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Protistology ■ 47
D. Méndez-Sánchez. To IRCN-BC for the grant to D. Méndez-Sánchez for a short stay at Laboratory of Protozoology OUC. Special thanks to Miss Chundi Wang and Mr. Zhishuai Qu (Laboratory of Protozoology, OUC) for their technical assistance in the molecular procedures and staining protocols respectively. We thankful to people from Tziscao, Chiapas, who allowed us recollected samples.
DIVERSITY OF THECAMOEBID AMOEBAE (AMOEBOZOA: DISCOSEA: THECAMOE-BIDAE) Mezentsev Y.S.
Saint-Peterburg State University [email protected]
Amoebae of the family Thecamoebidae are widely distributed in the different environments: salt and fresh water and soil. These protists are relatively easy to isolate and cultivate. They are relatively easy determining to genus, or even to species, thus representing a convenient object for many kinds of studies. The frequency of occurrence of species is very different: there are rather common ones like Thecamoeba quadrilineata, T. similis and T. orbis as well as numerous species known from few findings or never re- isolated since initial description. Many of these species were studied only at the light-microscopic level and require investigation with modern methods, including electron microscopy and molecular studies (this especially concerns the genus Thecamoeba and genera ofunclear systematic position like Pseudothecamoeba and Thecochaos). Our studies show that "hotspot" of Thecamoeba diversity is terrestrial habitats — soil, grass, dry leaves and surface of trees. During our studies we isolated 20 strains of Thecamoeba; some were identified as known species (Thecamoeba aesculea, T. similis, T. quadrilineata and T. terrícola) and 6 strains, which represent new species of the genus Thecamoeba. We have found two new strains of amoebae belonging to the genus Sappinia. In contrast, amoebae of the genus Stenamoeba were never found in terrestrial samples. Our data shows that species diversity of thecamoebid amoebae remains considerably underexplored.
Supported with Russian Science Foundation grant 14-14-00474.
ESTABLISHING CRYPTOSPORIDIUM AS A
MODEL FOR STUDYING THE BIOLOGY
AND EVOLUTION OF APICOMPLEXANS
AND UNIQUE ORGANELLES
Miller C., Brown I., Howard M., Michaelis M.,
Tsaousis A.
University of Kent, UK
Cryptosporidium spp. display a number of unusual traits, distinct from other apicomplexans; notably a smaller genome and the absence of an essential organelle, the apicoplast. There are also intra-species differences, such as a reduced mitochondrion in C. parvum and C. hominis, in size and functions. Studying these organisms holds great potential for our understanding of how the apicomplexans diverged. Studies of Cryptosporidium are inhibited by a limited selection of in-vitro culturing systems, with short lifespans and production volumes. Using a novel cell line for propagation we have seen a significant increase in parasite production volume as well as a longer lifespan, which have enabled the development of many new techniques for studying Cryptosporidium and its cell biology and biochemistry. Amongst these new techniques is the development of NMR and Mass spectrometry protocols, which have been optimised to examine the metabolic flux of the parasite, as well as investigating the role of the mitosome. We have also acquired many electron microscopy (EM) images ofthe parasite during its life cycle, identifying details of structures at levels previously unseen in the literature. Furthermore, using Immuno-EM we have shown the first experimental evidence of the function of the mitosome in C. parvum, with the localisation of iron sulphur cluster biosynthesis proteins.
DIVERSITY AND TEMPORAL DYNAMICS OF CRYPTOMYCOTA AND APHELIDA, TWO OVERLOOKED GROUPS OF PARASITES IN FRESHWATER ECOSYSTEMS Moreira D.1, Jardillier L.1, Bertolino P.1, Karpov S.A.2, Lopez-Garcia P.1
1 - Ecologie Systématique Evolution, CNRS - University Paris-Sud, Orsay, France
2 - Zoological Institute, Russian Academy ofSciences, St. Petersburg, Russian Federation [email protected]
Basal-branching fungi, especially chytrids, are important heterotrophic members of aquatic microbial food chains, especially in freshwater. More recently, two additional groups very distantly related to fungi, Cryptomycota (or Rozellomycota) and Aphelida, have also been found to be ubiquitous members of freshwater microbial communities. Together with the Microsporidia, they form the recently described superphylum Opisthosporidia. All known opisthosporidian species are parasites of very diverse eukaryotic hosts. Remarkably, both cryptomycetes and aphelids are able to feed by phagotrophy, a chief difference with microsporidia
48 • "PROTIST—2016
and true fungi, all of them osmotrophic. Despite the ubiquity of cryptomycetes and aphelids in diverse environments, little is known about their abundance and temporal dynamics. We have carried out a 2-year monthly survey of eukaryotic plankton diversity in five contrasted freshwater ecosystems (one brook, one small lake, and three shallow ponds) using massive 18S rRNA gene amplicon sequencing to compare cryptomycetes and aphelids with the much better-known chytrids. OTU analysis reveal that cryptomycetes and aphelids are less diverse than chytrids (556, 313, and 1274 OTUs, respectively) although in some moments cryptomycete+aphelid combined OTU number can exceed that of chytrids. Cryptomycetes show stable low numbers of sequences (<1% of total sequences) all along the year, in contrast with aphelids, which exhibit a more heterogeneous dynamics with recurrent abundance peaks in early autumn (>4% of sequences), when they become even more abundant than chytrids. These results suggest that cryptomycetes and aphelids are important overlooked members of freshwater ecosystems that most likely control other eukaryotic populations through their parasitic activity.
APPLICATION OF RECOMBINANT ANTIGENS FOR THE SERODIAGNOSIS OF TOXOPLASMOSIS
Mota Catia, 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 [email protected]
Toxoplasma gondii is an opportunistic apicomplexan protozoon that can cause devastating disease in immunosuppressed patients and congenital infection. The diagnosis of toxoplasmosis is usually done by observing the parasite in biological samples or by the detection of specific IgM and IgG against T. gondii antigens in the patient's serum. The improvement of toxoplasmosis diagnostic techniques and the differentiation between the infection stages can be achieved using recombinant antigen. This study aims to use micronemal protein MIC3 (elicits a strong specific host immune response) recombinant antigens in the serodiagnosis of toxoplasmosis. Toxoplasma RNA was isolated using the Tri-Reagent method and a PCR was performed using primers for MIC3 nucleotide sequence. The bands corresponding in size to the recombinant plasmid were purified and cloned into the cloning vector pLATE 28 and in the expression vector
pLATE 31. An expression study was performed in different E. coli BL21 (DE3) strains: Star, XJB, RIPL and PlysS. These strains were transformed with the recombinant vector pLATE 31 in order to obtain clones. Three random clones were therefore selected and induced with IPTG. The result ofthe induction was observed on a SDS-PAGE electrophoresis. The recombinant protein was purified by high-affinity chromatography with immobilized nickel ions and subsequently analyzed by ELISA, SDS-PAGE electrophoresis and quantified by Nanodrop 1000. Preliminary results show that the best E. coli strains for expression are BL21 (DE3) RIPL and BL21 (DE3) PlysS, based on SDS-PAGE analysis. The optimization of the ELISA assay is in progress. Acknowledgments: Supported partially by FCT ref:VIH/SAU/0019/2011.
THE RAPUNZEL TINTINNID - REDESCRIPTION OF TINTINNOPSIS SUBACUTA JORGENSEN, 1899 (ALVEOLATA, CILIO-PHORA, SPIROTRICHA) Muhlthaler A., Kagerer M., Agatha S. Dept. Ecology and Evolution, University ofSalzburg, Salzburg, Austria [email protected]
Tintinnids contribute distinctly to the microbial biomass in the marine plankton. Since the species have specific requirements concerning physico-chemical conditions and food items, reliable identification is indispensable for assessing their role in the food web. About one thousand extant tintinnid species are known, whose descriptions are exclusively based on the features of their loricae (houses); merely in about 30 species, cell characteristics have been studied. Since lorica shape and size are affected by environmental conditions and might show a polymorphism in the cell cycle, the tintinnid classification is artificial. Investigations of the cell, especially of the ciliary pattern and nuclear apparatus (generative micronuclei and somatic macronucleus nodules) are, however, supposed to provide features for a natural classification; these characters are revealed by protargol (silver proteinate) staining. Tintinnopsis subacuta was collected from surface waters of the Indiana River at the Atlantic coast of Florida (USA) and stained with protargol. Cell and lorica morphology were investigated under a compound microscope at up to 1250* magnification. The lorica is 55-119 ^m, on average 79 ^m long and consists of a cylindroidal collar about 34 ^m across and a subspherical bowl about 45 ^m wide. The lorica wall has agglutinated mainly mineral particles. The contracted cell
