N. gruberi, N. philippinensis, N. clarki, Acanthamoeba polyphagia and Vermamoeba vermiformis. A Korean isolate (confirmed by PCR as A. polyphagia) was isolated from Yeoju sample and have been subcultured in Nelson's and PYG liquid medium with 10% FBS at 30 °C incubator. In the in vitro cytotoxicity test, Korean isolate (tentative A. polyphagia) showed high cytotoxicity as much as reference amoebae, A. polyphagia and A. castellanii. This study will be useful, in the further study, for the detailed seasonal detection of free-living amoebae from Korean hydrosphere.
MORPHOLOGICAL AND GENETIC DIVERSITY OF OPISTHOSPORIDIA: NEW APHELID PARAPHELIDIUM TRIBONEMAE GEN. ET SP. NOV
Karpov S.A.12, Tcvetkova V.S.2, Mamkaeva M.A.2, Torruella G.3, Timpano H.3, Moreira D.3, LopezGarcia P.3
1 - Zoological Institute, Russian Academy ofSciences, Universitetskaya nab. 1, St. Petersburg 199034, Russian Federation
2 - St. Petersburg State University, Universitetskaya nab. 7/9, St. Petersburg 199034, Russian Federation
3 - Unité d'Ecologie, Systématique et Evolution, UMR CNRS 8079, Université Paris-Sud, 91405 Orsay cedex, France
sakarpov4@gmail.com
Aphelids are a poorly known group of parasitoids of algae that have raised considerable interest due to their pivotal phylogenetic position. Together with Cryptomycota and the highly derived Microspo-ridia, they have recently re-classified as the Opistho-sporidia, which constitute the sister group to the fungi within the Holomycota. Despite their huge diversity, as revealed by molecular environmental studies, and their phylogenetic interest, only three genera have been described (Aphelidium, Amoeboaphelidium, and Pseudaphelidium), from which 18S rRNA gene sequences exist only for Amoeboaphelidium and Aphelidium species. Here, we present the molecular phylogeny, life cycle and ultrastructure of new freshwater aphelid, which is a new genus and species
Paraphelidium tribonemae. Molecular phylogenetic analysis of 18S rRNA gene sequences ofthis parasite indicates that P. tribonemae defines a distinct cluster which is only distantly related to Amoebaphelidium and Aphelidium, what confirms the wide genetic diversity ofthe aphelids. P. tribonemae has amoeboid opisthokont zoospores which are twice smaller than morphologically similar zoospores of Aphelidium aff. melosirae. Nonetheless, although the morphology of Paraphelidium species is generally similar to that of Aphelidium representatives, molecular phylogenetic analyses unambiguously show, that the two clades of strains are distantly related and must define two distinct genera.
We thank RFBR grant No. 15-29-02734 for financial support, and for cultivation of strains and access to the EM facilities the Center for Culturing Collection of Microorganisms and the Research Resource Center for Molecular and Cell Technologies (RRC MCT) of Research park of St. Petersburg State University correspondingly.
AN EVOLUTIONARY TRANSITION OF CHLOROPLAST DEGRADATION IN EU-GLENOIDS: HETEROTROPHIC DIGESTION TO SECONDARY PLASTID SENESCENCE Kashiyama Y.12, Kawahara J.1, Maruyama M.1, Kayama M.1, Nakazawa M.3, Tanifuji G.4, Yoko-yama A.5, Ishikawa T.6, Tamiaki H.2, Suzaki T.7
1 - Fukui University of Technology
2 - Ritsumeikan University
3 - Osaka Prefecture University
4 - National Museum of Nature and Science
5 - National Institute for Environmental Studies
6 - Shimane University
7 - Kobe University chiro@fukui-ut.ac.jp
Establishments of regulatory mechanisms for the integrated endosymbiont must have been key steps in organelle acquisitions, where both endosymbiont and host processes cooperated to evolve new functions. Specifically important for the chloroplast is to establish regulatory mechanisms for chlorophyll-related metabolisms since the phototoxicity of chlorophyll would cause fatal damages. In particular, regulated degradation of chloroplasts or chloroplast dismantlement is critical not only for recycling nutrients but also for disposing the phototoxic chlorophylls safely. Unlike land plants, little has been understood for algae on chloroplast dismantlement and associated chlorophyll catabolism. We previously reported that the phototrophic euglenoid accumulates 132,173-cyclopheophorbide enols (CPEs) within their cells; CPEs are non-phototoxic catabolites
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of chlorophylls. A phototrophic euglenoid Euglena gracilis exhibited that chloroplasts underwent dismantled when incubated in the dark, leaving brown-colored granules. The brown granules were massive structures of membranes generated after degraded thylakoids. Chlorophylls are converted to CPEs in an early stage of dismantling, and proteins were removed during the dismantling. Macroautophagy of chloroplasts was not apparent, and the dismantling chloroplasts seem not to undergo acidification; instead, large acidic vacuoles commonly formed beside the dismantling chloroplasts. This would be comparable to digestion of chloroplasts by phycophagic euglenoids, where also accumulated CPEs. However, the digestive degradation proceeds within acidified phagosome hence being apparently non-homologous to the dismantlement in the phototrophs. We infer that the CPE-metabolism, which has not been reported for green algae, might have been inherited from ancestral heterotrophs to phototrophs, and is now adapted into a novel mechanism to dismantle the secondary chloroplasts.
COMPARISON OF FRESH WATER CILIATED PROTIST COMMUNITIES FROM TWO LOCATIONS ALONG NAJAFGARH DRAIN, DELHI, INDIA, AND THEIR CORRELATION WITH PHYSIO-CHEMICAL PARAMETERS Kaur Harpreet1, Rani Pooja2, Kaur Surinder1, Narula Laxmi1, Kamra Komal1, Lal Rup2, Warren Alan3
1 - Ciliate Biology Lab, SGTB Khalsa College, University of Delhi, Delhi 110007
2 - Department of Zoology, University of Delhi, Delhi 110007
3 - Natural History Museum, London, SW75BD, UK komalkamra@gmail.com
There are 18 sewage drains traversing the National Capital Region (NCR) of Delhi, India, that flow into the River Yamuna. Najafgarh Drain is the largest with a catchment area of around 400 km2. It picks up domestic and industrial wastes from 38 secondary drains coming from over 2/3 of the city. It carries a total flow of over 2100 million litres per day ofwhich 30% is treated. Sewage treatment plants and effluent treatment plants discharging into the drain are inadequate and not working to full capacity. Ciliated protist communities were assessed (with appropriate morphological, morphometric and morphogenetic descriptions) at two locations along the drain: the mid-point of the drain (D1) and at the end of the drain before it empties into the river Yamuna (D2). Various physico-chemical parameters were also
measured. Sampling was carried out weekly for 3 weeks during the pre-mon-soon season. Compared to D1, substantially higher levels of free CO2, chloride, phosphates and hardness were observed in D2 indicating the water here is more polluted. The ciliate communities at the two sites differed significantly. The results of detailed analyses, showing correlations between physico-chemical parameters (including heavy metals), and ciliate communities, will be presented. The significance of such data collected over a period of time along the drains, the river, and at water treatment plants, shall benefit ecologists in developing strategies to help mitigate river pollution as ciliates are known to clarify waste water and act as bio-indicators of specific pollutants.
OLIGOTROPHIC LAGOONS OF THE SOUTH PACIFIC OCEAN ARE HOME TO A SURPRISING NUMBER OF NOVEL EUKARYOTIC MICROORGANISMS
Kim E.1, Sprung B.2, Duhamel S.3, Filardi C.1, Shin M.4
1 - American Museum of Natural History
2 - University of Pennsylvania
3 - Lamont-Doherty Earth Observatory, Columbia University
4 - University of Ulsan ekim1@amnh.org
The diversity of microbial eukaryotes was surveyed by metagenomic sequencing from tropical lagoon sites of the South Pacific, collected through the American Museum of Natural History (AMNH)'s Explore21 expedition to the Solomon Islands in September 2013. The sampled lagoons presented low nutrient concentrations typical of oligotrophic waters, but contained levels of chlorophyll a, a proxy for phytoplankton biomass, characteristic of meso- to eutrophic waters. Two 18S rDNA sites that include the V4 and V8-V9 regions were amplified from the total ofeight lagoon samples and sequenced on the MiSeq system. After assembly, clustering at 97% similarity, and removal of singletons and chimeras, a total of 2,741 (V4) and 2,606 (V8-V9) operational taxonomic units (OTUs) were identified. Taxonomic annotation of these reads, including phylogeny, was based on a combination of automated pipeline and manual inspection. 18.4% (V4) and 13.8% (V8-V9) of the OTUs could not be assigned to any of the known eukaryotic groups. Of these, we focused on OTUs that were not divergent and possessed multiple sources of evidence for their existence. Phylogenetic analyses of these sequences revealed more than ten branches that
