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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
Protistology ■ 49
measures 30^28 ^m and is attached to the bottom of the lorica by a contractile peduncle. The somatic ciliary pattern is of the most complex type, i.e., it comprises a ventral, dorsal, and posterior kinety as well as a right, left, and lateral ciliary field. The ventral kinety has associated an extraordinary ciliary tuft of cell length that extends outside the lorica posteriorly, resembling the golden hair let down from the tower by Rapunzel; T. subacuta is unique in this respect. The right and left ciliary fields are composed of about 11 ciliary rows each, the lateral field consists of invariably 15 rows. While the majority of tintinnids have only two macronucleus nodules, T. subacuta has 4—34, on average 14 nodules. Financially supported by FWF Project P28790.
PROTISTAN VERSUS CYANOBACTERIAL PICOPHYTOPLANKTON PRODUCTION AND GRAZING MORTALITY IN SEVASTOPOL BAY AND ADJACENT WATERS (THE BLACK SEA)
Mukhanov V.S., Rylkova O.A., Sakhon E.G. A.O. Kovalevsky Institute of Marine Biological Research, Russian Academy of Sciences, Nakhimov av. 2, Sevastopol, 299011, Russia [email protected]
Seasonal dynamics of abundance, specific growth rate, daily production and grazing mortality of the major picophytoplankton components, eukaryotic protists and prokaryotic cyanobacteria, were studied at three stations in Sevastopol bay and adjacent waters (the Black Sea) in 2014 by flow cytometry and dilution method. In the shallow coastal waters, protistan picophytoplankton (PP) dominated (64 ± 23 (SD) %, n=26) the community in terms of abundance (annual average of 16.3 ± 12.4 * 103 cells ml-1), with the latter increasing along the nutrient and pollution gradient from the coastal waters outside the bay (7.3 ± 5.4 * 103 cells ml-1) to the eastern corner ofthe bay (28.7 ± 11.4 * 103 cells ml-1). PP demonstrated significantly lower specific growth rates (0.20 ± 0.19 d-1) and significantly higher daily grazing mortality (4.0 ± 5.8 ^g C l-1 d-1) than cyanobacterial picophytoplankton (0.70 ± 0.46 d-1 and 1.1 ± 1.1 ^g C l-1 d-1, respectively) while the protistan and cyanobacterial daily productions did not differ significantly (paired t-test, p>0.05, n=26). Matter flows through both the community components were comparable to or even exceeded their biomass stocks that indicated high biomass turnover rates. Thus, the protistan component has been shown to play a major role in the community functioning in the Black Sea coastal waters.
THE SPECIALIZATION OF THE PROTO-MITOCHONDRION AS A RESPIRATORY ORGANELLE
Muñoz-Gómez S.A., Roger A.J., Slamovits C.H. Centre for Comparative Genomics and Evolutionary Bioinformatics, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada. [email protected]
The ancestor of mitochondria was an alpha-proteobacterium whose exact phylogenetic and phenotypic nature remains obscure. Therefore, the proximate selective force that drove the initial endosymbiosis is unknown, even though the ultimate selective advantage was undoubtedly greater efficiency in energy production through aerobic respiration. The specialization of the proto-mitochondrion as the respiratory organelle of eukaryotes required the host to exert increased control over the biogenesis of the newly evolving organelle. Among the several adaptations that transformed the ancestral endosymbiont into a respiratory organelle, two evolutionary innovations were of major importance. The first major innovation was the evolution of mitochondrial cristae to make respiratory sub-compartments. Cristae likely evolved from precursor structures in alpha-proteobacteria. Later molecular innovations further modified cristae to improve their respiratory function. This required the expansion of MICOS (Mitochondrial contact site and Cristae Organizing System) and the evolution of the capability of the ATP synthase complex to form multimers. The second major innovation was the evolution of the ability ofthe host to control the overall morphology, positioning and distribution of mitochondria within the cell. These adaptations optimized bioenergetic output in response to host needs. This was made possible by the origin of mitochondrial fusion, as well as the establishment of interactions between mitochondria and diverse endomembranes and the cytoskeleton. I discuss a detailed evolutionary scenario for the evolution of these two major adaptations in the context of the co-evolutionary integration of mitochondria and their host.
THE ULTRASTRUCTURE OF AMOEBOID
FLAGELLATES AMASTIGOMONAS (CERCO-
ZOA, RHISARIA)
Mylnikov A.A., Mylnikov A.P.
Papanin Institute for Biology of Inland Waters,
Russian Academy of Sciences, Borok, Russia
The cytoskeleton of three amoeboid flagellates Amastigomonas spp. dwelling in freshwater (one
