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64 • "PROTIST—2016
maria.rautian@mail.ru
BACKGROUND. P. bursaria belongs to one of the best studied Ciliate's genus Paramecium. This species diverged basically from other paramecia. They possess symbiotic Chlorella in the cytoplasm. These Chlorella were attributed to three Chlorella species — Ch. vulgaris, Ch. variabilis and Micractinum reisseri according to their phylogeny inferred from ITS1-5.8S-ITS2-5'LSU rDNA sequence and their sensitivity to specific viruses (PBCV). Morphospecies P. bursaria consist of 5 syngens — reproductively isolated intraspecies groups. Previously we have shown that syngens were represented by different branches on phylogenetic trees. The purpose of our investigation was to study phylogeny of P. bursaria and their symbiotic Chlorella using the same set of strains collected in different geographic regions. RESULTS. Gene RuBisCo have been used as molecular marker for Chlorella phylogeny. The tree consists of three separate branches. Diversity in each branch is negligible. Our data support strong correspondence between P. bursaria syngens and branch of Chlorella, while weak correlation between Chlorella type and its geographic location. CONCLUSIONS. According to our results we have concluded that successful symbiosis between P. bursaria ancestor and Chlorella has been established at least three times. The last time it had been happened before syngens R1 and R2 have diverged. We propose the strategy of evolution and geographic dispersion of this symbiotic system. Scientific research was performed at the Center for Culturing Collection ofMicroorganisms and Center for Molecular and Cell Technologies of Research park of St. Petersburg State University. Supported by RFBR grant 13-04-01714.
THE PLASTID GENOME OF POLYTOMA UVELLA IS THE LARGEST KNOWN AMONG NON-PHOTOSYNTHETIC ALGAE Reves-Prieto A.12
1 - Biology Department. University of New Brunswick, Fredericton
2 - Integrated Microbiology Program. Canadian Institute for Advanced Research. areyes@unb.ca
The polyphyletic assembly Polytoma and the species of the genus Polytomella represent different lineages within the Order Chlamydomonadales (Chlorophyta) that lost photosynthesis independently. The Polytomella clade is one of the two known groups of plastid-bearing eukaryotes that have lost completely the plastid genome (ptDNA)
after the loss of photosynthesis. Before the present report it was unknown if the absence of genome in the Polytomella plastids was a shared condition with species of the Polytoma genus. We present here the ptDNA of Polytoma uvella UTEX 964, which is the largest (circa 230 Kb) reported among non-photosynthetic algae. The ptDNA of P. uvella has lost all genes related with the photosynthetic function and its residual coding capacity is similar to ptDNAs from distantly related colorless chlorophytes, such as the pathogens Prototheca and Helicosporidium. The gene repertoire of the P. uvella ptDNA comprises mostly transcription and translation players and just few conserved proteins (e.g., FtsH-like, Ycf1-like and ClpP). Regardless of the substantial gene loss, the P. uvella ptDNA shows evidence of expansion due to the accumulation ofintergenic short repeated sequences (average length of 2.9 Kb). It is plausible that recombination-based DNA repair mechanisms are responsible of the genomic expansion of the P. uvella ptDNA.
SELECTION AND CHARACTERIZATION OF SINGLE CHAIN VARIABLE FRAGMENT (SCFV) ANTIBODIES AGAINST PNEUMOCYSTIS JIROVECII FROM PHAGE DISPLAY LIBRARIES
Ribeiro Marta, Cardoso Fernando, Matos Olga Medical Parasitology Unit, Group of Opportunistic Protozoa/HIVand Other Protozoa, Global Health and Tropical Medicine, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Portugal omatos@ihmt.unl.pt
Pneumocystis pneumonia (PcP) is an infectious disease caused by Pneumocystisjirovecii, an atypical fungus. PcP remains a major cause of respiratory illness among immunosuppressed patients. Current PcP diagnosis is based on the detection of P. jirovecii in respiratory specimens, obtained by invasive methods such as bronchoalveolar lavage, followed by cytochemical staining, immunofluorescent staining with monoclonal antibodies (IF/Mab) or PCR. Therefore, the possibility of an early diagnostic method allowing the use of biological specimens obtained non-invasively, is highly desirable. Rapid diagnostic tests (RDTs) using gold nanoparticles (AuNPs) allow a more sensitive, fast and cheap diagnosis, to be used in developing countries. The goal of this work is to develop an immunochromatographic RDT for the detection of P. jirovecii in non-invasive specimens like serum. In this test, spherical AuNPs are conjugated with a multi-epitope synthetic recombinant antigen (msr) which will allow the detection of circulating anti-P.
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jirovecii antibodies in sera. In order to obtain the highest amount of pure antigen, the expression vector pLATE 31, which contains the coding sequence for the MSG g antigen, was isolated and cloned in E. coli XJb (DE3). Extraction and purification through affinity chromatography with immobilized metallic ions, ELISA, SDS-PAGE and Western-Blot, were performed with the objective to obtain the maximum quantity of antigen and determine its purity. The antigen was then used to form bionanoconjugates with AuNPs, previously functionalized with several types of ligands, which will be the core of the immunochromatographic RDT with potential for point-of-care diagnostics. Acknowledgments: Partially supported by Gilead GENESE-PGG/001/2014.
CHANGES IN CILIATES COMMUNITY ACROSS AN EUTROPHICATION GRADIENT IN A LARGE TEMPERATE ESTUARY Rubin E., Santoferrara L., Zhang H., McManus G. Department of Marine Sciences, University of Connecticut, Groton, CT, USA ewelina.rubin@uconn.edu
The ciliate community composition in Long Island Sound, USA, was investigated using high throughput sequencing (Miseq, Illumina) ofvariable region 4 (V4) of the small subunit ribosomal gene (V4-SSU). Due to its proximity to heavily populated areas, the western Sound (WLIS) is eutrophic, and strong gradients have been observed down-estuary (east to west) in nutrients, chlorophyll, and plankton biomass. We sampled six stations on the long axis of the estuary, at two depths (surface and 6 m), in the summer of 2015. Analysis of ciliate V4-SSU sequences grouped into operational taxonomic units (OTUs) corresponded to the eutrophication gradient, with higher OTU-richness found in WLIS compared to the east. The majority of ciliates were classified into Choreotrichia and Oligotrichia, contributing together 65% to 91% of all ciliate OTUs. Litostomatea and Prostomatea contributed from 4 to over 30% of the OTUs. The relative proportion of Oligotrichia was higher near the surface and increased in the eastern, more pristine stations. The Choreotrichia showed the opposite pattern. The relative proportion of Oligotrichs and Choreotrichs across the eutrophication gradient in Long Island Sound was correlated with water transparency and it could be partially explained by variation in abundance of major phytoplankton groups in the western, central and eastern Long Island Sound.
TERRESTRIAL ALGA KLEBSORMIDIUM IN THE LIGHT OF THE HYPOTHESIS „EVERYTHING IS EVERYWHERE, BUT THE ENVIRONMENT SELECTS" Rysanek D.1, Rindi F.2, Holzinger A.3, Elster J.4, Skaloud P.1
1 - Department ofBotany, Faculty ofScience, Charles University ofPrague, Benatska 2, CZ-12801, Prague, Czech Republic
2 - Dipartimento di Scienze della Vita e dell'Ambiente, Universita Politecnica delle Marche, Via Brecce Bianche, IT-60131, Ancona, Italy
3 - University of Innsbruck, Institute of Botany, Functional Plant Biology, Sternwartestrase 15, A-6020 Innsbruck, Austria
4 - Centre for PolarEcology, Faculty ofScience, University of South Bohemia, Ceske Budejovice and Academy of Sciences of the Czech Republic, Institute of Botany, Phycology Centrum, Trebon 37901, Czech Republic rysadavid@seznam.cz
Baas Becking's hypothesis "Everything is everywhere, but the environment selects" first mentioned in 1934 still contains three current controversial issues in protists: disunity in the description of diversity, distribution patterns, and the ecological preferences. We examined a very common, globally distributed terrestrial algal genus Klebsormidium and tested (i) whether there is any biogeographical pattern of this microorganism, (ii) if there is a comparable diversity in the polar and temperate regions, and (iii) a potential role of ecological speciation processes on diversification.Our analyses revealed the presence of two different distribution patterns which are supposed to characterize both macroorganisms and protists. We demonstrated an unlimited dispersal and intensive gene flow within one of the inferred lineages (superclade B). However, the majority of Klebsormidium clades showed rather a limited distribution. In addition, we detected a significant decrease of species richness towards the poles, i.e. the macro-ecological pattern typical for macroorganisms. Species within a single protist genus may thus exhibit highly contrasting distribution patterns, based on their dispersal capabilities, which are usually shaped by both intrinsic and extrinsic factors. In addition, we determined a distinct ecophysiological differentiation among distantly and closely related lineages, thereby corroborating our hypothesis that the sympatric speciation of terrestrial algae is driven by ecological divergence. We clearly showed that pH is a critical ecological factor that influences the diversity of autotrophic protists in terrestrial habitats.
