5 (1) 991 (2007) Protistology

Abstracts of V European Congress of Protistology and XI European Conference on

CHARACTERIZATION OF MICROSPORIDIAN sporidiosis. Finally, we have recently identified and METHIONINE AMINO PEPTIDASE TYPE 2 cloned the Enterocytozoon bieneusi MetAP2 gene, (METAP2): A THERAPEUTIC TARGET allowing studies on this noncultivatable J. Aclvarado1, R. Upadhya2, A. Nemkal2, H. Zhang2, pathogen. Supported by NIH AI31788 and AI 069953. S. Almo1, L.M. Weiss3 1 Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY10461, USA; 2 Department of Pathology, Albert Einstein ABUNDANCE, DYNAMICS AND SUC College of Medicine, Bronx, NY10461, USA; 3 Departments of CESSION IN SOIL Pathology and Medicine, Albert Einstein College of Medicine, Bronx, M.S. Adl NY10461, USA. Email: [email protected] Department of Biology, Dalhousie University, Canada. Microsporidia are parasites of all classes of vertebrates Email: [email protected] and most invertebrates. They have recently emerged as Soil are responsible for a significant portion important infections in various immunosuppressed of the bacterivory and cytotrophy. They also contribute patient populations. Current therapies for microspo to other trophic functional groups. According to ridiosis include benzimidazoles, which bind tubulin empirical models, soil protozoa are an important rate inhibiting assembly, and fumagilin, which regulating component of decomposition food webs. The binds and inhibit Methionine Aminopeptidase Type 2 activity of these protozoa varies with local abiotic (MetAP2). We have initiated a program to define the conditions on the short term, and there are seasonal unique structural features of microsporidian MetAP2 and succession patterns on the long term. We have using Encephalitozoon cuniculi (Ec) MetAP2. Despite shown that day to day abundance variations, in response extensive efforts using E. colibased expression systems, to weather, fluctuate by up to one thousand times. In no active/soluble material could be obtained. However, forest litter, we have shown there are successional baculovirusdriven expression in Sf9 cells yielded patterns with seasons, and with litter chemistry. multiple milligrams per liter of EcMetAP2. This Over longer periods, we observed increase in species material was enzymatically active and has been diversity over a 50 year chronosequence, in agriculture characterized. In addition, this material yielded fields. These fields were transferred from tillage to no structures of the native enzyme and the EcMetAP2 till management, so the soil profile was undisturbed and fumagilin complex at a 0.21 nm resolution. Residues allowed to develop. Although abundance fluctuations contacting fumagillin that are not conserved between were governed by abiotic changes, longer term suc human MetAP2 and EcMetAP2 are D256 and H210, cessional and diversity changes were correlated with and these differences give clues as how to modify fuma changes in the profile due to organic matter accumu gillin in order to enhance its specificity for EcMetAP2. lation. Similarly, postmining sites under remediation To further expedite and facilitate the discovery of safe showed similar trends of diversity changes with soil and effective MetAP2 inhibitors, we have also engine profile development. These studies are being extended ered Saccharomyces cerevisiae to be dependent on at a forested site under conservation that offers several EcMetAP2 for its growth, where EcMetAP2 is harbored soil profile chronosequences. This study site allows on an episomal uracil selectable tetracycline regulated comparison of soil protozoa community structure with plasmid. By comparing yeast dependent on human and plant species composition, along with different soil microsporidian MetAP2 in these assays we are able to profiles and organic matter content, due to past land screen for new compounds for the treatment of micro use history, at the same climatic region. The aim is to

© 2007 by Russia, Protistology 10 · Abstracts of V European Congress of Protistology · 2007 conduct sufficient studies to distinguish between the fragments, it becomes surrounded by plant rhizosphere effect, the soil profile organic matter, argyrophilic vesicles (probably autophagosomes). The the mineral chemistry, as they interplay and affect soil performs three typical maturation community structure changes on the long term. divisions. While the mates are still connected, the syn karyon divides twice: one derivative becomes a micro REDESCRIPTION OF STENOSEMELLA NIVALIS nucleus, one a macronuclear anlage, and two disinte (CILIOPHORA, SPIROTRICHA, TINTINNINA) grate. Likely, the somatic bristle rows are reduced from BASED ON LIVE OBSERVATION, PROTARGOL seven to four in each partner, i.e., the first becomes IMPREGNATION, AND SCANNING ELECTRON imperceptible on the ventral side during pair formation, the second and third when the contact of the partners S. Agatha becomes more intimate. During the second maturation University of Salzburg, Department of Organismal Biology, Salzburg, division, somatic anlagen become recognizable as in Austria. Email: [email protected] ordinary ontogenesis; their fate remains obscure. Although Stenosemella nivalis (Meunier, 1910) Kofoid During prophase I, the partners become dimorphic: the & Campbell, 1929 is widely distributed in coastal collar membranelles of the more anteriorly located waters, its cell and morphology were poorly conjugant arrange around the pair's anterior end, for known. The specimens were collected in the pelagial of ming a membranellar zone for both partners; its buccal the Irish Sea near the Isle of Man during spring. The membranelles as well as the collar and buccal membra investigations comprised live observation, protargol nelles of the more posteriorly located conjugant dis impregnation, and scanning electron microscopy. × appear. Simultaneously, an oral primordium originates Lorica ~ 53 40 µm in size, amphoriform, incrustrated on the ventral side of both partners, probably generating by quartz particles, except for hyaline collar with ~ 7 only the collar membranelles. Halteria resembles the ellipsoidal windows; occasionally, with agglutinated choreotrichid ciliate Pelagostrobilidium in the inter second collar anterior to the hyaline one. Cell in × locking arrangement of the partners, while the dimor extended state ~ 55 18 µm, attached to bottom of phism and the shifting of the partners as well as the lorica by ~ 7 µm long stalk; in protargol preparations, × common membranellar zone are like those in the Sti specimens usually contracted and only 34 21 µm in chotrichida. Since data on the Oligotrichida are not size. Nuclear apparatus composed of two ellipsoidal available, the apomorphic character states are unknown. macronuclear nodules and usually two globular Study supported by the Austrian Science Foundation micronuclei. Capsules (probably extrusive ) (FWF; projects P17752B06 and P19699B17). in striae (beaded strands) on the collar membranelles and tentaculoids (fingerlike cytoplasmic processes) DIVERSITY OF EPIBIONT OF VARIOUS originating from the intermembranellar ridges. Somatic ORGANIC SUBSTRATES IN MEXICO ciliary pattern is complex and comprises the right, left, M.A. AladroLubel, R. MayenEstrada, M. ReyesSantos and lateral ciliary fields, as well as the monokinetidal Laboratorio de Protozoologia, Departamento de Biologia Comparada, ventral, dikinetidal dorsal and dikinetidal posterior Facultad de Ciencias, Universidad Nacional Autonoma de Mexico. kineties. Oral apparatus is transversely orientated at the Email: [email protected] apical cell end. Adoral zone of membranelles is closed, The total number of free living and symbiotic ciliates composed of one buccal and ~ 18 collar membranelles. reported from Mexico is 952 species. The epibiont Endoral membrane probably is of monostichomonad ciliates (106 species) represent 11.13% of the total type. Oral primordium originates in the subsurface species richness. Results are based on studies of ciliate pouch posterior to lateral ciliary field. Study supported diversity on different organic substrates during several by the Austrian Science Foundation (FWF; project years using optical and scanning electron microscopy. P17752B06). On the leaves of the angiosperms Thalassia testudinum, Phyllospadix sp. (marine), Halodule beaudetti, Rhizo CONJUGATION IN HALTERIA GRANDINELLA: phora mangle roots (brackish) and freshwater , 77 THE MYSTERY OF ITS GENEALOGY PERSISTS species of epibiotic ciliates have been identified. On S. Agatha, W. Foissner seven species of brackish , 47 ciliate species have University of Salzburg, Department of Organismal Biology, Salzburg, been observed, and on filamentous freshwater algae we Austria. Email: [email protected] found seven species. The most studied invertebrate has The conjugants are isomorphic and fuse partially to a been the freshwater crustacean Cambarellus patzcuaren homopolar pair. The partners become ventrally concave, sis (a decapod), on which distribution of 28 species of obtain an interlocking arrangement, and one is shifted epibiotic ciliates has been studied. On Hyalella azteca slightly posteriorly. The pair almost achieves the size (freshwater amphipod) and other crustaceans, five and outline of the morphostatic specimen. Before the species have been reported, and on Penaeus (brackish Protistology · 11 decapod) only one species. Of the seven groups of wild rodents and domestic to serve as potential epibiotic ciliates (apostomatids, folliculinids, licnopho reservoirs of opportunistic Microsporidia in HIVinfec rid, peritrichs, prostomatids, stichotrich and sucto ted , we examined farm animals and rodents in rians), the ciliated were the most diverse group, semidesert areas of Azerbaijan: in vicinity of Baku City represented by 62 species (58.5%). Acineta tuberosa, megalopolis, and in GobustanAbsheron and Davachi campanula and Platycola decumbens are the regions. Fecal samples from 144 rodents, 20 cows and three common species reported from angiosperms, algae buffaloes, 20 horses, 20 donkeys, 30 sheep and goats and Cambarellus patzcuarensis. were collected. Samples were kept in 70% ethyl alcohol. Fixed fecal smears were analyzed under fluorescent FAUNA OF FREELIVING CILIATES IN AZERBAI . Positive samples were double checked with JAN: MODERN STUDIES AND PERSPECTIVES PCR. Microsporidialike objects were found in calco I.Kh. Alekperov fluorstained fecal smears of 22 Meriones libycus and 1 Institute of NAS of Azerbaijan. Email: [email protected] red fox (Vulpes vulpes). Fecal samples of Microtus socia At present, fauna of ciliates in Azerbaijan includes ca. lis, Allactaga mayor, Allactaga elater were free from any 1300 species. It is known that 620 ciliate species are spores of Microsporidia. Additionally, we did not detect from the Caspian Sea, 780 species from inland waters, any spores in fecal samples from farm animals. PCR and about 550 species from soil samples from different based method failed to detect microsporidia in positive places of Azerbaijan. A lot of species have been found samples. Supported by US Collaborative Program, Post in two or three different biocenoses. The analysis of the Doctoral Fellowship Ref. Nr 051134445 INTAS published data and our own experience showed that the foundation. ciliate fauna consists of three main groups: the first group of eurybiont cosmopolitan species includes about CHANGES IN RAT HEPATOCYTE PLOIDY AND 25% of ciliates widely distributed worldwide. The HYPETROPHY AFTER CRYPTOSPORIDIAL GAST second group, about 20%, is stenoterm (coldresistant ROENTERITIS ( PARVUM, or heatloving) species which are distributed in various SPOROZOA, ) regions with similar ecological conditions. The third O.V. Anatskaya, N.V. Sidorenko, A.E. Vinogradov, and the greatest group (55% or more), are stenobiont T.V. Beyer ciliate species, some part of which might be endemic. Institute of Cytology RAS, St. Petersburg, Russia. On my opinion, for the full statistically significant Email: [email protected] investigation of ciliate fauna of any region of interest, Using the laboratory model of mild, moderate, and not less than 15 years should be spent, because usually acute intestinal in suckling (1014 during the first years the researchers register ciliate days old) and weaned (1822 days old) rats, we tested species belonging only to the first and second group. the extent of cryptosporidial impact on hepatocytes The real estimation of ciliate fauna of any region (first (where no cryptosporidian development occurs) at the of all, the soil species as inhabitants of the most peak of infection and after recovery from disease. Light heterogeneous environment) is possible only after long and confocal microscopy and image analysis were term regular observations, when the number of rare applied for studying changes in cell morphology. Protein stenobiont species increases from year to year. For amount and cell ploidy levels were measured by means example, despite of previous longterm research of absorption and fluorescent cytophotometry. Liver performed by Agamaliev, we found about 180 new ciliate hypertrophy and obvious growth retardation were species in the Caspian Sea. remarkable outcomes at the 4th day of cryptosporidian infection. Our results show that cryptosporidiosis is able to provoke a burstlike premature hepatocyte polyplo PILOT STUDY ON MICROSPORIDIAN INFECTI idization and hypertrophy (in proportion to parasitic ON OF TERRESTRIAL VERTEBRATES IN AZER load). In 14 and 22 days old infected rats, the percentage BAIJAN of hepatocytes with multiplicated , including M.A. Aliyev Institute of Zoology, Azerbaijan National Academy of Sciences, 4n, 4nx2, 8n, and occasionally 16n cells, were Department of Protozoology, Baku, Azerbaijan. Email: [email protected] substantially raised above control values and were Microsporidia is a group of obligatory intracellular pa similar to those in 1 to 23 month old uninfected rats rasitic protozoans of high practical significance. Infec respectively, i.e. rats after transition from fluid (milk) tion of HIVpositive individuals with microsporidia, to solid food and liver parenchyma reorganization. At especially with the species of genera Encephalitozoon day 21 after cessation of diarrhea, the difference in and Enterocytozoon, can radically complicate the hepatocyte ploidy still persisted, whereas the difference process of the main disease, since microsporidia may in cell protein content almost completely smoothed out. cause opportunistic infections. To determine ability of This is the first documented evidence of serious patho 12 · Abstracts of V European Congress of Protistology · 2007 logical changes in hepatocytes of early postnatal rats rized by presence of hydrosulphuric layer and absence experimentally infected with the intestinal pathogen C. of fish. A. salinus is a dominating species of zooplankton parvum. The observed alterations in hepatocytes suggest (up to 100%). Till now, despite of high prevalence of that cryptosporidiosis modulate liver ploidy through yet peritrich epibionts of calanoid copepods, the ciliates unknown mechanisms, and to predict some presumably have not been considered as a component of fauna of negative consequences of cryptosporidiosis in later these lakes. Identification and a morphological charac of infected rats. Supported by CRDF Project RUB2 terization of peritrichs were performed by live observa 002707SP05 and RFBR. tions in light microscope equipped with Lumenera INFINITY digital camera. Infestation density, load, PHYLOGENETIC RELATIONSHIPS WITHIN mean number of zooids per on the host (adults, PENICULINE CILIATES AND THE copepodites and nauplii of A. salinus) and morfometric OF characteristics of sp. were registered. Zooids I. Andreoli, S.I. Fokin, F. Verni, G. Petroni were 37.8±1.3 µm long and 24.8±1.0 µm wide. Colonies Universitа di Pisa, Dipartimento di Biologia, Unitа di Protistologia contained up to 15 bellshaped zooids (in the lake Shira). Zoologia, Pisa, Italy. Email: [email protected] Zooids in the lake Shunet were 39.1±1.6 µm long and The peniculine ciliates Frontonia are usual members of 26.7±1.5 µm wide; number of zooids per colony was 2 the pelagic and benthic fauna in both freshwater and 15. Infestation rates were high. In the lake Shira, 18.4% marine environments. The comprises about thirty of nauplii, 53.4% of copepodits and up to 66.7% of species but the validity of some of them still has to be adults were infected in summer; 39.3% of copepodits, confirmed. Despite the high number of species, few up to 34.2 % of adults were infected in winter. In the lake molecular data are available for this genus. Here we Shunet, 52.6% of nauplii, 82% of copepodits, and 80 present 18S rRNA molecular data of nine different, 87.5% of adults were infected in summer; in total, 98 morphologically recognizable, Frontonia species. Five 100% of calanoid copepods were infected in winter. species were classified as Frontonia leucas, Frontonia Peritrich epibions were localized on the cephalothorax, salmastra, Frontonia minuta, Frontonia atra and Fron abdomen, swimming legs, antennae, or on the whole tonia fusca. The identification, or de novo description, surface of the host. of the remaining species is still ongoing. The phylo genetic analysis based on 18S rRNA data showed that PLASTICITY IN THE DICYEMID ME the genus Frontonia is paraphyletic, with the re SOZOANS presented by genera Apofrontonia and H. Awata, T. Noto, H. Endoh branching from within. In particular, Frontonia is split Division of Life Science, Graduate School of Natural Science and into three subgroups. One of these (Cluster 1) is Technology, Kanazawa University, Kanazawa 9201192, Japan. associated with Apofrontonia dohrni, despite the Email: [email protected]u.ac.jp significant morphological differences. Preliminary Dicyemid mesozoans are obligate parasites that inhabit analyses suggest that the genus Paramecium associates the cephalopod renal appendage. They have a simple to both Apofrontonia dohrni and Frontonia spp. Cluster body construction: one large cylindrical axial cell 1. Despite the observed molecular differences, all the contains intracellular stem cells (called axoblasts) from analyzed species display the typical grossmorphologi which embryos are derived, and is surrounded by some cal features of Frontonia. According to these data, many 30 ciliated peripheral cells. Previously, it was shown distinctive traits of the genus Frontonia could be plesio that numerous unique DNA sequences are first ampli morphic characters of limited systematic value. A fied and then eliminated during early somatic cell deve taxonomic revision of the genus will be necessary in the lopment, in the form of extrachromosomal circular next future. DNAs, leading to genome reduction. Here, we demon strate using in situ hybridization and incorporation of THE EPISTYLIS SP. (CILIATA, PERITRICHA) AS BrdU that other DNA elements, such as single copy EPIBIONTS OF CALANOID COPEPODS ARCTO genes and repetitive sequences, have very different fates. DIAPTOMUS SALINUS (DADAY) IN BRACKISH Single copy genes represented by btubulin are initially LAKES IN KHAKASIA amplified, presumably via endoreduplication, but T.N. Anufrieva subsequently decrease in copy number through develop Siberian Federal University, Krasnoyarsk, Russia. ment, suggesting that a whole genome is initially ampli Email: [email protected] fied and then the amplified DNAs are simply diluted in We examined Epistylis sp. associated with calanoid successive cell divisions, with little DNA replication. copepods Arctodiaptomus salinus in brackish lakes in In contrast, tandemly repetitive sequences are main 20052007. The lakes Shira and Shunet are characte tained even in terminally differentiated somatic cell Protistology · 13 nuclei. Taking the increasing intensity of in situ hybri (120140 species per site). By contrast, the point diver dization after somatic cell differentiation, incorporation sity (estimated as average number of species per station of BrdU, and a general correlation between nuclear for one sampling session) turns to be positively related content and cell size in consideration, those repetitive with absolute latitude (i.e., samples taken at higher sequences must be selectively endoreplicated in the latitudes tend to bring more species). We suggest that peripheral , concomitant with the increase marine benthic ciliates demonstrate many macroecolo of cell size. Similar genome plasticity is also observed gical patterns that consistently contradict the regula in mitochondrial genome, where initial amplification rities commonly obtained for macroorganisms. Thus, of mtDNA and subsequent copy number reduction even if ciliates have biogeography, it is very specific and occur, accompanied by minicircle formation in somatic quite different from that of macrobiota. mitochondria. Biological significance of the different behaviors of these DNA elements is discussed as a FACTORS INFLUENCING THE DIVERSITY AND unique to . DISTRIBUTION OF CERCOMONADS (CERCO ZOA) AT DIFFERENT PHYLOGENETIC SCALES: MACROECOLOGICAL PATTERNS IN DIVERSITY UBIQUITOUS AND ECOLOGICALLY DIVERSE OF MARINE BENTHIC CILIATES HETEROTROPHIC ZOOFLAGELLATES WHICH A.I. Azovsky1, Yu.A. Mazei2 YET SHOW CLEAR PATTERNS OF COMMUNITY 1 Moscow State University, Biology Faculty, Dept. of Hydrobiology, DIFFERENTIATION 2 Moscow, Russia, Penza State Pedagogical University, Dept. of D. Bass, A.T. Howe, C. Yackulic, H. Withers, Zoology and , Penza, Russia. Email: [email protected] The data on occurrence of over 1230 species of marine E.E. Chao, T. CavalierSmith University of Oxford, Department of Zoology, Oxford, UK. benthic ciliates compiled from more than 170 literature Email: [email protected] sources were used for studying the worldwide distribu We created and sequenced 18S rDNA libraries for tion and diversity of the group. Among 14 regions that Cercomonas and Eocercomonas from around the world, were distinguished, the Northeastern Atlantic is the with particular emphasis on local patterns of diversity richest one (totally 608 species), then follow the at a grassland sampling site near Oxford, UK. Individual Caspian, Baltic and Black seas (516, 456 and 434 spe soil samples (<1 gram soil) from the Oxford site each cies, respectively); Northwestern Atlantic, North Pacific contained between four and 20 genetically distinct and Antarctic regions have the lowerrange richness cercomonad 18SrDNA sequences (18Stypes), which (226, 218 and 94 species). Thirty one percent of species accounted for up to 66% of the total diversity detected were found in one region only; the others, however, in the 10m x 10m grassland site at a single timepoint. mainly had wide geographical distribution covering We then used this approach to compare assemblages of both hemispheres. There is no tendency to narrowing cercomonad 18Stypes over larger geographical the occupancy ranges for species found at high latitudes. distances, at various timepoints, and from different Comparison of faunistic composition reveals that ecological conditions. In these cases the overlap specieslevel similarity between regions roughly cor between sample sets in terms of shared 18Stypes was responds to their geographical position. This correspon significantly less than that seen within sets collected dence, however, disappears when similarity is estimated proximally in space and time. Thus we show that at the levels of genera or higher taxa, though ciliofaunas cercomonad communities become more dissimilar with of the Northwestern Atlantic, North Pacific and parti increasing geographical and temporal distance, even cularly Antarctic show noticeable specificity in their though our previous studies have shown that most, if composition. Regionalscale diversity (RSD) depends not all, 18Stypes can be found on all continents with neither on the total area, nor on the total coastline sufficient sampling effort. Cercomonad assemblages are length, and does not show any latitudinal gradients. At also influenced by ecological conditions: we recovered the same time, it highly correlates with investigation cercomonad sequences far less frequently from marine effort (estimated as total time of studies carried out in habitats and acid soils than elsewhere, and sets of soil the region). Besides, RSD correlates with average and permanently submerged sediment libraries sampled salinity (negatively, which is unusual!). Localscale at the same place and time were quantitatively more diversity (LSD, estimated as species richness at the similar within than between sets. We suggest that many selected wellstudied sites) does not depend on RSD habitats hold a large pool of dormant or relatively rare values, possibly indicating the high speciessaturation cercomonad strains and that observable differences in level of local ciliate communities. Also, RSD values do cercomonad assemblages between sites are determined not show any latitudinal trends. Moreover, regionave by a combination of environmental fluctuations and raged LSD values demonstrate striking resemblance stochastic effects of space and time. 14 · Abstracts of V European Congress of Protistology · 2007

IN SEARCH OF NUCLEAR ACTIN IN PARAME . Some plesiomorphies of the hypotrichs CIUM CAUDATUM are: (1) cirri present; (2) body dorsoventrally flattened; K.A. Benken, E.V. Sabaneyeva (3) body flexible; (4) frontalventraltransverse cirri SaintPetersburg State University, Department of Cytology and originate from 6 anlagen; (5) caudal cirri present; (6) Histology, St. Petersburg, Russia. Email: [email protected] cortical granules present; (7) parental adoral zone not For a long time actin was regarded only as a cytoplasmic reorganised during cell division; (8) lack of kineto protein. However, recent years have been marked by desmal fibre in interphasic specimens; (9) benthic; (10) increasing interest to its nuclear functions. Actin is telomeric repeat sequence TTTTGGGG. For more believed to be an important constituent of the nuclear apomorphic and plesiomorphic features and discussion matrix required for chromatin remodeling and (including convergences and alternative hypotheses) see . Actin has been found in the nuclei of poster and Monographs on hypotrichs by Berger (1999, several popular model objects, such as Xenopus oocytes Monographiae Biol., 78: 11080: oxytrichids), Berger and slime molds Dictyostelium and Physarum. Though (2006, Monographiae Biol., 85: 11303: urostyloids), nuclear matrix might be of fundamental importance in and Berger (2007, Monographiae Biol., in press: organization and functioning of the amplyploid macro amphisiellids). Financial support was provided by a nucleus, little if any is known about nuclear actin in grant (APART; Austrian Programme for Advanced Ciliates. The present study is the first attempt to de Research and Technology; Project 10940) of the monstrate the presence of actin in the nuclei of Parame Austrian Academy of Sciences, Vienna. cium caudatum. To reveal actin whole cells and paraffin embedded sections were treated with TRITCphallo idin or polyclonal antibodies to paramecium actin 11. MYOSIN II AND THE OF UNIKONTS Antibody treatment failed to detect actin 11 in the nuclei C. Berney, T. CavalierSmith which evidences for the absence of this form of actin in University of Oxford, Department of Zoology, Oxford, UK. the nuclei. Conversely, TRITCphalloidin staining was Email: [email protected] very intensive in both macro and micronucleus, and Based on the taxonomic distribution of myosin was mostly associated with chromatin, as shown in the combinations and the presence of myosin II in alternative sections stained with DAPI. Nucleoli in the particular, it has recently been suggested that Amoebo macronucleus and the achromatin cap in the micronuc zoa and share a common ancestor, in leus seem to lack actin. Our data argue for the presence accordance with their close relationship in several of Factin in the nuclei of P. caudatum. Further inves multigene phylogenies. They are grouped together in tigations of actin in the nuclear apparatus of ciliates are the unikonts, while all other would comprise underway. the . However, clear evidence for the monophyly of is yet missing. Thus, although some THE GROUNDPATTERN OF THE HYPOTRICHA members of this are known to belong to (SPIROTRICHA, CILIOPHORA) unikonts, the phylogenetic position of many lineages H. Berger of lobose amoebae remains unclear. Besides, relation Consulting Engineering Office for Ecology, Salzburg, Austria. ships between the amoebozoan lineages defined on the Email: [email protected] basis of morphology and smallsubunit ribosomal RNA The Hypotricha Stein (= Stichotrichia Small & Lynn) gene (SSU rDNA) phylogenies are still unresolved. In are a major group of the spirotrichs. The spirotrichs this study, we use myosin II to try and elucidate some show, mainly according to molecular data, the following of these important evolutionary questions. The presence structure: (euplotids + (oligotrichs + hypotrichs)). The of myosin II was tested in about 30 lobose amoebae, groundpattern (= groundplan) of an evolutionary unit representing almost all previously defined lineages of is of fundamental importance for the establishment of Amoebozoa, and a taxonomically rich phylogeny of a phylogenetic system. It is the combination of all types unikonts was produced. Our results show that (1) all of features (e.g., morphological, ontogenetic, ecological, members of the phylum Amoebozoa belong to uni molecular) of the stemspecies from which the mono konts, ruling out the possibility that the root of the phylum evolved, that is, it is a summary of apomorphies tree lies among Amoebozoa; (2) myosin II and more or less young plesiomorphies. Some supposed supports the monophyly of Amoebozoa; (3) relation apomorphies of the hypotrichs are: (1) 18 frontalvent ships among lobose amoebae as inferred from myosin raltransverse cirri; (2) 3 dorsal kineties; (3) 2 macro II sequences are in striking accordance with the SSU nuclear nodules; (4) contractile near proximal rDNA phylogeny; and (4) a specific duplication of end of adoral zone; (5) 2 undulating membranes (?); myosin II known in metazoans occurred before the (6) oral primordium originates on cell surface; (7) divergence of and ichthyosporeans, parental somatic ciliature completely replaced during supporting their close relationship to animals. Protistology · 15

ULTRASTRUCTURAL INVESTIGATION OF CHLA abnormalities. Initially was diagnosed as bacterial me MYDOMONAS CARRIZOENSIS AND THE VARIA ningitis then treated with vancomycin and cefotaxime BILITY OF TRADITIONAL MORPHOLOGICAL without success. CSF (cerebrospinal fluid) was purulent CRITERIA OF CHLAMYDOMONAS with counts of 47000 cells/mm3, glucose of zero, O.N. Boldina polymorphonuclear leukocytes (90%), Gramstain was Komarov Botanical Institute RAS, Algological Department, St. Peters negative for . Light microscopy examination of burg, Russia. Email: [email protected] CSF revealed the presence of amoebic trophozoites of The cell structure of an unicellular Chlamy freeliving amoebae (FLA), next patient was treated domonas carrizoensis Deason et Bold (strain 46.72 SAG) with amphotericin B, rifampin, fluconazole, trimetho described as the member of Pleiochlorisgroup has been primsulfamethoxazole. However, in spite of treatment, studied by light and transmission electron microscopy. the evolution was fulminant. CAT showed hemorrhage As a rule the cells of C. carrizoensis are cylindrical, subachnoidea massive and important cerebral edema ellipsoidcylindrical or ellipsoidegg, slightly asymmet and died 11 days after the onset of symptoms. Culture rical, 1421 µm in length, 512 µm in width. The 2 of CSF was negative for FLA. Sample of 500 ml of contractile are located at the edge of 2 flagella, thermal water where the child swam, was culture in identical or slightly shorter than cell length. The cell NNE medium at 37°C. After 18 hours, amoebic wall consists of 2 layers and bears a prominent keel trophozoites morphologically identified as sp. papilla. Under highly invaginated plasmalemma a great (Nw), were observed. Western blot assay between Nw number of homogeneous electron dense globules have and N. fowleri (ATCC30808) was made. The clinical been observed. The nucleus is of the complex chromo manifestations, morphological characteristics of the center type. Dictyosomes, vacuoles of 3 types and small amoebae and the results of Western blot, are compatible profiles of mitochondria are located perinuclearly. The with Naegleria fowleri, however the isolate was not is cupshaped, highly lobed, massive, with pathogenic in mice. Even thought Mexico is not con some perforations. It is formed by short stacks of 3 to 8 sidered as endemic zone of meningoencephalitis, there (15) thylakoids, stroma, containing some plastoglobu have been reported several cases of this pathology. It is les, starch grains and ribosomes. The real chloroplast important to consider it since the initial diagnosis in perforations have not been observed at the ultrastruc order to determine the real incidence of this pathology tural level. The stigma is formed by 3 rows of pigmental in our country. globules. The number of varies from 1 to 3 or extremely rare more. The pyrenoids are located in LOCOMOTORIAL BEHAVIOR OF PSAMMO thylakoid lobes by such way that the cells of C. car PHILIC CILIATES rizoensis can be refered to any of 3 groups of Chlamy V.V. Borisov, I.V. Burkovsky domonas: Chlorogoniella, Agloe and Pleiochloris. The Moscow State University, Faculty of Biology, Department of pyrenoids C. carrizoensis have the same structural type Hydrobiology, Moscow, Russia. Email: [email protected] as the representatives of Agloe (C. radiata, strain 47.72 We investigated locomotion of 11 species of psammo SAG) and Pseudagloe (C. mutabilis, 34.72 SAG) which philic (sanddwelling) ciliates, using framebyframe together demonstrate the nearest to Chloromonas video analysis. Twodimensional trajectories were localizations in different kinds of phylogenetic trees regarded as composition of arcs and straight segments (Proeschold etal., 2001; Nozaki et al., 2002; Pocock et (long lasting elements) and locomotorial patterns which al, 2004). determined their arrangement (short lasting elements). Long lasting elements of threedimensional trajectories PRIMARY AMOEBIC MENINGOENCEPHALITIS were regarded as helixes with straight or bend axis. IN MEXICO: CASE REPORT Difference in locomotorial behavior between some of P. BonillaLemus1, D. HernandezMartinez1, the species was observed. The greatest velocities were M. OmanaMolina1, C. Melchor2, A. Esparza2 typical for species which inhabit mainly surface layers 1 National Autonomous University Mexico, FESIztacala, of sea sand. Angles of contingence of arcs were Environmental Project, Mexico, 2 Infectology Hospital, Medical Center La Raza, IMSS, Mexico, Mexico. correlated with vertical distribution of ciliates. Smaller Email: [email protected] angles of contingence were more common for ciliates A 5year old male, immunocompetent with history of from deeper layers of sand. Fractal dimensions of contact with thermal water in a recreative centre 9 days trajectories were also negatively correlated with depth before the onset of symptoms: fever (3839°C), of distribution maxima. Difference in locomotion increased intracranial pressure, headache, nausea, between species with similar nutrition characteristics vomiting, somnolence, anisochoria, restlessnnes, was observed. We suggest that there is no strict tachycardia, generalized seizures and mental status connection between locomotion and nutrition of 16 · Abstracts of V European Congress of Protistology · 2007 ciliates. It was found that the values of locomotion DUODENALIS WB C6 INDUCES EARLY characteristics of species with different morphology CASPASEDEPENDENT APOPTOSIS IN HCT8 overlap strongly. It suggests the absence of strict mor CELL LINE phological determination of locomotorial behavior for O. Brandonisio1, P. Cavallo2, A. Cianciulli2, V. Mitolo2, ciliates. Smaller velocities, smaller reorientation angles, V. Lacasella2, M.A. Panaro2 and smaller fractal dimensions of trajectories that are 1 Department of Internal Medicine, Immunology and Infectious 2 characteristic to ciliates from deeper sea sand layers, Diseases, University of Bari, Italy, Department of Human and Histology, University of Bari, Italy. are consistent with spatial restriction of interstitial Email: [email protected] capillary system in natural habitats. We suppose also Giardia duodenalis is a flagellated protozoan which that environmental conditions of psammophilic ciliates causes enteritis in humans and animals. Giardia from deeper sand layer do not require considerable trophozoites adhere to epithelial cells of the duodenum, displacements and reorientations, as in case of super causing diarrhoea and often malabsorption, whose ficial species. Difference in locomotion of psammo pathogenesis remains poorly investigated. Apoptosis is philic ciliates could be the evidence of various locomo a cell program executed by intracellular cysteine torial tactics for exploring habitats and food seeking. proteases (caspases) activated through an extrinsic (caspase8mediated) and an intrinsic (caspase9 THE COMMENSAL SESSILE PERITRICHOUS mediated) pathways, which converge to activate the CILIATES (CILIOPHORA, PERITRICHIA) ON downstream effector caspase3, responsible for cell MOLLUSCS FROM WATER BODIES OF UKRA death. Several enteric pathogens, including protozoan INE AND THE MASURIAN LAKES OF POLAND parasites, are able to induce enterocyte apoptosis. In E.G. Boshko particular, some strains of G. duodenalis were reported Schmalhausen Institute of Zoology, Kiev, Ukraine. to induce enterocyte apoptosis, but the mechanism Email: [email protected] The material was collected in Ukraine during 19812006 involved in this process has not been clarified. There in fresh and brackish water bodies in the drainage basins fore, the aim of this work was to assess whether G. of the rivers Dnieper, South Bug, Dniester and Danube, duodenalis strain WB clone C6 is able to induce apo and in the Black Sea (in total, ca. 2500 molluscs were ptosis in the human intestinal epithelial cell line HCT investigated). In Poland (the Masurian lakes) material 8, and to investigate the role of caspases in this process. was collected in 1989; 45 molluscs were examined. The Results demonstrated that after 16 h from infection, the commensal species which were specific to the particular parasite is able to induce the morphological features of host taxa were attached directly to mollusc bodies. Totally cells undergoing apoptosis, assessed by using the 12 species of Mantoscyphidia were observed on gastro fluorescent dyes YOPRO1 and propidium iodide, and pod molluscs in Ukraine: M. physarum, M. limacina, confirmed by DNA fragmentation analysis, detection M. hydrobiae, M. acanthophora, M. radixi, M. theodoxis, of active caspase3 and degradation of the caspase3 and 6 more species that will be described as new for substrate PARP. Furthermore, G. duodenalis infection science. On gastropod molluscs from the Masurian induces activation of both caspase9 and caspase8 lakes of Poland, M. physarum (on Physa fontinalis and mediated apoptotic pathways, downregulation of the Theodoxus fluviatilis), M. radixi (on Lymnaea ovata) and antiapoptotic protein Bcl2 and upregulation of the Mantoscyphidia sp.1 (on L. stagnalis) were collected. proapoptotic Bax. Overall, these data suggest that in Two species of sessile commensal peritrichs from genera HCT8 cells G. duodenalis WB C6 is able to induce early Mantoscyphidia and Epistylis were found on bivalve caspasedependent apoptosis, which may contribute to molluscs. M. sphaeriidarum was found on the leg of the pathogenesis of giardiasis. Sphaerium rivicola from the Upper and Middle Dnieper River. E. borysthenicus was widely distributed on the COMPARATIVE MORPHOLOGY OF PHAGOTRO molluscs Anodonta spp. and Unio spp. in most of PHIC investigated water bodies of Ukraine and on the Unio S.A. Breglia, B.S. Leander University of British Columbia, Zoology Department, 6270 University sp. in the Masurian lakes. The ciliates were located Blvd., Vancouver, BC V6T1Z4, Canada. mostly on the edge of mantle and on the inhalant Email: [email protected] siphon. It should be noted that E. entzii, E. balatonica Euglenids form a diverse group of protists adapted to a (attaching to E. entzii stalk), gracilis, and wide range of aquatic environments. Members of the (rarely) O. plicatilis, which previously had been known group have different modes of nutrition, including from Hungarian unionid molluscs (Stiller, 1931, 1935, phagotrophy, and phototrophy. Most 1941), were universally observed on the inner periostra research on euglenids has been done in photosynthetic cum of unionids during our investigation. species, such as gracilis, because they can be Protistology · 17 cultured more readily and are significant as indicators papilio, H. elegance, petricola, Nebela tincta for the health of freshwater . However, major, vulgaris, Euglypha ciliata, leidyi phagotrophic species found in marine environments were the dominants. At the slope of the boggy lake shore, comprise the majority of euglenids. Phagotrophic (i.e. the driest niche was inhabited by Assuina muscorum, bacterivorous and eukaryovorous) euglenids have an Nebela tincta, and the wet zone by Cyclopyxis eurystoma, elaborate feeding apparatus for capturing prey cells that C. arcelloides, Trinema enchelys, Hyalosphenia papilio; together with the ability to accommodate large prey among the submerged mosses in boggy water zone particles by means of a plastic pellicle, allowed an Arcella arenaria, A. discoides, A. conica, A. gibbosa were ancestral eukaryovore to acquire by secon dwelling. The highest heterogeneity of species structure dary endosymbiosis. Aside from a few relatively well along the moisture gradient was observed in the hum studied phagotrophic species (e.g. Entosiphon spp. and mock, the lowest in the flat ravine in the boggy forest. spp.), the overall diversity and mechanisms The observed patterns show that variation in the of feeding in phagotrophic euglenids is poorly under structure of protozoan communities can be observed at stood. This basic knowledge is critical for reconstructing scales measured in centimeters. the earliest stage of euglenozoan evolution and the origins of complex ultrastructural systems in the group. SPATIAL AND TEMPORAL PATTERNS OF THE My research focuses on characterizing new species of STRUCTURE OF MARINE INTERSTITIAL CI phagotrophic euglenids using electron microscopy and LIATE COMMUNITIES molecular phylogenetic approaches. I will present I.V. Burkovsky1, Yu.A. Mazei2 molecular and comparative ultrastructural data on three 1 Moscow State University, Dept. of Hydrobiology, Moscow, Russia, 2 new species of phagotrophic euglenids: (1) a freshwater Penza State Pedagogical University, Dept. of Zoology and Ecology, Penza, Russia. Email: [email protected] sp. with a novel eukaryovorous feeding strategy, The ciliate community structure in the White Sea (2) a freshwater sp. with a novel corkscrew estuary was studied from 1991 to 2006. Altogether 143 shaped , and (3) a marine, facultative anae ciliate species were identified. Two aspects of spatial robic eukaryovorous covered in episymbiotic variability of the community structure were analyzed: bacteria. organization of an elementary (low energy sandy littoral of a shallow bay), and distribution of PATTERNS OF THE COM community along the salinity gradient (a system of MUNITY STRUCTURE ALONG THE MOISTURE alternative communities). Within the elementary GRADIENT: MICROSPATIAL VIEW 1 2 ecosystem, ciliates displayed aggregative distribution O.A. Bubnova , Yu.A. Mazei due to the complex of factors. At relatively large scales 1 Penza Regional Center for Ecological Monitoring, Penza, Russia, 2 Penza State Pedagogical University, Dept. of Zoology and Ecology, (tens to hundreds of meters) spatial heterogeneity of Penza, Russia. Email: [email protected] the community was determined by abiotic conditions Variations in structure of the testate amoebae commu (type of sediments, amount of organic carbon, Eh, etc.). nities among four moisture microgradient series (moss At the lower scales (centimetersdecimeters), the role hummocks, shore of boggy lakes, etc.) were investigated of biotic interactions (competition, predation, and food in different bogs in the Middle Volga Region in 2006. availability) increased. Along the salinity gradient, the Each of the four gradient series contained 10 to 12 local community was found to be unified, continual, and two communities. Sixty six species and subspecific forms polar, i.e. it contained marine and brackishwater sets of testate amoebae were identified. Communities' of species. A relative and fuzzy border between the two structure revealed similar trends in all gradients series: poles was located in brackish waters of the estuary, abundance and the species richness increased from dry characterized by the greatest alterations in redox to wet habitats, whereas the species diversity (in terms potential and salinity which reached occasionally the of Shannon index) remained at the same level. Three critical level of 38‰. Two aspects of temporal community types were distinguished along all micro variability were studied as well: seasonal changes, and spatial gradients. In the moss hummock of the long term (16year period) pattern. The dynamics of oligotrophic bog, the driest habitat was occupied by ciliate community showed clear annual cycle. Comp xerophilous species: Euglypha laevis, Corythion dubium, lexity of the community increased from the late winter Trinema enchelys, Assulina muscorum, A. seminulum. to the early autumn. Increase in available energy in The slope of the hummock was inhabited by Archerella spring presumably triggered this process. In early flavum, Phryganella hemisphaerica, Ph. acropodia, autumn, amount of light lessened and temperatures fell Euglypha ciliata glabra. In the wet habitats at the base down, which probably caused simplification of the of the hummock, hydrophilous species Hyalosphenia community structure. Alterations in longterm dyna 18 · Abstracts of V European Congress of Protistology · 2007 mics of the community structure were characterized by of the group and support multiple origins of hyper both stochastic and trended changes, alongside with mastigote cell form and the monophyly of the order stability of some community parameters. An obvious Trichonymphida. Our results allow us to present a new, stability in species richness and evenness was recorded integrated view of character evolution in these groups. during longterm observations, which suggested certain constancy in general organization of the community A CENTRIOLAR SYNAPOMORPHY FOR CER niche. On this stable background abundance and COZOA AND AN OVERLOOKED CILIARY/ biomass as well as trophic type, size, and species CENTRIOLAR RETICULUM WIDESPREAD IN structure are more variable. All trended changes of PROTISTS ciliate community structure, probably, reflect changes T. CavalierSmith of littoral ecosystem: silt accumulation and redox Department of Zoology, University of Oxford, Oxford, UK. potential decrease. Email: tom.cavalier[email protected] The cercozoan zooflagellate Sainouron has a remar MORPHOLOGICAL EVOLUTION, , kably clear hublattice structure that extends from the AND PHYLOGENY OF AND distal part of the centriolar lumen into the transitional : NEW INSIGHTS FROM THE GUT region of the . Both distal and proximal to this is COMMUNITY OF THE WOODFEE a smooth ciliary/centriolar membrane reticulum. I shall DING COCKROACH CRYPTOCERCUS PUNCTU argue that the centriolar part of the hublattice structure LATUS is the first clear ultrastructural synapomorphy for the K.J. Carpenter, P.J. Keeling phylum . A ciliary or centriolar reticulum is Univeristy of British Columbia, Department, Vancouver, apparently also present throughout the Cercozoa, but Canada. Email: [email protected] both structures were previously overlooked. However, Parabasalids and oxymonads are two of the most unlike the hub lattice itself, the ciliary reticulum is not unusual, evolutionary divergent, and morphologically a synapomorphy for Cercozoa, being apparently in spectacular of all but remain poorly studied, association with a wide variety of transition region partly due to difficulty in cultivation. Members of Para structures in many bikont protists, present but previous basalia have evolved enormous structural complexity ly overlooked because of its inconspicuousness, asym and bizarre morphologies, while oxymonads apparently metry, and the lack of prior expectation of there being lack normal aerobic mitochondria or any derivative any intraciliary membranes. I shall discuss the possible , use a noncanonical genetic code, and have cell biological and evolutionary significance of these a unique singlestep . Most parabasalids and all and other ciliary features of Cercozoa. oxymonads are found exclusively in symbiotic asso ciation with animals, particularly and wood ULTRASTRUCTURAL UNITY OF CERCOZOA: eating cockroaches, where they aid in digestion of THE CENTRIOLAR/TRANSITIONAL HUBLAT lignocellulose, a mutualism of enormous ecological TICE, CENTRIOLAR RETICULUM, AND CILIARY importance. We examined members of the gut protist CORTICAL FILAMENTS, WITH POSSIBLE RO community of the woodfeeding cockroach, Cryptocer LES IN NONCONVENTIONAL CILIARY BE cus puntculatus with light microscopy, transmission and HAVIOUR, USING SAINOURON ACRONEMATICA scanning electron microscopy, as well as molecular SP. N., A ZOOFLAGELLATE WITH REMARKABLY techniques to improve our knowledge of morphological SIMPLIFIED AND GEOMETRICALLY DEFINED character evolution in these groups, their associations ULTRASTRUCTURE, AS MODEL with symbiotic bacteria, and phylogenetic affiliations. T. CavalierSmith, R. Lewis, E.E. Chao, B. Oates, Surface morphology of members of the D. Bass family Saccinobaculidae allies it with families Pyrso Department of Zoology, University of Oxford, Oxford, UK. nymphidae and Oxymonadidae, according with Email: [email protected] previously published molecular results. Unlike other Sainouron is a genus of very poorly known but common oxymonad families, Saccinobaculidae apparently lack soil zooflagellates. We sequenced the 18S rDNA and bacterial surface symbionts but have a welldeveloped made the first ultrastructural study of Sainouron glycocalyx and other features that may represent acronematica sp. n. Our phylogenetic analyses indicate synapormorphies of oxymonads as a whole. Exami that it is specifically related to an unusual nongliding nation of members of the genera Eucomo , Proleptomonas, which groups with hetero nympha, , Barbulanympha, Urinympha, mitid Cercozoa. Proleptomonas has a previously unno and Leptospironympha reveal previously unrecognized ticed unusual cortical nuclear skeleton like Sainouron's classes of bacterial surface symbionts and ultrastructural and a number of other similarities, so we now classify features, while molecular results clarify the evolution them together in a new family Sainouridae to distin Protistology · 19 guish them from typical gliding heteromitids. S. acrone singlet , although its ciliary membrane matica has a unique ultrastructure. Each centriole has may extend well beyond any axoneme. Both centrioles two fibrous rhizoplasts and a novel centriolar reticulum, have a distal/transitional lattice, with a slender hub in with a putative role in centrinbased ciliary bending. the ciliated one. At least two Golgi dictyosomes and a The cell has only three cytoplasmic microtubules, all microbody are attached to the nuclear envelope, which organelles being connected by specific links to them or has slender microinvaginations and probably a cortical the nucleus, whose cortex has unusual strengthening lattice. Bacteria are ingested apically, probably via a tubular inner membrane invaginations and fibrous rudimentary , and digested posteriorly in honeycomblike lattice. The posterior centriole bearing association with numerous mitochondria with flat the motile cilium (with probable receptor) has a novel cristae. transitional hublattice, and remarkable dense spiral fibre linking its anterior rhizoplast and triplets, and the COMPARATIVE ULTRASTRUCTURE OF THE sole ciliary microtubular root with two microtubules: FLAGELLAR APPARATUS OF SPECI mt1, underlying the plasma membrane, initiates at the ES (PELOFLAGELLATA, PELOBIONTIDA) 1 2 spiral; mt2, laterally attached to mt1 and nucleus, on L.V. Chystjakova , A.V. Goodkov 1 Biological Research Institute of St. Petersburg State University, the anterior rhizoplast. The anterior younger cilium, Invertebrate Zoology, St. Petersburg, Russia, 2 Institute of Cytology an immotile stub with novel submembrane skeleton, RAS, St. Petersburg, Russia. Email: [email protected] lacks axoneme, microtubular root, rhizoplast, hub Protists attributed currently to the genus Pelomyxa lattice, reticulum, and spiral and becomes the motile (pelomyxoids), are amoeboid, usually multinucleated, posterior one every cell cycle. We conclude that despite micro or anaerobic organisms with numerous flagella. being previously overlooked, the hublattice structures Flagella of all but one Pelomyxa species, are nonmotile. and mt1related roots are phylogenetically widespread Ultrastructure of ten species of this group was described. in Cercozoa and probably represent synapomorphies The structure of the flagellar apparatus of pelomyxoids for the phylum. varied. The axoneme had a nonstable set of micro tubules. The transition zone contained a transition HELKESIMASTIX MARINA SP. N. (CERCOZOA, cylinder and/or an electrondense column. The basal MONADOFILOSA): A GLIDING ZOOFLAGEL body of some species differed from kinetosomes of LATE LINEAGE OF NOVEL ULTRASTRUCTURE most protists. The basal body was often associated with T. CavalierSmith, B. Oates, R. Lewis, E.E. Chao, the following microtubular structures: (1) radial D. Bass microtubules; (2) the lateral rootlet; (3) the basal bundle Department of Zoology, University of Oxford, Oxford, UK. of microtubules. Each species had a particular Email: [email protected] composition of microtubular rootlets. All the species H. marina differs from H. faecicola in that the former had radial microtubules. In some pelomyxoids they was isolated from marine sediment and is unable to grow formed a conelike structure. In others radial micro in nonmarine media, whereas H. faecicola was isolated tubules were organized in a bundle which lay along the from faeces and grew well in nonmarine media. cell surface. In the cytoplasm of some species we In the absence of previous ultrastructural work the revealed free kinetosomes associated with microtu taxonomic position of Helkesimastix has been altogether bular rootlets, which were structurally different in each obscure, though it has been suggested to be related to species. Structural variability of pelomyxoid flagellar cercomonads. However, Helkesimastix differs radically apparatus may be explained by reduction of some of its from cercomonads, lacking their complex microtubular elements in connection with the loss of flagellar motility. ciliary roots, and our phylogenetic analyses based on Flagellar apparatus of pelomyxoids demonstrated some 18S rDNA demonstrate that it does not group with structural similarity with that of mastigamoebids.The them, being either a novel major lineage within work was supported by the Russian Foundation for Monadofilosa, or specifically related to Aurigamonas Basic Research (Project 050448166). solis and Cercobodo agilis, which form a sister clade to heteromitids. Longitudinal cortical microtubules MOLECULAR COLDADAPTATION OF AN AN emanate from a dense apical centrosomal plate to which TARCTIC CILIATE the tip of the pyriform nucleus and two subparallel A. Claudio1, P. Bill2, P. William2, K. Wuthrich2, 1 centrioles attach by amorphous fibres. A slender striated P. Luporini 1 rhizoplast connects the centrioles: one attached to the Dipartimento di Biologia molecolare, cellulare e animale, University of Camerino, 62032 Camerino (MC), Italy, 2 The Scripps Research centrosomal plate by a complex dense fibrous root bears Institute, La Jolla, CA 92037, USA. Email: [email protected] the long 9+2 posterior cilium; the other has no complex A huge variety of ciliates inhabit the freezing coastal root and no axoneme or just nine very short disorganized waters of Antarctica. species are easy to collect 20 · Abstracts of V European Congress of Protistology · 2007 and expand into stable laboratory cultures. Therefore, trophozoites were irradiated for 10 min and cysts for 20 they are excellent material to study how life adapted to min with 600700 nm light (50 mW cm2), at photosen cold at the molecular and cellular levels. Most research sitiser concentrations in the range 0.55.0 µM and interest was initially focused on E. focardii, because of previous incubation of 1 hour in the dark. The location its strictly psychrophilic behaviour associated with a of RLP068 inside nonirradiated trophozoites is suppression of the heatshock response, and its unique particularly evident in the and structural and functional features of the tubulin/ becomes more diffuse in photosensitised cells. RLP068 microtubule system. E. nobilii, a species that is closely appears to readily cross the external wall of the cysts, in allied to E. raikovi inhabiting temperate waters (Dini spite of its relatively large molecular weight, and to & Di Giuseppe, personal communication), has now localise in various subcellular sites, including the double been found to provide unique opportunities to study the cystic wall. The ultrastructural studies on trophozoites adaptive modifications that permit the activity of its showed a cellular disorganisation, consisting in a large waterborn signal pheromones at freezing temperatures. vacuolisation and subsequent mitochondria alteration Like E. raikovi, E. nobilii constitutively secretes a large with loss of cristae, according to the photosensitiser family of protein pheromones, three of which were concentration and incubation time. These data isolated and purified in amounts sufficient to be encourage a future use of photosensitiser and light structurally characterized by NMR spectroscopy. Their against . threedimensional conformations show a compact threehelix bundle core that is in common with E. PROTISTAN MICROFOSSILS IN TRIASSIC raikovi pheromones, and two extended not structured AMBER FROM THE DOLOMITES (ITALY) regions that are unique to E. nobilii pheromones, one O. Coppellotti1, A.R. Schmidt2, E. Ragazzi3, located at the amino terminus and the other linking the L. Guidolin1, G. Roghi4 first and second helices. The evolution of these two 1 University of Padova, Department of Biology, Padova, Italy, 2 regions is likely to be functionally significant for cold Universitat zu Berlin, Museum fur Naturkunde der Humboldt, Berlin, adaptation, as they appear to substantially improve, Germany, 3 University of Padova, Department of Pharmacology, 4 locally and globally, the flexibility of the molecular Padova, Italy, CNR, Institute of Geosciences and Earth Resources, Padova, Italy. Email: [email protected] backbone of E. nobilii pheromones. In the last few years the largest known deposit of Triassic amber was discovered near the town of Cortina ACANTHAMOEBA PALESTINENSIS PHOTOSEN d'Ampezzo in the Italian Dolomites (Southern Alps). SITISED INACTIVATION AND THE NEED OF Thousands of millimetresized dropshaped pieces of NEW TREATMENTS OF PATHOGENIC PROTO amber per square metre were preserved in a paleosol, ZOA which belongs to the Upper Triassic Series and is about O. Coppellotti, S. Ferro, L. Guidolin, G. Jori 230 million years old. The tiny drops of amber were University of Padova, Department of Biology, Padova, Italy. Email: [email protected] removed from the sediment, ground and polished using Acanthamoeba palestinensis was chosen as a model for a series of wet silicon carbide papers and observed in photoinactivation experiments, using a combination of water on microscopic slides under an interference a photosensitiser, visible light, and molecular oxygen, contrast lightmicroscope. The amber inclusions are with the aim of decontaminating waters from pathogenic representatives of a complex and diverse biocoenosis protozoa and, subsequently, of treating diseases caused consisting of both prokaryotic (filiform cyanobacteria, by pathogenic amoebae. Indeed, some freeliving soil rodshaped and coccoid bacteria) and eukaryotic and water amoebae, such as species of the genera (fungi, microalgae, protozoa) as well Acanthamoeba, Naegleria and Balamuthia, are recog as spores, pollen grains and tissues of higher plants. Here nized etiologic agents of mostly fatal amoebic encepha we present some species of testate amoebae of the litis in humans, with immunocompromised and families , such as hirsuta, and immunocompetent hosts among the victims; Acantha Difflugiidae, such as Difflugialike specimens, some of moeba spp. are also agents of amoebic keratitis. The life which are preserved in dividing stage. Several specimens cycle of Acanthamoeba includes an active feeding of Ciliophora were also observed: one could be attri trophozoite and a dormant cyst. In our experiments buted to the genus (Colepidae), others were both trophozoites and cysts undergo extensive inactiva identified as members of Colpodidae. All protozoa are tion upon photosensitisation by a tetracationic Zn(II) well preserved and represent the earliest evidence of phthalocyanine (RLP068). The two forms exhibit nonmarine testaceans and ciliates. The amberfossils similar dependency on photosensitiser concentration, are similar or identical to some extant species, showing although cysts require a significantly longer irradiation that little or no morphological changes took place in time in order to produce a similar degree of inactivation: protozoa from the Triassic to the Recent. Investigations Protistology · 21 using transmission electron microscope techniques are Cytology RAS, St. Petersburg, Russia. in progress and could reveal important features of the Email: [email protected] fine structure of the microinclusions. Project on studies of prevalence and clinical manifes tation of microsporidiosis and cryptosporidiosis in HIVinfected patients has been recently initiated in St. INFANTUM IDENTIFICATION Petersburg City Hospital No 30. Stool samples from 55 DURING LEISHMANIA/HIV COINFECTION IN patients with chronic diarrhea were collected and SOUTHERN EUROPE: STUDY OF 972 STRAINS frozen. Seventeen stool samples were examined for ISOLATED BETWEEN 1986 AND 2005 presence of cryptosporidia and microsporidia. Neither J.P. Dedet1, F. Pratlong1, C. Chicharro2, M. Gramiccia3, staining with CarbolFuchsin, nor PCR amplification, C. Canavate2, L. Campino4, J. Alvar2 or indirect fluorescent assay (IFA) with antiCrypto 1 Universitй Montpellier 1, Montpellier, France, 2 Instituto de Salud Carlos III, Madrid, , 3 Istituto Superiore di Sanita, Roma, Italy, sporidium parvum monoclonal antibodies (mabs) re 4 Instituto de Higiene e Medicina Tropical, Lisboa, Portugal. vealed presence of cryptosporidia. Three samples were Email: parasito@univmontp1.fr positive for the microsporidium Enterocytozoon bieneusi Thanks to the existence of three main Cryobanks and by PCRbased detection method. Infection of one Identification Centres of France (Montpellier), Italy sample was confirmed also by Calcofluor staining, by (Roma) and Spain (Madrid), which use the same IFA with E. beineusispecific mabs, and by staining with reference identification technique of starch gel FITCconjugated polyclonal antibodies. The PCR electrophoresis for 15 enzymatic systems, a series of amplified region of E. bieneusi DNA was about 400 bp Leishmania isolates have been identified in patients with in length and contained ITS and part of SSU ribosomal Leishmania/HIV coinfections. The majority of the gene. It was sequenced and revealed 100% identity to isolates came from four countries of southwestern E. bieneusi, isolate Peru 13, Genbank Accession # Europe: France, Italy, Portugal and Spain. Between EF014429. Sequencing of amplicons obtained from 1986 and 2005, 972 strains isolated from 741 coinfec other PCRpositive samples, as well as search for ted patients were identified as L. infantum. Out of 27 infection in the rest 38 collected samples, is in progress. zymodemes detected, two were predominant in the Also analysis of 51 clinical reference forms (CRFs) western part of the Mediterranean Basin, namely based on patients' medical charts, is underway. These MON1 and MON24. Several zymodemes usually CRFs contain anonymous information on HIV causing CL in non immunosupressed patients are infection stage, CD4 count, immunological status, responsible directly for VL when infecting HIVpositive presence of other opportunistic infections, antiretroviral patients. Some zymodemes can be found only during therapy, as well as epidemiological data obtained Leishmania/HIV coinfection. Enzymatic polymor through questionnaires, and will hopefully help to phism is high in Spain and Italy, and is based on the understand pathogenesis and routs of transmission of large number of rare zymodemes. With the exception the studied pathogens. New patients are being of Portugal, the numbers of strains isolated from permanently included in the survey. Supported by grant Leishmania/HIV coinfected humans in the countries No. RUB2002707SP05 from the U.S. Civilian of Southern Europe have dramatically decreased since Research and Development Foundation. 2001, which presumably reflects introduction of highly active antiretroviral therapy (HAART) in treatment of THE ROLE OF ACTIN IN TRANSPORTATION OF AIDS. Another reason of such a decrease in some HOLOSPORABACTERIA THROUGH THE CYTO countries could also be diffuse application of molecular PLASM OF PARAMECIUM CAUDAUTM DURING techniques in the hospitals for diagnosis purpose, INFECTION PROCESS making unnecessary in vitro isolation of the parasite. M. Derkacheva1, I. Skovorodkin2 1 Department of Cytology and Histology, Faculty of Biology and Soil 2 OCCURENCE OF MICROSPORIDIA AND CRYP Sciences, St. Petersburg State University, Russia, Institute of Cytology, Academy of Sciences of Russia, Laboratory of Cytology of TOSPORIDIA IN STOOL SAMPLES FROM HIV Unicellular Organisms, St. Petersburg, Russia. POSITIVE PATIENTS WITH CHRONIC DIAR Email: [email protected] RHEA IN BOTKIN CITY HOSPITAL (ST. PETERS The endocytobiotic system ParameciumHolospora BURG, RUSSIA) represents by itself the welldeveloped model for study A.V. Demyanov1, O.I. Sokolova2, V.A. Cama3, of hostsymbiont relationships. Bacteria of the genus A.A. Yakovlev4, L. Xiao3, Y.Y. Sokolova5, S.O. Skarlato5 Holospora are obligate endonucleobionts of the ciliates 1 Institute of Pure Biopreparations, St. Petersburg, Russia, 2 Medical of genus Paramecium; the bacteria are showing species 3 Department of St. Petersburg State University, St. Petersburg, Russia, specificity as well as nuclear specificity. Our research is Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta GA, USA, 4 St. Petersburg City Hospital for focused on study of the process of the bacterial transport Infectious Diseases No. 30, St. Petersburg, Russia, 5 Institute of from the food vacuole of P. caudatum into the "target" 22 · Abstracts of V European Congress of Protistology · 2007 nucleus (H. obtusa to the macronucleus and H. night. These features have adaptive significance under undulata to the micronucleus). The electron micro the conditions of long summer day and might have been scope observations showed that actin of the host cell important for the formation of modern (Holocene) may play a role in this process (Gortz, Wiemann, 1989; flora of volvocine algae in high latitudes of the Northern Fokin et al., 2002). To prove it we use in our work the hemisphere. According to molecularphylogenetic method of genetical transfection of P. caudatum with data, the family Volvocaceae originated about 50 million linear vectors containing actinGFP fusion constructs. years ago (in early Cenozoic). Therefore, Codonoptimized GFP gene (Takenaka et al., 2001) evolution predominantly occurred under the warm was fused through the 7 amino acid linker (Wassmer et climate conditions, such as in Eocene and Miocene. al., 2005) to the 5' end of the actin of P. caudatum Even during winter the temperature in high latitudes (AB070223). This actin is a homologue of actin 1 might have been favorable for Volvox vegetation. isoform 1 (AJ537442) of P. tetraurelia. Vector was linea However, our experiments show that under the diurnal rised and microinjected into P. caudatum macronucleus. light/dark regime of 8 h 16 h (instead of the routine In uninfected cells this actin isoform forms comet tails 16 h 8 h regime) development in cultures of V. carteri at food vacuoles and accumulates in patches at the is blocked, whereas V. aureus is able to complete the surface of food vacuoles. Also it forms a fine ring on the asexual life cycle. These data imply that evolutionary surface of some food vacuoles and we can observe it in reorganizations of Volvox development, which are the area of cytopharyngal fibers and cytoproct area. It primarily connected with changes in the rate and light/ is very similar to the distribution of its homologue in P. dark control of gonidial divisions, might have occurred tetraurelia (Sehring et al., 2007). In infected cells of P. as an adaptation to warm and short winter day in high caudatum this actin was observed to take part in a latitudes in the past. specific exit of Holospora spp. to the cytoplasm from the food vacuole. We also were able to see a regular layer MECHANISMS OF MACROPHAGEMEDIATED of actin distributed over the surface of bacteria in the INHIBITION OF ENCEPHALITOZOON CUNICULI host cytoplasm. Comet tailshaped actin filaments were E.S. Didier1, L.C. Bowers1, P.J. Didier2 also observed at the surface of the bacteria. Thus, we 1 Tulane National Primate Research Center, Division of Microbiology, confirm that actin of P. caudatum plays a significant Covington, LA, USA, 2 Tulane National Primate Research Center, Division of Comparative Pathology, Covington, LA, USA. role in transport of Holospora through the cytoplasm Email: [email protected] during the infection process. Supported by RFBR grant Macrophages host the replication of microsporidia but 070400662. also can become activated to kill these intracellular organisms. Murine peritoneal macrophages and EVOLUTION OF DEVELOPMENT, PHOTOBIO RAW264.7γNO/ cells activated by treatment with LPS LOGY AND BIOGEOGRAPHY OF VOLVOX (10 ng/ml) and IFNγ (100 u/ml) in vitro destroyed A.G. Desnitskiy intracellular Encephalitozoon cuniculi via reactive Biological Institute of St. Petersburg State University, Department of Embryology, St. Petersburg, Russia. Email: [email protected] oxygen intermediates (ROI) and reactive nitrogen In cultures of Volvox carteri, V. spermatosphaera and intermediates (RNI). To further evaluate this in vivo, several other species of Volvox (as well as in more mice unable to generate ROI (B6.129S6Cybbtml), RNI primitive colonial Volvocaceae) a long period of light (B6.129SF2/J mice given 2.5% aminoguanidine in dependent gonidial growth is followed by a series of drinking water), or both ROI+RNI (B6.129S6Cybbtml rapid consecutive divisions, which may occur in given aminoguanidine), along with WT (B6.129SF2/ darkness. This type of light/dark control is ancestral for J) mice, were infected with 5×107 E. cuniculi. All mice the family Volvocaceae. By contrast, in V. aureus, V. survived at least 6 weeks, but the percent of infected globator, V. tertius and several other Volvox species a peritoneal macrophages after 2 weeks of infection was period of lightdependent growth is followed by a series significantly higher in the mice deficient for ROI (2.1+/ of slow and lightdependent divisions. This type of light/ 0.29; P<0.001), RNI (1.6+/0.73; P<0.05), and dark control is derived. Data on the geographical ROI+RNI (2.4+/0.37; P<0.001) compared with WT distribution of all 18 species of Volvox have been mice (0.85+/0.09). By 4 weeks, no microsporidia were summarized. An attempt was made to trace a correla observed in the peritioneal macrophages of the WT or tion of their latitudinal distribution with the type of ROIdeficient mice, but were observed in the mice light/dark control during asexual development. In high deficient for NOI and ROI+RNI. Addition of ferric latitudes of the Northern hemisphere (northward of 50 citrate to BALB/c peritoneal or RAW264.7γNO/ 57° north) only 3 abovementioned derived species of macrophages in vitro increased replication of E. cuniculi Volvox occur, in which slow gonidial divisions start in to >50% of controls. Furthermore, ferric citrate the morning and are temporarily blocked during the reversed the inhibition of E. cuniculi infection by Protistology · 23 activated macrophages from 24.5% (+/7.7) of controls inferred to be a common event in protists, since the in the absence of exogenous ferric citrate to 56.02% (+/ same 18S rRNA has been recorded in both 6.4) in the presence of 500 µM ferric citrate in a dose the Arctic and Antarctic populations of all the analyzed dependant manner. These results suggest that activated bipolar morphospecies. We addressed this problem in macrophages employ mechanisms that, in addition to taxa comprising the genus Euplotes, a cosmopolitan, ROI and RNI, may also utilize iron sequestration for ubiquitous protistan ciliate. Four morphospecies were inhibiting E. cuniculi. analysed: E. euryhalinus, Euplotes sp., E. focardii and E. nobilii. In assigning strains to the appropriate mor MICROSPORIDIOSIS: CURRENT STATUS phospecies, the intra (inside and between strains) and 1 2 E.S. Didier , L.M. Weiss interpopulations variability was considered. Twelve 1 Division of Microbiology, Tulane National Primate Research Center, 18703 Three Rivers Road, Covington, LA 70433, USA, 2 Departments characters, most commonly used for morphological of Medicine and Pathology, Albert Einstein College of Medicine, 1300 distinctness among Euplotes taxa, were chosen. Data Morris Park Avenue Room 504 Forchheimer, Bronx, NY 10461, USA. produced by the pursued morphometric procedure were Email: [email protected] analyzed using the multivariate technique of discrimi The phylum Microsporidia includes approximately nating functions. The 18S rRNA, EF(Elongation 1200 species that infect members of all animal phyla, Factor )1α1 and EF1α2 genes, as well as the Internal and 14 of these can infect humans. Interest in these Transcribed Spacers (ITS) regions of the rDNA locus, organisms grew tremendously during the past 20 years all characterizing the nuclear genotype, plus the after being associated as a cause of persistent diarrhea mitochondrial 16S rRNA gene were selected. They were and systemic disease in persons with AIDS. Increased amplified and directly sequenced, in order to establish awareness and improved diagnostics have broadened both the phylogenetic relationships among populations our knowledge about the wide demographic, geogra phic, zoonotic, and environmental range of the species and the occurrence of their interbreeding. Euplotes of microsporidia that infect humans. Identification of euryhalinus included two Arctic population groups, Ar1 microsporidia in water sources also led to their inclusion and Ar2, and only one Antarctic, An. The Ar1 and Ar2, on the National Institutes of Health Category B 18S rRNA differed for 7 mutations, whereas pathogen list and the EPA microbial contaminant the Arctic genotype of Ar1 was shared with the Antarctic candidates list of concern for foodborne and waterborne An. Yet, these last population groups (Ar1 and An) transmission. The completion of the Encephalitozoon differed in their ITS (3 mutations) and 16S rRNA (87 cuniculi genome has led to new insights into the mutations) genotypes. The fruitful breedings inside the molecular phylogeny and biology of the microsporidia. Arctic (Ar1 and Ar2) and Antarctic (An) population Additional genomic data on other microsporidia is groups did not follow the interpolar mating tests: mixed becoming available and providing additional insights strains failed to mate or if mating occurred, as in mixture into the biology of these organisms. This presentation between Ar1 × An strains, conjugating mates belonged will provide an overview of microsporidiosis in humans to Antarctic representatives of the An population group. and highlight new research findings on these pathogenic A far larger number of mutations at selected loci protists. occurred between Arctic and Antarctic population groups of Euplotes sp., as well as between Antarctic and BIPOLAR DISTRIBUTION OF PROTISTAN SPE equatorial population groups of E. focardii (this latter CIES IS NOT A ROSE morphospecies has no Arctic representatives). In both F. Dini, G. Di Giuseppe morphospecies, conjugating strains of the Antarctic Universita di Pisa, Dipartimento di Biologia, Pisa, Italy. population groups failed to mate with conjugating Email: [email protected] Literature records the bipolar distribution of some representatives of population groups of the Arctic or terrestrial and marine species. Even protistan represen equatorial regions, respectively. Also in E. nobilii, Arctic tatives join to this restricted club. Most discussions focus and Antarctic populations cluster in different groups, whether bipolar species are isolated inside their own showing a number of mutations, larger or smaller, highlatitude areas, or whether trans?tropical shifting whatever gene is considered. Nevertheless, a strain of occurs that allows intermixing between the gene pools an Arctic population group, shared one of the 3 of northern and southern populations. Such a traffic mutations at the 18S rRNA locus, differentiating this could be favoured by the planktonic life style or group from an Antarctic one. Interpolar crosses intrusion into a transient microhabitat (detritus involving this Arctic strain produced a hybrid although particle), so as to be conveyed by the oceanic currents, poor viable progeny, suggesting that an irreversible or by cystforming ability to be dispersed by some evolutionary divergence between the two polar interpolar traveller. Recently, intermixing has been populations has not yet occurred. 24 · Abstracts of V European Congress of Protistology · 2007

VIRUSES ECOTYPES IN COLLECTION OF CILIA description. In 2004, the BIOSOPE oceanographic TES WITH DOUBLE VIRAL INFECTION cruise provided an opportunity to obtain material from P.V. Dobrynin1, T.H. Hoang1, D.A. Elster1, some of the same areas between the Marquise and D.V. Karelov2, A. V. Migunova1 Easter Islands where Agassiz and Carnegie expeditions 1 SaintPetersburg State University, St. Petersburg, Russia, 2 Saint had collected their samples. Many of unusual and Petersburg Nuclear Physics Institute, St. Petersburg, Russia. ornate morphologies, as well as an extraodinary species Email: [email protected] This work is dedicated to the study of PBCV virus richness described by Kofoid and Campbell were ( virus, family Phycodna confirmed. The marked diversity of tintinnid ciliates viridae, genus Chlorovirus) from the triple symbiotic may be indicative of a very foodpoor environment, in system of P. bursaria. We tested the media from P. which competitive interactions between species are rare. bursaria cultures isolated from the geographic zones, in which water samples were known to be infected with UNIQUE FEATURES OF MICROSPORIDIA PHY viruses of southern and northern ecotypes, for presence SIOLOGY CAUSED BY MINIMIZATION OF PARA of both viral ecotypes. We demonstrated that after long SITE CELL MACHINERY 1 2 1 term cultivation, only the viruses with the ecotype V.V. Dolgikh , G.V. Beznoussenko , Yu.S. Tokarev , 1 1 2 corresponding to the host survived. To I.V. Senderskii , A.M. Naumov , A.A. Mironov 1 identify the viral ecotype in water samples from Laboratory of Microbiological Control, AllRussia Institute for Plant Protection, Academy of Agricultural Sciences, St.Petersburg, Russia, 2 Vietnam, we amplified pol gene (760 b.p. of length) Department of and Oncology, Consorzio Mario Negri using PCR with degenerated primer. Amplification of Sud, Santa Maria Imbaro (Chieti), Italy. Email: [email protected] two DNA samples from Vietnam (viruses of northern The ultrastructural, biochemical and mainly genomic ecotype from the NamDin and KuangBin rivers), as studies of microsporidia demonstrate profound mini well as from Georgia, Tadjikistan, and Japan (viruses mization of parasite cell machinery. At the same time of southern ecotype) provided similar results. Amplifi the loss of many organelles, metabolic pathways and cation has been preceded in three investigated samples genes by microsporidia was accompanied by acquisition from Vietnam, which confirms that the virus was of unique physiological features not found in other present in samples. The electrophoresis of Asian eukaryotic cells. Our study was focused on two points: (Chinese, Japanese) viruses' amplificates shows that it (1) absence of transport vesicles in the parasite cell side is closelyrelated to the southern viruses (PBCV1). by side with presence of genes potentially involved in Sequensing of clone DNA was performed. Chinese vesicle formation and fusion with target membranes; viruses (from ciliate clones E12 and B7) and Japanese (2) a very scanty repertoire of enzymes involved in the viruses (from KZ13) are placed in southern cluster, last steps of glycolysis suggesting unique mechanisms but inside one they are located closely to the most for pyruvate converting and electron transport. northern viruses (NewYork origin) in dendrogramm Heterologous expression of proteins of the microspori of relationship made using the alignment data. dium Paranosema locustae, as well as immunocytoche mical and biochemical approaches was used to REVISITING KOFOID AND CAMPBELL TIN investigate these questions. Supported by RFBR grant TINNIDS (PLANKTONIC CILIATES) OF THE 050449616. SOUTH PACIFIC J.R. Dolan PROBLEMS OF IDENTIFICATION OF PILISUC University of Paris VI & CNRS, Zoological Station, Villefranchesur TORID CILIATES (APOSTOMATEA, PILISUCTO Mer, France. Email: dolan@obsvlfr.fr RIDA) Tintintinnid ciliates of the plankton are one of the most I.V. Dovgal speciesrich groups among all protist groups. Charles I.I. Schmalhausen Institute of Zoology, Kiev, Ukraine. Kofoid and Arthur Campbell are responsible for a large Email: [email protected] part of the tintinnid species catalogue, they recognized The pilisuctorid ciliates of the order Pilisuctorida are 724 species overall. From material gathered during 2 parasites of crustaceans. At present the order involves cruises, the Agassiz Expedition in 19041905 and the three families: Conidophryidae, Ascophryidae and Carnegie Expedition in 19281929, they described 221 Askoellidae, each comprising a single genus with small new species. Most of the new species were found in the number of species. In spite of poor species richness, South Pacific, an area poorly sampled then, as well as several problems concerning systematic and nomencla now. Based on their data, speciesrichness appeared to ture of this group of ciliates arise. Moreover, even species be extraordinary, and today it provides evidence of a identification is problematic, and often is based rather strong latitudinal gradient in tintinnid diversity. on host specificity than on the specific characters of However, many tintinnids pictured in their monographs the parasite. Our investigations showed that host have remained apparently unrecorded since their specificity of pilisuctorid is not so strict, as it had been Protistology · 25 assumed earlier, and methods of specific identification some viruses, bacteria, and protozoans. Membrane rafts based on morphology are needed. The keys for are small and dynamic regions enriched in sphingoli identification of pilisuctorid species, which are based pids, cholesterol, ganglioside GM1 and protein markers on the macronucleus morphology and mode of like flotillins (1 or 2) forming flatter domains, or attachment of these ciliated to the host body, were caveolins (1, 2 or 3), which are characterized as stable developed for the above mentioned problemsolving. flaskshape invaginations. We explored whether membrane rafts participate in the entry of T. cruzi's FIRST REPORT OF ACTINOSPOREANS IN RUS trypomastigotes into murine macrophages. Transient SIA depletion of macrophage membrane cholesterol by A.S. Dudin application of methylbetacyclodextrin, and treatment State Research Institute on Lake and River Fisheries, Department of with filipin caused reduction of trypomastigote Fish Diseases, St. Petersburg, Russia. Email: [email protected] internalization. Treatment with a crescent concentra During 20052006, actinospore infection of oligocha tion of cholera toxin B that binds GM1, demonstrated etes was consistently revealed at five freshwater sites of the ability to inhibit parasite entry. Using immunofluo St. Petersburg and Leningrad district. Totally 36000 rescence microscopy we observed a colocalization of specimens of two common oligochaete species, Tubifex GM1, flotilin1 and caveolin 1 in T. cruzi's parasitopho tubifex and Limnodrilus hoffmeisteri, were collected in rous vacuole. Together our results suggest that mud and examined. By using the "cellwell plate membrane rafts are involved in the process of T. cruzi method" (Yokoyama et al., 1991) five types of triactino invasion of macrophages. Supported by CNPq, myxon, one type of raabeia, and one type of siedleckiella FAPERJ and CAPES. were identified. Three different triactinomyxon types were isolated from T. tubifex. First type was revealed in CORRELATION BETWEEN CORTEX STRUC midstream of River Okhta, with maximal prevalence TURE AND MOTILITY TYPE OF GREGARINES BY of 0.4% in June 2006 samples; the second type was THE EXAMPLE OF THREE UROSPORA SPECIES discovered in the mouth of same river (0.44%); and A. Dyakin, G. Paskerova the third type in a small cold water spring inflowing in SaintPetersburg State University, St. Petersburg, Russia. Lake Mozhayskoe (0.5%). From L. hoffmeisteri Email: [email protected] collected in midstream of River Slavyanka, the fourth We have studied Urospora travisiae, U. ovalis, and U. type of triactinomyxon was isolated (0.08%). The fifth chiridotae (Apicomplexa, Eugregarinida, Urostildae) type of triactinomyxon was released from the unidenti from polychaetes and holothurians of the White Sea. fied immature oligochaete collected in midstream of Trophozoites of U. travisiae inhabiting the celom cavity River Okhta (prevalence up to 0.2% in the September of Travisia forbesii have a Vlike body, with a tip for 2006 sample). Actinosporean of raabeia type was attaching to the host tissues, and two beadlike rays. isolated from L. hoffmeisteri collected in the mouth of Trophozoites glide by one or both leading rays. River Okhta (0.6%). Actinosporean of siedleckiella type Numerous epicytic folds extend from the attaching tip was released from T. tubifex collected in midstream of to distal ends of rays. The structure of cortex is typical River Okhta (0.6%). Five detected types (three of gregarines. Trophozoites of U. ovalis inhabiting the triactinomyxon, one raabeia and one siedleckiella) were celom cavity of T. forbesii are nonattached and oval in different from actinosporea forms hitherto described shape. They move by metaboly. The cell surface of the and probably are new. This is the first report on the noncontracted part of the parasite body is covered with occurrence of members of Actinosporea in Russia. typically structured epicytic folds, whereas that of the contracted part is covered with superfolds bearing PARTICIPATION OF HOST several epicytic folds. The skittlelike trophozoites of RAFTS IN CRUZI INVASION U. chiridotae inhabit the bloodvessels of Chiridota laevis PROCESS attaching to the vessel wall by narrow ends. Gregarines J. Dutra, E. Barrias, W. de Souza, T. Carvalho are immobile. Their surface is covered with numerous Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil. Email: [email protected] cytopilia, which are cylindrical, narrowing to the distal Establishment of the infection by , end, variable in size and armed with numerous the ethiologic agent of Chagas' disease, depends on a microtubules. The pellicle forms cylindrical invagina series of events involving adhesion of the parasite to the tions into cytoplasm, at the bottom of which micropores cell surface receptors, recruitment of additional are formed. The data obtained suggest a correlation receptors to the infectionsite, a reorganization of the between cortex structure and type of motility of gre membrane and, in particular, the parasitophorous garines: the presence of typical epicytic folds corres vacuole formation. Distinct microdomains in the ponds with gliding; formation of superfolds corresponds plasma membrane are responsible for the invasion of with metaboly; disappearance of folds corresponds with 26 · Abstracts of V European Congress of Protistology · 2007 immobility. The mode of motility is probably adaptation families Centropyxidae, Cyclopyxidae, Euglyphidae, of gregarines to parasitizing in a particular hostal Trinematidae were predominant in the species list. The biotope. most peculiar structure of species community was observed in broom bush, where Plagiopyxis penardi and ACANTHOCYSTIS TURFACEA CHLORELLA VIRUS Euglypha strigosa glabra dominated, as well as in cherry FROM FRESHWATER PONDS OF SAINTPE tree bush, where Centropyxis aerophila sphagnicola was TERSBURG especially abundant. In other biotopes Centropyxis D.A. Elster, P.V. Dobrynin, A.V. Migunova sylvatica was a part of the dominant species complexes, SaintPetersburg State University, St. Petersburg, Russia. as well as Cyclopyxis kahli in the meadowsteppe, Email: [email protected] Centropyxis aerophila sphagnicola in the steppe Since 1984 we have been carrying out the laboratory meadow, and Centropyxis aerophila in the blackthorn studies of the tripletic simbiosis Paramecium bursaria bush, bird cherry tree and aspen forests. Thus, a few Chlorella virus. The related ATCV (Acanthocystis dominant species formed diversity of communities due turfaceaChlorellavirus)type viruses were first found to rearrangement of the same set of dominant species. in Germany by Bubeck and Pfitzner. We were interested Species richness, species diversity, and abundance of if we could find this type of viruses in freshwater samples amoebae decreased along the soil profile, whereas in SaintPetersburg. Twelve freshwater samples were Pielou evenness index seemed to be rather constant both taken from the ponds of SaintPetersburg in order to between different biotopes and along the soil profile. the presence of the ATCVtype viruses. One ml of The cherrytree bush and bird cherry tree forest the sampled water was added to the suspension (9×107 exhibited the maximal species richness. The lowest cells/ml) of the virussensitive cells of Chlorella, the number of species was observed in the meadowsteppe, of the heliozoan Acanthocystis turfacea. the driest habitat. The largest abundance (more than This culture (SAG 3.83) was originally obtained from 400 ind. per gram of dry soil) was recorded in the cherry the Gettingen University collection, and currently is tree bush where population of Centropyxis aerophila maintained in the Algae collection of the St. Petersburg sphagnicola reached the highest abundance. Micro State University (CALU). Lysis of Chlorella cells was spatial heterogeneity (within a single biotope) was rather observed in 3 samples after a week of cultivation, which low. On average, only 35% of total species richness in a suggested occurrence of the ATCVtype virus. To biotope was affected by the betadiversity component confirm this, and in order to isolate the local virus (i.e. differences between samples within one biotope). populations, we cloned the halfliquid mineral medium On the contrast, mesospatial heterogeneity within with the virussensitive Chlorella (SAG 3.83. culture). "Osrovtsovskaya foreststeppe reserve" region was To multiply the received clones, one week later we higher: approximately 65% of the regional species placed the appeared virus plaques into the liquid richness was affected by the betadiversity component. suspension of the virussensitive Chlorella cells (SAG 3.83. culture). Lysate was filtered through the nucleo HOLOSPORACEAE, RICKETTSIALES, AND MI pore filter (d=0.4 µm) and stored at 4°C. As a result we TOCHONDRIAL ORIGIN: RICKETTSIAE IMPORT got 6 clones of the ATCVtype virus. MITOCHONDRIAL PORIN V.V. Emelyanov TESTATE AMOEBAE FROM SOILS OF FOREST School of Biology, University of Newcastle, Newcastle upon Tyne NE1 STEPPE ECOSYSTEMS 7RU, UK. Email: [email protected] Huge numberof protists are well known to contain E.A. Embulaeva, Yu.A. Mazei Penza State Pedagogical University, Dept. of Zoology and Ecology, different endosymbiotic bacteria. From an evolutionary Penza, Russia. Email: [email protected] perspective, the most interesting among them are α Species composition and community structure of testate Proteobacteria in view of their apparent connection to amoebae from black earth soils in foreststeppe zone mitochondrial origin. Phylogenetic data based on was studied in "Osrovtsovskaya foreststeppe reserve" rRNAs, whose genes reside in mitochondrial genomes, (Middle Volga region). Different biotopes representing and chaperonin 60 (Cpn60), encoded by a nuclear gene, all types of foreststeppe transition were studied: indicate that mitochondria and Rickettsiaceae assemb meadowsteppe, steppemeadow, broom bush, cherry lage, i.e. true rickettsiae, share last common ancestor tree bush, blackthorn bush, bird cherry tree forest and (LCA) to the immediate exclusion of a group of aspen forest. Thirtytwo species and forms were rickettsialike , or family Holosporaceae. identified. Centropyxis sylvatica was the most common These in turn diverge after freeliving αProteobacteria. species, which inhabited all biotopes studied. Other Holosporaceae encompasses mostly endosymbiotic common species were Cyclopyxis kahli, Trinema bacteria of protists such as Acanthamoeba, paramecia complanatum, and Centropyxis aerophila. Members of and acidophilic Protozoa. Rickettsiaceae (rickettsiae Protistology · 27 and anaplasmas) and Holosporaceae are classified with insertional mutants defective at regulatory steps of the the order Rickettsiales. The above data reveal a adaptive responses to hypoosmotic shock. Two osmo paraphyletic nature of Rickettsiales and strengthen an regulatory mutants, osr1 and osr2, were isolated on the idea that organelle has derived from an already reduced basis of the requirement for growth in medium of high rickettsialike endosymbiotic bacterium whose closest osmotic strength. In normal medium of lowosmotic extant relatives are the species of Holosporaceae. Two strength the mutant cells swell and burst. The mutants major evolutionary events that transformed endosym exhibited defects in the contractile vacuole cycle. Wild biont into were the acquisitions of ATP/ type recombinant progeny were obtained from the ADP carrier (AAC) and protein import machinery. crosses among two mutants, indicating that these two Earlier on I suggested that both systems have already mutations implicated two different genes. Insertional been present in the above LCA and inherited by mutagenesis has thus permitted the generation of novel Rickettsiaceae vertically. Phylogenetic analyses of non osmoregulatory mutants that will be of value in mole mitochondrial type AAC are consistent with this view, cular dissection of the osmoregulatory system in the the classic endosymbiont theory, and suggest that it may unicellular organisms. have first appeared in Holosporaceae during mitochon drial origin to provide the host with respirationderived CHEMOTAXIS TO AMMONIUM AND AMMO ATP. It was shown that the species of the genus NIUM TRANSPORT IN CHLAMYDOMONAS 1 1 2 Rickettsia, but not a facultative intracellular α E.V. Ermilova , M.M. Nikitin , E. Fernandez 1 Proteobacterium Bartonella henselae import mitochon Department of Microbiology, Biological Research Institute of St. Petersburg State University, St. Petersburg, Russia, 2 Department of drial porin VDAC1. Cell fractionation experiments Biochemistry and , University of Cordoba, 14071 suggest that porin localizes to Bayer's adhesion zones, Cordoba, Spain. Email: [email protected] which may thus be a prototype of mitochondrial contact Ammonium is the preferred nitrogen source for most sites between inner and outer membranes which contain unicellular organisms, including the soil green alga VDAC1 (membrane heredity). The more porin is Chlamydomonas reinhardtii. Ammonium transport is a contained in rickettsial cells, the easier they are key process in its . The current evidence permeabilized, suggesting a functionality of mitoporin strongly suggests that eight of the CrAmt1 genes encode in Rickettsia. It is thought that Rickettsiaceae do possess the highaffinity transport systems (HATS) for ammo some primitive protein import machinery, yet having nium in Chlamydomonas cells. These represent the some components in common with sophisticated largest set of Amt1 genes described so far in any . mitochondrial protein import. The data presented may The importance of ammonium as a nitrogen source also also shed light on a nature of obligate rickettsial sym means that many motile microorganisms have evolved biosis. Supported by the European Commission under an additional adaptation such as chemotaxis that allows Marie Curie Incoming International Fellowship, grant them to move towards ammonium. Chlamydomonas MIF1CT2006039819. vegetative cells are attracted to ammonium, which in the absence of nitrogen source initiate the program of INSERTIONAL MUTAGENESIS GENERATES sexual differentiation. A new methylammonium OSMOREGULATORY MUTANTS IN CHLAMYDO resistant mutant hat1 has been isolated by insertional MONAS REINHARDTII mutagenesis. This mutant was affected at multiple loci E.V. Ermilova, T.V. Lapina, Zh.M. Zalutskaya and, at physiological level, seems to be affected in the Department of Microbiology, Biological Research Institute of St. activity of HATS component for ammonium/methyl Petersburg State University, St. Petersburg, Russia. ammonium. The transcription of the CrAmt1.(18) Email: [email protected] genes in hat1 strain appeared to be normal except for a Chlamydomonas cells have developed elaborate and slight and general lower expression than in the wild type sensitive protection systems that enable them to rapidly that was significant for Amt1.5, 6 and 8. Treatment with signal, respond and adapt to osmotic changes. However, the potassium channel inhibitor tetraethylammonium genes and proteins responsive to osmotic shock, (TEA) blocked chemotaxis to ammonium/methylam especially to hypoosmotic shock, are practically monium and [14C]methylammonium uptake. Our unknown in C. reinhardtii. Existance of organisms with results suggest that hat1 mutant could be affected at mutations at the loci responsible for the specific steps genes encoding regulatory elements of AMT1 activity of metabolism is a valuable tool of functional genomics. and that chemotaxis in response to ammonium/methyl Heterologous integration occurring during transforma ammonium is mediated by ammonium transporters tion with a selectable marker in C. reinhardtii has been sensitive to TEA inhibition related to one class of potas used to generate osmoregulatory mutants. A wildtype sium channels voltageindependent nonselective strain was transformed with the plasmid bearing the cation channels. Supported by the grant N051000008 paromomycinresistant AphVIII gene to generate 8004 from INTAS. 28 · Abstracts of V European Congress of Protistology · 2007

A MODEL FOR THE MORPHOGENESIS AND herd the cumulative prevalence of E. bieneusi was 100% EVOLUTION OF THE EUGLENID PELLICLE since all 30 calves shed spores at some time during the H.J. Esson study. Of 990 specimens collected, 234 were infected University of British Columbia, Department of Botany, Vancouver, with microsporidia (23.5%). Differences in prevalence Canada. Email: [email protected] of infection appeared to be related to the age of the ani The articulating protein strips of the euglenid pellicle mals. The prevalence was lower in preweaned calves are novel cytoskeletal elements that vary considerably (less than 8 weeks of age) (33.3 %) than in postweaned in number, surface patterns and ultrastructure. While calves (312 months of age) (100%) and heifers (1324 pellicle characters have been used previously to make months of age) (70%). Four genotypes of E. bieneusi inferences about euglenid evolutionary history, taxon were identified. EB2 was the most prevalent genotype sampling is poor and little is known about the develop (100%), while EB4, EB3, and EB1 were found in 30, ment and evolution of the characters themselves. I will 10, and 0.3%, respectively. None of the infected animals present comparative data that provide insights into the had diarrhea or appeared ill. Based on previous reports developmental processes involved in pellicle duplication genotypes EB14 have been found only in cattle and and maturation during . These data support a appear to present no danger to humans. model of pellicle morphogenesis and evolution that explains the observed variation in characters like strip CILIATES AS NATURAL HOSTS OF MANY NOVEL number, strip reduction and rows of pellicle pores. For RICKETTSIALIKE BACTERIA instance, examination of dividing cells of Euglena F. Ferrantini1, S.I. Fokin1,2, C. Vannini1, L. Modeo1, gracilis has shown that the posterior whorls of strip F. Verni1, G. Petroni1 reduction in phototrophic euglenids are each composed 1 Department of Biology, University of Pisa, Pisa, 56126, Italy, 2 of strips that were synthesized during different rounds Biological Research Institute of St. Petersburg State University, St. of cell division. During subsequent cell divisions, the Petersburg, 198504, Russia. Email: [email protected] Rickettsialike organisms (RLOs) are Gramnegative component strips of a single whorl of reduction increase prokaryotes known as obligate intracellular parasites of in length to form the next posterior whorl of reduction. arthropods, annelids, mollusks and vertebrates; some These results demonstrate that strips of the euglenid species are human pathogens. Recent studies reported pellicle are "multigenerational" and variation in whorled the presence of these organisms also in protists, although patterns of reduction is the result of evolutionary no specific researches were accomplished up to now. modifications in the relative timing of strip development In this study we assess the presence of RLOs infecting (i.e. heterochrony). Moreover, examination of the some brackishliving ciliate protists, by using the "full highly differentiated strip lengths in the benthic marine cycle rRNA approach" (16S rDNA characterization Euglena obtusa indicate that pellicle pores and use of specifically designed oligonucleotide probes are formed in the most mature strips and that parent for in situ detection) and TEM techniques to perform strip identity (i.e. maturity and length) plays an ultrastructural analysis. At present, six kinds of RLOs important role in determining the identity of nascent were identified in five different ciliates, namely strips. Pseudomicrothorax dubius (), minus (Heterotrichea), Euplotes octocarinatus (Spirotri LONGITUDINAL STUDY OF MICROSPORIDIA IN chea), Paramecium cfr. multimicronucleatum (Oligohy CATTLE ON A DAIRY FARM menophorea) and Diophrys oligothrix (Spirotrichea), R. Fayer, M. Santin, J.M. Trout which harbors two different symbionts. Phylogenetic USDA, ARS, Beltsville, Maryland, USA. Email: [email protected] analysis based on 16S rDNA sequences revealed that Feces from 30 purebred Holstein female cattle on a the symbionts of S. minus and Paramecium cfr. multi dairy farm in Maryland were examined consecutively micronucleatum are associated to the genus Rickettsia at weekly, biweekly, or monthly intervals from 1 week (fam. Rickettsiaceae); the symbionts of E. octocarinatus to 24 months of age for the presence of microsporidian and one of that of D. oligothrix, despite the host spores. Fecal specimens were sieved and subjected to difference, form a monophyletic clade that probably density gradient centrifugation to remove debris and to represents a new genus within Rickettsiaceae; the concentrate spores. The presence of spores was deter symbiont of P. dubius and the other symbiont of D. mined by PCR/gene sequence analysis of the internal oligothrix form distinct basal within Rickettsi transcriber spacer (ITS) region of E. bieneusi rDNA aceae: they can also represent new genera. In some which has extensive genetic diversity. It has been used cases, ultrastructural analysis showed peculiar morpho to indicate that some genotypes have zoonotic potential logical features. These preliminary results revealed an whereas others possibly represent hostspecific genotypes unexpected, intriguing phylogenetic and morphological or even different species from E. bieneusi. In this dairy diversity among the RLOs; the frequency of occurrence Protistology · 29 of such kind of association suggests that protists could The mitochondrial () genome of Trypanoso play the role of natural reservoir for some potentially matidae has a unique structure. It is composed of hazardous pathogens. thousands of interlocked circular molecules, maxicircles and minicircles. Minicircles (100010000 copies in a EVOLUTIONARY PATHWAYS IN FRUITING kinetoplast) are represented by different sequence AMOEBAE () classes coding for guide RNAs. Usually a few classes A.M. FioreDonno1, J. Pawlowski2, S. Baldauf3, are much more abundant than the others. Minor T. CavalierSmith1 minicircle classes may be eliminated after long mainte 1 Department of Zoology, University of Oxford, South Parks Road, nance of cells in culture, or, on the contrary, amplified Oxford OX1 3PS, UK, 2 Department of Zoology and Animal Biology, after acquisition of drug resistance. In contrast, maxi University of Geneva, 30, Quai EAnsermet, 1211 Geneva 4, circle population has been supposed to be homogeneous Switzerland, 3 Department of Biology, University of York, Box 373, Heslington, York YO10 5YW, UK. and stable. We have demonstrated that maxicircle Email: afiore[email protected] population in clonal cultures of Leishmania major is The phylum Amoebozoa includes numerous aerobic composed of at least 10 major and minor sequence amoebae that lack cytoplasmic microtubules and the classes. Each class has a specific number of long repeats clade , comprizing the freeliving aerobic in the noncoding region. Nearly all investigated clones Mycetozoa (slimemolds) and the amitochondrial (10 of 11) have an identical set of major maxicircle (e.g. , ), which classes, but some minor classes appear to be absent in typically have a microtubular skeleton. Mycetozoa are some clones. Leishmania major is a digenetic parasite common soil organisms with a life cycle having a trophic with two life cycle stages: promastigotes (with active stage of amoebae or plasmodia alternating with a mitochondria) and amastigotes (anaerobic). We have dispersal phase culminating in the formation of often demonstrated that amplification of minor maxicircle stalked fruiting bodies. Traditionally they include three classes and/or elimination of major classes always major groups: , Dictyostelia, and Protostelia. occurs during promastigoteamastigote transformation We have examined the phylogenetic relationships (after infection of hamsters with clonal promastigote among Conosa using sequences of 18S rRNA and cultures). Remarkably, patterns of maxicircle amplifica protein synthesis elongation factor genes. Previously the tion and elimination differ from clone to clone. position of fruticulosa, which has a Amastigotepromastigote transformation is accompa like myxogastrids but singlespore nied by complete reversion to the promastigotespecific sporangia on stalks like protostelids, has been very pattern of maxicircle classes in all clones. The signifi unclear; some taxonomists put it in Myxogastria and cance of these drastic genome rearrangements remains others in Protostelia. We find that it is clearly sister to unknown. Our results provide a new outlook on the other Myxogastria, not to any more typical protostelids. kinetoplast genome structure and functioning. We also find strong support for a new clade, grouping Myxogastria (including Ceratiomyxa) with Dictyostelia. DIVERSITY AND GEOGRAPHIC DISTRIBUTION Protostelia appear to be split into two groups, and there OF SOIL PROTOZOA fore might be polyphyletic; however weak bootstrap W. Foissner1, A. Chao2 support and some differences among trees make further 1 Universitaet Salzburg, FB Organismische Biologie, Salzburg, Austria, data essential to test further the possible monophyly of 2 National Tsing Hua University, Institute of Statistics, HsinChu, Taiwan. Email: [email protected] Protostelia and Mycetozoa. However the position of Soil is inhabited by most main groups of protists, ranging Ceratiomyxa and grouping of Myxogastria and Dictyo from heterotrophic to autotrophic species, the latter stelia seems very robust. We will discuss the characters being especially frequent in the litter layer. Two groups that appear to be most meaningful and possible evolu of soil protists have been investigated in considerable tionary scenarios. detail, viz., testate amoebae and ciliates. However, still all data refer to morphospecies because molecular THE KINETOPLAST GENOME OF LEISHMANIA investigations are difficult in the soil habitat. Data on MAJOR CONTAINS SEVERAL MAXICIRCLE are still rare, but one species, Hemimastix CLASSES UNDERGOING DIFFERENTIAL amphikineta, is a highly conspicuous flagship with re AMPLIFICATION AT THE AMASTIGOTE STAGE stricted Gondwanan distribution. About 600 testate P.N. Flegontov1, E.S. Gerasimov1, E.N. Zhirenkina2, amoebae species have been recorded from terrestrial E.N. Ponirovsky2, M.V. Strelkova2, A.A. Kolesnikov1 habitats globally, and rather many of them are palaeo 1 Moscow State University, Department of Molecular Biology, Moscow, endemics (break of Pangaea) or continental endemics, Russia, 2 Sechenov Moscow Medical Academy, Martsinovsky Institute of Medical Parasitology and Tropical Medicine, Department of Medical for instance, the genera Apodera, Certesiella, Lamtopyxis Protozoology, Moscow, Russia. Email: [email protected] and Matsakision. Many of these are size flagships and 30 · Abstracts of V European Congress of Protistology · 2007 thus provide indisputable evidences for a restricted S.S. Kutorga (18051861), who compiled the "Natural distribution of protist morphospecies. Over 1000 species History of " (1839). Then he switched comp of soil ciliates have been reported. We show by faunistic letely to geology. However, one of Kutorga's students, and statistical analyses restricted distribution patterns, L.S. Cienkowsky (18221887) was a real protistologist. especially restricted Gondwanan/Laurasian occurrence. He discovered and described several dozens of protists In sum, protist biogeography is similar to that of plants and traced life cycles of many of them. He payed and animals, but with an increased proportion of particular attention to the cyst formation in ciliates and cosmopolites, favouring the moderate endemicity phenomenon of symbiosis in lower organisms. His model proposed by Foissner. Supported by FWF grants protistological ideas and works inspired a number of 19699, 15017 and by the Taiwan National Science students and followers. Among of them A.S. Famintzin Council, projects NSC942118M006001, 952118 (18351918), plant physiologist, was always interested M007003. in protists. He probably inspired for the field his student K.S. Merezhkovsky (18551921) although the latter A UNIQUE ASSOCIATION BETWEEN BACTERIA graduated from the Zoological Department. Merezh AND CILIATES: SILICIOUS RESIDUES FROM kovsky was the one to study protists most systematically FOOD BACTERIA ARE THE MAIN COMPONENT in Russia during that period: in the beginning ciliates OF THE CYST WALL OF MARYNA UMBRELLATA (18771886) and then . In his own turn, (CILIOPHORA, ) Merezhkovsky started to teach W.T. Schewiakoff in St. W. Foissner, B. Peukert, W.D. Krautgartner Petersburg University (18811884). Later Schewiakoff Universitaet Salzburg, FB Organismische Biologie, Salzburg, Austria. (18591930) graduated from Heidelberg University Email: [email protected] under the supervision of O. Butschli (1889). In 1896 Maryna umbrellata is an about 150 µmsized, globular Schewiakoff presented in St. Petersburg the monogra ciliate typically inhabiting ephemeral pools. It belongs phy "Organisation and systematics of Infusoria Aspi to the class Colpodea, family Marynidae. Maryna rotricha ( auctorum)", which was a brilliant umbrellata can quickly encyst and excyst and feeds on conclusion of a series of monographs started by F. Stein. bacteria. The resting cyst, which has an average From this point all St. Petersburg's protistological diameter of 105 µm, has a 10 µm thick wall. The interior "forces" grew up from Schewiakoff directly (S.I. Metal half of the wall is of ordinary fine structure, while the nikoff, S.V. Awerinzev, A.S. Schepotiev, I.K. Dembovsky) exterior half consists of countless, minute globules with or from his best student, V.A. Dogiel (18821955): G.N. a size of 0.5 2 µm. Transmission electron microscopy Gassovsky, A.A. Strelkov, G.I. Polyansky, E.M. Cheisin, (TEM), Xray analysis, and treatment with hydrofluoric and finally, L.N. Seravin and I.B. Raikov. acid (HF) reveal the globules to be composed of amorphic silicon. The silicon globules are recognizable HOLOSPORA AND HOLOSPORALIKE ENDOCY in the interphase specimens, where they appear as TOBIONTS IN CILIOPHORA minute, strongly sparkling "crystals" when observed with S.I. Fokin1, F. Ferrantini2, C Vannini2, H.D. Goertz3, interference contrast. Detailed TEM investigations F. Verni2, G. Rosati2, G. Petroni2 showed that the silicon globules are not produced by 1 Biological Research Institute of St.Petersburg State Univer the ciliate, but taken from certain food bacteria sity.198504, St.Petersburg, Russia, 2 University of Pisa, Department containing minute silicon spheres (proven by TEM, X of Biology, 56126, Pisa, Italy, 3 Biological Institute, University of ray, and HF!). These spheres are agglomerated by the Stuttgart, 70569, Germany. Email: [email protected] ciliate and extruded during the very early phase of The life cycle of highly infectious endocytobionts (Eb) encystment. To our best knowledge, such mechanism as Holospora includes infective (I) and reproductive (R) (slave silicon spheres) has not been described in any stages. These Eb are widespread in several Paramecium other protist, but likely occurs in several species of the spp. However, Eb with similar life cycle are also present family. Supported by the FWF, grants P19699 and P in some natural populations of other ciliates: , 15017. Frontonia, , Trithigmostoma, , Prorodon, , and Vorticella. SOME FACES OF RUSSIAN PROTISTOLOGY IN Such Holosporalike Eb are, as a rule, represented by µ ST. PETERSBURG small Rforms (13 m) with homogeneous cytoplasm, µ S.I. Fokin whereas the Iforms are often much larger (1030 m) Biological Research Institute of St.Petersburg State University, Russia. and differentiated into the cytoplasmic, the periplasmic Email: [email protected] and the recognition tip zones. Three Holosporalike Eb The first Russian protozoological research dates back were newly discovered in native populations of S. minus, to the late XVIII century. But visible protistological line F. leucas and F. salmastra in Italy. Those Eb populated was started from the Russian follower of Ehrenberg the macronucleus of ciliates. Both Eb from Frontonia Protistology · 31 spp. manifested FISH signal using Holosporaspecific The pelobionts (Pelomyxidae + Mastigamoebidae) are probes. The Eb from S. minus also belong to alpha a group of mostly freeliving amoeboid and flagellated proteobacteria. The Eb from F. leucas were typical protists which inhabit microoxic environments. The Holospora with dimensions: 12.5×0.91.0 µm (R) and group can be defined by reference to ultrastructural 512×0.70.8 µm (I). Eb from F. salmastra had size 2 characters: a single basal body and absence of Golgi 4×1.31.5 µm (R), but Iforms could reach 830×25 dictyosomes and cristate mitochondria. A major µm with peculiar spindleshape form. The Eb of S. impediment to progress in determining the phyloge minus differed from "classical" Holospora: 36×23 µm netic position of pelobionts is the fragmentary nature (R) and 58×45 µm (I) and had some kind of extrusive of structural data for the group and taxonomic tangle. device. Aposymbiotic cells can be experimentally In the present work the original ultrastructural data from infected by the homogenate of infected ones, and only 10 studied Pelomyxa species (P. palustris, P. belevskii, Iformes could enter the macronucleus. Phylogenetical P. binucleata, P. corona, P. flava, P. gruberi, P. prima, P. position of the Eb from F. salmastra was confirmed by sigara, P.stagnalis and P. tertia) and 3 mastigamoebids 16S rRNA gene characterization. These findings for the species (Mastigamoeba aspera, M. setosa and first time definitely recorded at least two species of nitens) are compared to data from previously examined Holospora out of the Paramecium genus. pelobionts. The comparisons are documented and summarized as parsimony analyses. Our results show ANALYSIS OF THE βTUBULIN GENE FROM that pelobiontids extremely varied in their cellular VITTAFORMA CORNEAE SUGGESTS BENZIMI organization. The most considerable differences DAZOLE RESITANCE concerned the basal parts of flagella, nuclear organiza C. Franzen, J. Schoelmerich, B. Salzberger tion and cell surface of the pelobiontids. Cladistic University of Regensburg, Regensburg, Germany. analyses using morphological characters support the Email: [email protected]Regensburg.de monophyly of the pelobionts but do not favour mono Vittaforma corneae is a microsporidium causing corneal phyly of main genera of pelobionts. On the basis of these and disseminated infections in humans. Benzimidazoles data we now regard the genera Pelomyxa and Masti are widely used as antihelmintic drugs in veterinary and gamoeba as paraphyletic groups. The work was sup human medicine and as antifungal agents in agriculture, ported by the Russian Foundation for Basic Research and albendazole is one of the most commonly used (Project 050448166). drugs for treating microsporidiosis in humans. However, clinical data show poor response of Vittaforma infections to albendazole. We amplified, cloned, and sequenced INFECTION OF HOLOSPORA IS CONTROLED BY the βtubulin gene of V. corneae, and the sequence 89KDA PERIPLASMIC PROTEINS AND THE showed 72% similarity with the βtubulin of Enterocyto HOST ACTIN 1 1 1 2 zoon bieneusi and 72 to 74% similarity with βtubulins M. Fujishima , K. Iwatani , Y. Nakamura , Y. Kodama 1 Department of Environmental Science and Engineering, Graduate of Encephalitozoon spp. Analysis of the obtained sequ School of Science and Engineering, Yamaguchi University, Yoshida ence provides an explanation for the observed clinical 16771, Yamaguchi 7538512, Japan, 2 Department of Natural resistance of V. corneae to albendazole. The βtubulin Science and Symbiosis, Graduate School of Science and Engineering, gene encoded by the sequence has a substitution at Yamaguchi University, Yoshida 16771, Yamaguchi 7538512, Japan. Email: fujishim@yamaguchiu.ac.jp Glu (with glutamine), which is one of six amino acids 198 Holospora species are endonuclear bacteria symbionts reported to be associated with benzimidazole sensitivity. of the ciliate Paramecium species. Infectious forms of Benzimidazoles were assayed for antimicrosporidial this bacterium shows distinctive structure, one half of activity using an in vitro assay with V. corneae and which contains the cytoplasm and the other half of a Encephalitozoon cuniculi (as control). Microsporidian periplasmic lumen with an electrontranslucent tip. spores and drugs were added to monolayers of MRC5 When the infectious form is engulfed into the host cells on day 0, and the numbers of organisms were digestive vacuole (DV), the bacterium escapes from the measured on day 10. Albendazole expressed significant latter to appear in the host cytoplasm, migrates to the higher MIC50s for V. corneae than for E. cuniculi. target nucleus, and penetrates the nuclear envelope with Molecular data explain the clinicaly observed resistance the special tip ahead, but not with the other tip. To of V. corneae to benzimidazoles, that could be shown investigate the underlying molecular mechanism of this also in vitro. infection process, we raised a monoclonal antibody against the special tipspecific 89kDa protein of H. COMPARATIVE MORPHOLOGY OF PELOBIONTS obtusa, sequenced this protein partially and identified A. Frolov the corresponding complete gene. The deduced amino Zoological Institute RAS, St. Petersburg, Russia. Email: [email protected] acid sequence carries two actinbinding motifs near N 32 · Abstracts of V European Congress of Protistology · 2007 terminal of the protein. Indirect immunofluorescence EPIZOOTIOLOGY OF THELOHANIA SOLENOP microscopy shows that during the escape from the host SAE (MICROSPORIDIA) IN THE RED IMPORTED DV, the 89kDa proteins translocates from the inside FIRE ANT, SOLENOPSIS INVICTA to the outside of the tip. In the host cytoplasm, bacteria J.R. Fuxa, M.L. Milks, Y.Y. Sokolova, A.R. Richter keep the 89kDa proteins outside the tip. When the Department of Entomology, Louisiana State University, Baton Rouge, bacteria invades the macronucleus, the 89kDa proteins LA 70803 USA. Email: [email protected] are left behind at the entry point of the nuclear envelope. Epizootiology of the microsporidium Thelohania Monoclonal antibodies specific for P. caudatum actin, solenopsae was investigated in red imported fire ants, labeled bacterial 89kDa proteins in the host cytoplasm Solenopsis invicta. The microsporidium was detected at and penetrated the target nuclear envelope. Latrunculin 16% of 165 sites and in 10% of 1309 colonies surveyed B, an inhibitor of actin polymerization, inhibited the throughout Louisiana. Its distribution was clumped with bacterial infection. These results show that the 89kDa 32% of nests infected in northeastern Louisiana, 18% proteins and the host actin play a role in Holospora's in the southwestern corner, and 3% elsewhere. The migration through the host cytoplasm and the invasion microsporidium infected 2.3% of monogyne (single into the target nucleus. queen) and 55.9% of polygyne (multiplequeen) colonies. The highest prevalence rates were near marshes and waterways, and the lowest were in LIFE CYCLE AND PHYLOGENETIC POSITION agricultural and forested areas. Prevalence decreased OF NOCTILUCID (NOCTI as distance from commercial waterways and ports LUCA SCINTILLANS) increased. Occurrence of T. solenopsae was positively Y. Fukuda, H. Endoh Division of Life Science, Graduate School of Natural Science and correlated with the number of nests at a site and with Technology, Kanazawa University, Kanazawa 9201192, Japan. pH, calcium, and sodium content of the soil. Microspo Email: [email protected]u.ac.jp ridiuminfected colonies were less likely to have brood is an unarmed heterotrophic than healthy colonies. T. solenopsae epizootics were also dinoflagellate that inhabits world's oceans and is monitored over time. In a natural epizootic, 89100% sometimes responsible for harmful red tides. The life infected polygyne ants gradually disappeared, possibly cycle, in particular the developmental process after because they were at a competitive disadvantage to 15 gamete fusion, has not been fully elucidated. In 26%infected monogyne ants. The monogyne form did addition, the phylogenetic position of N. scintillans has not sustain the pathogen after polygyne ants disappea been still disputed because of two alternative views red. Longterm epizootics developed when the micro deduced from a few morphological characters and sporidium was released in two predominantly polygyne phylogenetic analyses of SSU rDNA. Here we show populations but not at two monogyne sites. Prevalence new details of various stages in a whole life cycle, leading peaked at >75% in both social forms; the form suffering to the idea that some characteristics in noctilucid higher prevalence decreased proportionally to the other. gametes might reflect the ancestral state of dinofla Prevalence averaged 4757% and did not vary seaso gellates. In fact, the gametes retain not only two flagella nally. The microsporidian rate of spread was 0.89.4 m/ that differ in length and motion (although the extent of month. T. solenopsae in these epizootics weakened ant differentiation is quite low), but also longitudinal and populations only sporadically, through decreases in transverse grooves, as is typical of , numbers of foragers, colony numbers, colony size, or indicating that dinoflagellatelike characteristics are brood. conserved only in the gametes, but not present in the specialized trophonts. This is also supported by our PREVALENCE OF CRYPTOSPORIDIA IN THE phylogenetic analyses using two proteincoding genes FARM ANIMALS OF AZERBAIJAN (betatubulin and hsp90), in which N. scintillans is one H.D. Gaibova, N.G. Iskenderova, M.A. Musaev of the most ancestral dinoflagellates, i.e. the next earliest Institute of Zoology, National Academy of Sciences of Azerbaijan, Baku, branch after marina within the dinoflagellates. Azerbaijan. Email: [email protected] Given the phylogenetic position of N. scintillans, its Various kinds of farm animals were examined in extremely specialised diploid trophont, and the pri Azerbaijan for presence of protozoan invasion by mitive dinoflagellatelike characteristics of its haploid observation of thin fecal smears stained by ZeelNeelsen zoospore, we conclude that noctilucids might be a technique with optical microscope. The extensivenesses possible evolutionary link between ancestral diploid of invasion (EI) with cryptosporidia amounted in cattle dinoflagellates and most other haploid core dinoflagel 20.8% (216 animals were examined), zebu 10% (30), lates. This implies that the transition from diploid to sheep 24.2% (335), goats 32.4% (127), buffalos haploid in trophonts occurred in the ancestor of core 20.2% (148), horses 17.4% (23), donkeys 5% (20). dinoflagellates, via neoteny of a noctilucidlike zoospore. In total 899 animals were examined in the farms situated Protistology · 33 in lowland, foothill and mountain areas of Azerbaijan. new species for the fauna of Ukraine. Three species The same animals were also examined for oocysts of (Acanthocystis myriospina, A. pectinata and Choanocystis in fecal smears and, excluding horses and aculeata) previously studied only by light microscopy, donkeys, for Cryptosporidium cysts in skeletal muscles. were examined by SEM. The joint invasion by Eimeria and Cryptosporidium was observed in all the studied kinds of animals. The highest DIVERSITY OF BACTERIAL SYMBIONTS IN values of EI were noted in the goats of the lowland farms PARAMECIUM (60.8%) and sheep of the foothill ones (53.3%). In cattle H.D. Goertz and buffalos in all farms, regardless of the altitude above Dept. Zoology, University of Stuttgart, Stuttgart, Germany. Email: sea level, the EI values were quite high: from 31.8 to [email protected]stuttgart.de 46.1%. The zebus manifested rather low EI 21.4%. In the middle of last century many bacterial symbionts The value of joint EI with cysts of and in Paramecium have been described; much information Cryptosporidium were 1.52 times higher than that with about them was reviewed by Preer, Preer and Jurand Eimeria and Sarcosystis. The Cryptosporidium oocysts (1974). However, since then many new symbionts were were found for the first time in the farm animals of found in Paramecium and in other ciliates. New Azerbaijan in the 1980s. In the subsequent 10 years of observations emphasize old views that interactions investigation significant prevalence of cryptosporidia between killer bacteria and paramecia are extremely in cattle, sheep and pigs was revealed. About one fifth complex, and it seems hardly acceptable to regard all of all the animals examined released oocysts. Till the these endocytobioses as examples of mutualism. Largely end of the 1990s, the trend to the reduction of EI with based upon rDNAsequences, the phylogenetic rela cryptosporidia in cattle, sheep and pigs was evident, tionships of some symbionts have been studied. The presumably due to dismantling of large livestock farms. phylogenetic position of the old kappaparticles, However, the present study demonstrated that all the Caedibacter taeniospiralis was found to be different from studied kinds of farm animals were invaded by that of other caedibacters showing that the genus is not cryprosporidia and EI of cattle with cryptosporidia monophyletic (Beier et al., 2002). As Caedibacter and rested at the same level. related bacteria have been found in protozoa other than ciliates, the question about host specificity of some of NEW DATA ON DIVERSITY AND DISTRIBUTION the symbionts is open. A low or even zero host specificity OF THE (CENTRO of intracellular bacteria may point to the potential risk HELIDA) IN UKRAINE for animals and humans. Most bacterial symbionts L.P. Gaponova known, however, appear to be host specific. As many I.I. Schmalhausen Institute of Zoology, Kiev, Ukraine. new symbionts of different bacterial taxa have been Email: [email protected] found in ciliates recently, we have to accept that many It is believed that the Centrohelida is a cosmopolitan if not most of the bacterial endocytobionts in ciliates group of protists. However their distribution and species and other protists are not yet known. The extended and composition in different regions of the world has not more systematic search for intracellular bacteria in yet been adequately investigated. Twentyfive species protists appears to be necessary. of heliozoans were registered in Ukraine. Only eleven species of heliozoans recorded in Ukraine have been COMMUNITY STRUCTURE OF BENTHIC FORA studied by electron microscopy (Mikrjukov, 1995, 1997, MINIFERA FROM A GLACIATED FJORD OF 1999); identification of other 14 species needs additional SPITSBERGEN confirmation. We collected fiftyfive samples in sub E.A. Golikova, M.A. Varfolomeeva, S.A. Korsun benthic water layers and inside clumps of aquatic SaintPetersburg State University, Department of Invertebrate Zoology, macrophytes from thirty freshwater reservoirs (shallow St. Petersburg, Russia. Email: [email protected] waters, rivers, streams, ponds, lakes and channels, etc.) Tidewater glaciers in subpolar fjords play an important in the environs of Kiev, Ukraine. Isolated heliozoans role in structuring the benthic communities. The were examined by light microscopy and scanning glaciers feed icebergs and meltwater in the fjords. electron microscopy (SEM) with special attention to Sequential change of the dominant species along the ultrastructure of the periplast and scales. So far 6 species fjord and decrease in the abundance have been identified and studied morphologically: seawards was recorded previously in Tempelfjorden Acanthocystis myriospina Penard, 1890, A. pectinata (Western Spitsbergen) (Korsun, Hald, 2000). In the Penard, 1889, Choanocystis aculeata (Hertwig et Lesser, present study we examined whether this pattern has 1874), Raphidocystis sp., Polyplacocystis ambigua (Pe been reproduced over years and whether the community nard, 1904) and P. coerulea (Penard, 1904). One species structure has changed following the surge of the glacier of Raphidocystis and two species of Polyplacocystis were in 2004. Sampling was performed on the transect of 9 34 · Abstracts of V European Congress of Protistology · 2007 stations along the fjord in July or August 20012005. necessary in one of the most ancient eukaryotes. Surface sediment samples were retrieved by RV Jan Moreover, the differences in the quantity of the Mayen using a 50x50cm boxcorer. Samples were transcripts show that they are regulated transcriptionally preserved in ethanol with Rose Bengal stain. Living during encystation, suggesting that they play an foraminifera were identified to the species level where important role in this differentiation process. possible, and counted. The species richness, the ShannonWeaver diversity index, and the total density MICROSPORIDIAN PARASITES OF of foraminifera increased seawards. Cluster analysis FROM THE VOLGA DELTA based on the absolute abundances revealed two groups A.K. Gorbunov of stations corresponding to the innermost and the outer Astrakhanskiy Biosphere Reserve, Astrakhan, Russia. parts of the fjord. At the innerfjord stations Elphidium Email: [email protected] excavatum f. clavata, Quinqueloculina stalkeri, Cassidu The study of infestation in water bodies of the lina reniforme dominated. However the density of E. Volga delta was based on light microscopy analysis of excavatum f. clavata, Q. stalkeri, as well as most other quantitative zooplankton samples collected during the taxa increased seawards. At the outerfjord stations years 19862006. In addition, qualitative samples were Nonionellina labradorica, Labrospira crassimargo and also taken and living material was examined. The most Recurvoides spp. dominated. Community structure was frequently found microsporidian parasites of rotifers stable during the whole period of observations, and no were Microsporidium asperospora (Fritsch, 1895). Mass significant effect of the glacier surging in 2004 could be infestation by M. asperospora was most common in B. detected from our data. calyciflorus. Severely infested rotifers were completely filled by these sausageshaped parasites. When the GENES OF THE MEVALONATE PATHWAY IN infested rotifers died, small round spores escaped from GIARDIA INTESTINALIS: HOW MANY DO EXIST, the body cavity of the rotifers through the head region WHICH ARE TRANSCRIBED AND HOW THEIR into the surrounding water. Single cases of infestation EXPRESSION CHANGES DURING ENCYSTA of Brachionus urceus, B. diversicornis, B. quadriden TION? tatus, Polyarthra longiremis, P. dolichoptera, Epiphanes M.V. Gomez, M. Wasserman brachionus, Conochilus unicornis, Conochiloides coeno Laboratorio de Investigaciones Basicas en Bioquimica, Deparment of basis, Sinantherina semibullata and some other rotifer Chemistry, National University of Colombia, Bogota, Colombia. species with the parasites similar to M. asperospora, were Email: [email protected] recorded. Another parasite, Microsporidium polygona The protist Giardia intestinalis is a human parasite which (Fritsch, 1895), was recorded in A. priodonta, A. causes diarrheal disease throughout the world. It is brightwelli and S. semibullata. Cysts similar to Bertramia frequently described as an ancient protist, member of beuchampi, as described by Stempell (1921), were found the primitive group . Thus, it becomes an in Trochosphaera solstitialis. Polyarthra luminosa was interesting model to search for the origin of eukaryotic infected with cysts resembling the ones of metchniko pathways. In the present study we looked for the genes vellids, possibly, of the genus Amphiacantha. Some other that encode the enzymes of the mevalonate pathway in protozoan parasites were observed in rotifers from the G. intestinalis, especially those specific for biosynthesis Volga delta, but those have not been identified. This of cholesterol, ubiquinone, dolichol and prenylated investigation showed that many species of rotifers proteins. Firstly, we made a bioinformatic search into inhabiting the Volga delta are subject to infestation with the parasite's genome using consensus sequences. We microsporidia. For most rotifer species only single cases found one gene of the dolichol biosynthesis, and four of the prenylated proteins biosynthesis. Their existence of infection were recorded through 20 years of observa in the parasite was proved by the polymerase chain tions. However, infestation of some abundant species reaction (PCR), and their transcription was evaluated was observed every year. by reverse transcription PCR (RTPCR). Finally, we PHOSPHOLIPIDS OF THE MICROAEROPHILIC studied the expression profile of the genes during PROTOZOA FLAGELLATE, VAGI encystation, a process in which a trophozoite becomes NALIS a cyst, with real time RTPCR. We found differences I. Guschina1, D. Lloyd1, J.L. Harwood1, B. Goldberg2, in the transcripts quantity during the different stages of and K. M. Harris1,2 encystation. In conclusion, the genes coding the 1 Cardiff School of Biosciences, Cardiff University, Wales, UK, 2 enzymes of the dolichol and prenylated proteins New York University, Department of Chemistry, New York, NY, USA. biosyntheses, as well as their transcripts were found in Email: [email protected] G. intestinalis, suggesting that these products which Lipid composition of the sexually transmitted flagellated participate in posttranslational modifications are protist, Trichomonas vaginalis, showed the following (% Protistology · 35 total) distribution of polar lipids: phosphatidylethanol the species level were found. The extensiveness of the amine (30%), phosphatidylcholine (9%), phosphatidyl amphibian invasion with the hemoparasites was 85.7% glycerol (7%), phosphatidylserine (7%), phoshpatidic and intensity 1038 hemoparasites per a blood smear. acid (3%), dimethylphosphatidylethanolamine (2%). Apart from that, coccidians (Apicomplexa, ) Two unusual constituents were acylphosphatidylglycerol of Haemogregarinidae and Lankesterellidae families (26%) and ceramide phosphorylethanolamine (7%). were found in the amphibian blood samples. Repre The two unusual lipids were subjected to both single sentatives of the genus Lankesterella were found within stage electrospray ionization mass spectrometry (ESI the Caucasus's territory for the first time. The multicel MS) and tandem ESIMS for structural analysis. For lular parasites, the larvae of the tissue nematodes acylphosphatidylglycerol, the peak at m/z 1013 gave microfillaria (Nematoda: Filariidae) were found as well. fragments at m/z 281, 283, and 255 representing stearic, Absence of hemoparasites in fish from the Ganyhchay oleic and palmitic acids, respectively. A fragment ion river may be due to the complex of abiotic and biotic at 749 corresponded to 16:0/18:1 phosphatidylglycerol, factors, characteristic for the mountain rivers, such as and those at 757 and 729 indicated neutral losses of lack of aquatic vegetation and speedy flow which blocks palmitic stearic acids respectively. For ceramide development and of the leeches Piscicola phosphorylethanolamine (m/z 661), palmitic acid was geometra, the main vectors of the fish hemoparasites. the amidelinked fatty acid of the phosphoceramide The amphibia had abundant hemoparasite fauna, (m/z 617), whereas stearic acid and ethanolamine were presumably, because of the semiaquatic lifestyle the other fragments detected. The structure finally facilitating contacts with the hemoparasite vectors: assigned corresponded to N(hexadecanoyl)sphing leeches and bloodsucker insects (mosquitoes, gnats 4enine1phosphoethanolamine. Cardiolipin was not etc.). detected on thin layer chromatography (TLC) plates, nor by ESIMS of total lipid or of eluates from TLC CIMES: CILIATES AS MONITORS FOR LATERAL plates, although an authentic standard gave the expected GENE TRANSFER negative ion (MH) at m/z 1470. These data are dis J.H.P. Hackstein cussed in relation to the unusual lifestyle of the organism Radboud University Nijmegen, The Netherlands. and its phylogenetic status. Email: [email protected] We had postulated that ciliates are ideal monitors to HEMOPARASITES OF FISH AND AMPHIBIA IN detect lateral gene transfer (LGT) from genetically THE GANYHCHAY RIVER (NORTHWESTERN modified organisms to ciliates thriving in the gastro AZERBAIJAN) intestinal tract of ruminants or cockroaches. Unique M.A. Guseinov nuclear dimorphism and the intriguing structure of the Institute of Zoology, Azerbaijan National Academy of Sciences, macronuclear genomes of ciliates should allow Department of Parasites of Aquatic Animals, Azerbaijan. excluding any artefacts due to contamination by traces Email: [email protected] The objective of our investigation was the study of the of DNA from GMO. This could be substantiated. hemoparasites of fish and amphibia in the Ganyhchay Nyctotherus ovalis and several rumen ciliates have been river (Alazan) that originates in the mountains of the used to study lateral gene transfer from bacterial and Greater Caucasus, flows through the territory of plant sources both for short term (3 years) challenges Georgia, and enters Mingechavir reservoir, the latter with transgenic Bt 176 maize and LGT in evolutionary being the dwelling place of valuable gamefish. Material time. While the short term experiments did not reveal for the study was collected in the upper reach of the any LGT from Bt 176 maize, we identified substantial river, at the border with Georgia, and in the lower one, amounts of LGT in evolutionary time. The bioin 5 kilometers from the point where it enters Mingechavir formatic analysis of more than 4000 cDNAs from reservoir. Forty six fish specimens (Varicorhinus capoeta, rumen ciliates and more than 4000 minichromosomes Barbus lacerta cyri, Chalcalburunus chalcoides, Abramis and 5000 cDNAs from N. ovalis revealed 148 bacterial brama orientalis, Cyprinus carpio, Lucioperca lucioperca, to ciliate transfers in the rumen ciliates, and about one Silurus glanis) and 14 specimens of Rana ridibunda were order of magnitude less in N. ovalis. Also, genes examined for hemoparasite invasion. Blood for obtained by LGT differed depending on the ciliate examination was taken by routine methods developed species. It will be discussed, whether different macro for fish and amphibia. All the examined fish specimen nuclear genome architecture, i.e. midichromosomes vs. were found to be free of hemoparasites. In the blood of minichromosomes, or dissimilarities in lifestyles could 12 amphibian specimens Trypanosoma loricanum, T. be responsible for these differences. In addition, we will neveulemairei, T. mega (Mastigophora: Trypanosomidae) discuss the significance of the frequent potential plant and also some trypanosomes that were not identified to ciliate transfers. 36 · Abstracts of V European Congress of Protistology · 2007

TRICHOMONAS VAGINALIS AND GIARDIA IN TOTAL INTRACELLULAR BACTERIUM SPECTRA TESTINALIS PRODUCE NITRIC OXIDE: DOES FROM DIFFERENT CLONAL CULTURES OF NITROSYLATION INDUCE CARBOHYDRATE ARCELLA SPP. COMPARED BY DGGE METABOLISM? Zs. Heeger1, B. Pollak1, J.K. Torok2, K. Marialigeti1 K.M. Harris1,2, B. Goldberg2, M. van der Giezen3, 1 Eotvos Lorand University, Department of Microbiology, Budapest, 2 G. Biagini4, D. Lloyd1 Hungary, Eotvos Lorand University, Department of Systematic Zoology and Ecology, Budapest, Hungary. Email: [email protected] 1 Cardiff University, Microbiology, Cardiff, Wales, UK, 2 New York University, Department of Chemistry, New York, NY, USA, 3 School Several species of intracellular bacteria which are of Biological and Chemical Sciences, Queen Mary, University of considered as endocytobionts of freeliving amoebae London, England, UK, 4 Liverpool School of Tropical Medicine, have already been reported in earlier studies; however, England, UK. Email: [email protected] our understanding of the total composition of bacterial The argininedependent production of nitric oxide species within one host organism is very poor. Further (NO) in Trichomonas vaginalis and Giardia intestinalis more, a comparison of total bacterial symbiont spectra was studied. Potentiometric measurement of whole cell among different host clones may also provide new suspensions of T. vaginalis, monitored by a NO electrode, significant data for symbiosis studies. In this experiment indicated that the basal production rate of NO (15.3 we examined the total bacterial spectra of different nmol min1/106 organisms) increased eightfold after 4 species of Arcella, a lobose testate . The host h starvation. G. intestinalis monitored by NO electrode cells were fed with pure Enterobacter aerogenes cultures produced NO, which was stimulated by the addition of in order to reduce the contamination by external arginine. When the granular fraction of G. intestinalis, bacteria and were washed in sterile water before mole which contained marker enzyme activity similar to that cular analyses. The 16S rRNA genebased molecular found in hydrogenosomal fractions of T. vaginalis, was technique was applied to reveal the complete bacterial monitored, NO was produced at a rate of 415.6 nmoles/ species composition and variability of Arcella cells. mg protein/min. Fluorimetric detection of both orga Molecular cloning was carried out to identify most of nisms by confocal microscopy, after preincubation the colonizing bacterial species, and denaturing with the NOspecific fluorogen 4amino5methyl gradient gel electrophoresis (DGGE) was used to amino2'7'difluorescein, indicated population hetero compare the symbiotic spectra of different host cells. geneity of NO production in freshly harvested organisms. We examined both different cultures kept up under Brightly fluorescent T. vaginalis (25%) showed fluores laboratory conditions for a longer period, and newly cent reaction product uniformly throughout the cytosol, established clonal cultures from several environmental whereas less intensely fluorescent organisms (70%) samples. showed organellar localization. G. intestinalis showed localization of NO production in the periphery of the cytosol; organisms stained with 5µM tetramethylrhoda TESTATE AMOEBAE (PROTISTA) COMMUNI mine ethyl ester showed localization in circular orga TIES IN CALLUNA VULGARIS LITTER DIFFER nelles with membrane potential along the periphery of WITH INCREASING ALTITUDE the organism, suggesting NO production by these mem T.J. Heger1, N. Derungs1, J.P. Theurillat2, E. Mitchell1 brane potential generating organelles. NO synthase 1 Swiss Federal Institute for Forest, Snow and Landscape Research, Ecosystem Boundaries Research Unit, Wetlands Research Group, activity measured in cellfree extracts by NO electrode Station 2, CH1015 Lausanne, Switzerland, 2 Centre alpien de and a colorimetric assay indicated the presence of both Phytogйographie, Fondation J.M. Aubert, CH1938 Champex, particulate and nonsedimentable activities in T. vagi Switzerland. Email: [email protected] nalis, and localized NOS activity to the granular frac Species richness of higher organisms decreases generally with increasing altitude and latitude, but it remains tion in G. intestinalis. The apparent KM for arginine for the nonsedimentable NOS in T. vaginalis was 2.5 × unclear whether this pattern also holds true for micro 104 moles/L, and for G. intestinalis 8.28 ×103 moles/ organisms. If this is the case, does the observed decrease L, respectively. We conclude that the granular fraction result from the effect of altitude or rather from declining of G. intestinalis contains nitric oxide synthase which diversity of habitats? To disentangle the effects of may be localized in redoxbalancing organelles. Bioin altitude and habitat diversity, we only considered com formatic searches confirmed the presence of two poten munities of a single habitat occurring along a large tial NOS genes in T. vaginalis and one in G. intestinalis, altitudinal gradient. We studied testate amoebae com which contain protein motifs typically associated with munities in Calluna vulgaris litter along the vertical NOS sequences. Bioinformatic searches also located transect crossing the forest and timberlines from 1800 nitrosylation sites in specific enzymes of bioenergetic to 2400 m in the Swiss Alps. The study area is charac pathways. Implications of NO production in the evolu terized by siliceous bedrock and by a timberline located tion, biology and pathogenicity of these parasites are at about 2200 m. The pH of each sample was also discussed. measured. A total of 31 testate amoebae taxa belonging Protistology · 37 to 14 genera were identified. The number of taxa MULTIPLE LOSSES OF SEX WITHIN A SINGLE observed per sample of 100 individuals varied from 12 GENUS OF MICROSPORIDIA to 21. The dominant species Corythion dubium, Assulina J.E. Ironside muscorum and Trinema lineare were present in all University of Wales, Institute of Biological Sciences, Aberystwyth, UK. samples. The sum of their relative abundance was always Email: [email protected] higher than 35%. Species richness varied only slightly Most asexual eukaryotic lineages have arisen recently across the samples, but nevertheless it decreased signi from sexual ancestors and contain few ecologically ficantly with the altitude. Although this trend was not distinct species, providing evidence for longterm strong, it supported the hypothesis about positive cor advantages of sex. Ancient asexual lineages provide rare relation between the testate amoeba diversity and exceptions to this rule and so can yield valuable temperature. In a canonical correspondence analysis, information relating to the evolutionary forces that the variation in the relative abundance of the testate underlie the maintenance of sex. Among the Microspo amoeba taxa was explained, firstly, by altitude, and, ridia there are many asexual species which have tradi secondly, by the litter pH. Presence or absence of forest tionally been grouped together into large, presumably was not significantly correlated with the composition ancient taxonomic groups. However, these putative of the testate amoebae communities; the altitudinal ancient asexual lineages have been identified on the trend is therefore gradual and does not reflect a thre basis of morphology, life cycles and small subunit shold effect. ribosomal RNA (16S rRNA) gene sequences, all of which are of questionable value in accurately inferring PHYLOGENY AND BIODIVERSITY OF HETERO phylogenetic relationships among Microsporidia. The MITID CERCOZOA: ABUNDANT, DIVERSE, AND hypothesis of a single, ancient loss of sex within the UBIQUITOUS ZOOFLAGELLATES REVEALED Nosema/Vairimorpha group of microsporidia was tested A.T. Howe, D. Bass, E.E. Chao, T. CavalierSmith using phylogenetic analyses based on alignments of University of Oxford, Department of Zoology, Oxford, UK. rRNA and RPB1 gene sequences from sexual and Email: [email protected] asexual species. Neither set of gene trees supported Heteromitids form a clade of diverse and abundant hete ancient asexuality, instead indicating at least two, recent rotrophic flagellates that includes Heteromita, Bodomor losses of sex within the Nosema/Vairimorpha group of pha, Allantion, Proleptomonas, and many unnamed and microsporidia. The absence of ancient asexual lineages previously unplaced taxa. They occupy freshwater and indicates that sex confers important longterm advanta soil habitats globally. However, intensive screening of ges even upon highly simplified eukaryotes such as ecologically and globally diverse DNA samples using microsporidia. The rapid evolution of microsporidian groupspecific primers suggests that heteromitids are life cycles indicated by this study also suggests that even not found in marine environments. We created 18S closely related microsporidia cannot be assumed to have rRNA gene libraries from individual (c. 1 gram) soil similar life cycles and the life cycle of each newly disco samples from a sampling site near Oxford. All samples vered species must therefore be completely described. tested contained heteromitid sequences. The number These findings are relevant to the use of microsporidia of genetically distinct lineages (18Stypes) in each soil as biological control agents, since several species under sample ranged from 12 to 40, and species richness consideration as potential agents have life cycles that estimators suggest that some such samples may harbour are incompletely described. more than 50 18Stypes. Our sampling of multiple 18S rDNA libraries from the UK, Australia, South America, PARASITIC SYSTEMS OF MICROSPORIDIA and Europe, reveals many novel heteromitid groups and I.V. Issi suggests that total global heteromitid diversity exceeds AllRussian Institute for Plant Protection, Microbiological Control 500 species. Community comparisons suggest that Laboratory, St.PetersburgPushkin, Russia. Email: [email protected] heteromitids are more homogeneously distributed on a Coevolution of microsporidia and invertebrates resulted global scale than Clade A cercomonads. Here we present in formation of two types of parasitic systems, which a new and highly sampled heteromitid phylogeny of over we refer to as "tight" (stable) and "loose" (unstable). 200 (mostly novel) sequences, and describe many new Microsporidia, infecting hosts with high abundance and strains. We show that the morphological diversity of population density like aquatic forms of dipterans, heteromitids is far greater than previously realized, and develop "tight parasitic systems". Stability of these demonstrate that robust species definitions must include systems is sustained by alteration of horizontal and a description of the mode of movement and behaviour vertical transmission of the parasites, both finely tuned of strains over time, as well as an 18S rDNA sequence to particular characters of the microsporidium and its signature unique to each strain. insect host, i.e., narrow specificity of the parasite, type 38 · Abstracts of V European Congress of Protistology · 2007

of the insect activities at the imaginal and larval stages, with 15 spores (as a result of asynchronous divisions in different susceptibility/resistance to infection expressed some species) were found. Spores come out from the by males and females, etc. Prevalence of infection in test and release small amoeboid cells young tropho such systems seldom exceeds 10%, and the number of zoites forming the tests. The formation of a new test members of the system is strictly limited. "Loose during asexual binary was also demonstrated. parasitic systems" emerged through interactions of Separate stages including copulation and spore forma microsporidia with the hosts with scanty natural tion were observed in the following species: C. orbicularis, populations and low densities. Wide spectrum of C. dubium, Trinema lineare, T. complanatum, T. systematically or ecologically related hosts, and diverse enchelys, Euglypha strigosa, E. cristata, E. ciliata, Nebela routes of horizontal and vertical transmission usually bohemica, N. tincta, Assulina seminilum, A. muscorum, maintain stability of these systems. Number of the Heleopera sylvatica, Arcella vulgaris, Tracheleuglypha members of such parasitic system may vary. Solar dentata. The EM studies of nuclear behaviour are in activity rhythms or human activity impact causing the schedule. outbreaks and increasing host population densities, favor development of pathogenic forms of microsporidia NEWLY DISCOVERED SYMBIOTIC ASSOCIA and emergence of epizooties with 100% infection TION BETWEEN AMOEBA AND prevalence. Epizooties of microsporidiosis may occupy L.O. Ivanova vast territories, including isolated spots. Such epizooties Biological Research Institute of SaintPetersburg State University, usually lead to elimination of the majority of the host Laboratory of Invertebrate Zoology, St. Petersburg, Russia. population together with the most highly pathogenic Email: [email protected] A large number of fungi are known to parasitize on microsporidia isolates, destabilizing thus the parasitic different protists: ciliates and there cysts, flagellates system. However, eventually the system recovers due to (especially algae), and amoebae. Here we provide the existence of low pathogenic forms of microsporidia. data on organization of newly discovered symbiotic Because pest outbreaks are considered as a result of association between a large naked lobose amoeba and anthropogenic pressure onto nature, expression of wide a fungus. In outward appearance the association looks epizooties of microsporidiosis may also be regarded as like an amoeba with a tuft of fungi hyphae protruding a consequence of human impact. from its surface. During amoeba locomotion, the tuft is located on the amoeba uroid. The tuft includes THE LIGHT MICROSCOPIC EVIDENCES OF several separate (up to 15) filamentous talli, each SEXUAL PROCESS IN LOBOSE AND FILOSE consisting of single frequently branched hypha 2.6 ?m TESTACEAN AMOEBAE wide and up to 300 ?m long. Each thallus forms the T.A. Iudina Herzen State Pedagogical University of Russia, St. Petersburg, Russia. lobed haustorium within the amoeba cell (in special Email: [email protected] invagination of amoeba plasmalemma), which also Life cycle stages of different terrestrian testacean amoe functions as a holdfast. In association with the amoeba, bae were investigated with light microscope in clonal the fungus produces zygospores with ornamented cultures and samples. Both, the lobose and filose testa thickened walls that arise from zygogamy, consisting in ceans have the same main stages: asexual part includes conjugation of equal parts of talli. After zygospores' trophozoite undergoing binary fission, precyst, and maturation, the whole tuft of fungi hyphae is detached resting cyst; sexual trophozoite copulation (hologamy), from the amoeba cell, leading either to amoeba's death zygocyst, spore, and trophozoite. The most complete or to its further freeoffungus endurance. The fate of life cycle has been shown for Corythion delamarei: the detached hypha tuft is unknown. Asexual reproduc trophozoites contact by pseudostomes; cytoplasms of tion most likely proceeds by means of detachment of both partners fuse; the nucleus and cytoplasm of one short parts of hypha from the tallus. Life cycle and cell move into the partner's test; the following nuclear morphological data suggest that the described fungus fusion gives a synkaryon; a zygote produces a thick wall belongs to a new genus within the family Cochlonema and becomes a zygocyst inside the test. At the next stage, tacea (Zygomycota). At least four species of amoebae zygocyst covering disappears, and the synkaryon of the family (Lobosea, Gymnamoebia) undergoes 2 meiotic divisions. This results in 4 separate form association with fungi. Three of them appeared haploid cells, which incyst inside the mother shell and to be new species of the genera Amoeba, typically produce 4 spores. Sometimes the zygote pro and , and another one is likely to be Amoeba duces more than 4 spores: the tests with 5, 6, 8 and even proteus. Protistology · 39

MACRONUCLEAR INTRA SPHASE CHECK 1 Moscow State University, Dept. of Invertebrate Zoology, Moscow, 2 POINT IN THERMOPHILA IN Russia, People Friendship University of Russia, Medical Faculty, Moscow, Russia. Email: [email protected] DUCED WITH LOW DOSES OF APHIDICOLIN, The recent studies suggest that inhibitory influence of ROLE OF "PSEUDO SPINDLE" IN MACRO T. cruzi on cancer growth has got a dual mechanism. NUCLEAR DNA SEGREGATION AND APOPTO The following observations show that direct selective TICLIKE DEGRADATION OF "EXTRUSION effect of live T. cruzi (and its preparations) on the cancer BODIES" cells in vivo is supplemented by indirect influence on A.W. Kaczanowski1, M. Kiersnowska1, M. Prajer2, the whole cancer process by means of immunological Sz. Kaczanowski3 mechanism: (1) organisms which survived the T. cruzi 1 Dept of Cytophysiology, Faculty of Biology, University of Warsaw, Poland, 2 Institute of Systematics and Evolution of Animals, Krakow, infection, were significantly protected against the trans Poland, 3 Institute of Biophysics and Biochemistry of Polish Academy planted tumor; (2) intentional immunization of mice of Sciences, Poland. Email: [email protected] with avirulent live trypanosomes resulted in reliable Low doses of aphidicolin induced cell division arrest in inhibition of sarcoma180 and Ehrlichadenocarcinoma T. thermophila. Cells advanced in oral morphogenesis growth; (3) preparation of lysed T. cruzi epimastigotes completed one cell division in the presence of the drug, was immunogenous, it possessed immunomodulatory while the other did not progress in cortical morphoge activity, and immunization with this preparation created nesis. They increased macronuclear DNA content (3 a definite degree of protection against tumor implanta × × 4 ), size (>2 cell surface) and number of ciliary basal tions. In all these experiments the antitumoral effect bodies during aphidicolin treatment. These data suggest coincided with appearance of mice' antibodies to T. an intra Sphase check point mechanism in Tetrahy cruzi, and it positively correlated with the level of con mena due to successive rounds of abnormal DNA sidered antibodies. The antitumoral effect is based on replication in presence of the drug. In silico of Tetrahy the known antigens community of T. cruzi and the host. mena genome many genes coding for PI3 kinases were This antigens community of T. cruzi and cancer cells found, but we were not able to specify direct homologs was shown by indirect immunofluorescence. Thus, the of genes coding for mammalian ATM and ATR participation of humoral immunity in T. cruzi anti proteins. The putative homolog of mammalian CHFR tumoral phenomenon is evident. Its role is rather protein, involved in G /M transition, was identified in 2 significant. In vivo effect of T. cruzi preparation is Tetrahymena. Cells shifted to the drugfree medium, accompanied by substantial pool of following antibodies resumed their divisions. Then a "central chromatin against T. cruzi: (a) spontaneous ones, discovered in the granule" appeared in the middle of dividing, elongating intact mice; (b) the ones induced by tumor transplanta "giant" macronucleus. This chromatin was first tion; (c) antibodies induced by T. cruzi itself, as a result surrounded by microtubule bands involved in the of injection with T. cruzi preparation. Occurrence of macronuclear division ("pseudo spindle") and next these antibodies is correlated with inhibition of cancer disposed as an extrusion body (EB), containing up to growth. The results presented are likely to make possible 1/3 of the previous macronuclear content. It suggests the oncoprophylactic use of T. cruzi extraordinary pro an active role of intra macronuclear "pseudo spindle" perties. both in segregation of the "normal" mini chromosomes into daughter macronuclei and segregation of the REGULATION OF AMINO ACID TRANSPORT BY defective chromatin into EBs. EBs underwent an CALCIUM ION AND SUGARS IN PARAMECIUM "apoptoticlike "degradation (as old macronuclei in SYMBIONT conjugants): they consisted of highly condensed Y. Kato, N. Imamura chromatin, were stained with the TUNEL method and Ritsumeikan University, Department of Bioscience and , showed DNA fragmentation in gel electrophoresis. EBs Kusatsu, Japan. Email: [email protected] were also stained in vivo with acridine orange, which is The green ciliate, Paramecium bursaria, has several a marker of autophagy. hundred chlorellas. Symbiont F36ZK isolated from Japanese P. bursaria F36 showed notable characteristics ON THE IMMUNOLOGICAL MECHANISMS OF on nitrogen utilization, i.e., the alga lacked nitrate TRYPANOSOMA CRUZI ANTITUMORAL PHENO reductase activity and had three constitutive amino acid MENON transport systems. Interestingly, the algal Ser uptake was V.D. Kallinikova1, E.G. Kravtzov2, Z. Batmunkh2, inhibited by polyvalent cations, especially Ca2+, E.N. Kosobokova1, L.V. Pakhorukova1, T.A. Ogloblina1, although the cation generally activates amino acid L.P. Karpenko2 transport in many organisms. The inhibition was 40 · Abstracts of V European Congress of Protistology · 2007

2+ noncompetitive, since Ca affected only the Vmax of Ser palintomy, suggesting reproduction of ciliates inside the transport. Effect of sugar on amino acid was also host body. Occurrence of large and wide specimens, evaluated, because symbionts were known to release which can be considered as protomonts, in the sugar. Uptake of Ser was increased by glucose even at examined population, confirms this suggestion. µ low concentration (EC50=3 M). Nonmetabolizable Presence of one small, pearshaped specimen, with glucose analogues also accelerated Ser uptake, regular pattern of kineties on its posterior end, indicating that sugar did not supply energy for transport. presumably a theront, supports the hypothesis that

The Vmax of Ser transport was doubled by treatment with reproduction of the ciliates parasitizing the inner organs glucose, however the response was sustained even when of their hosts may occur both in the external environ protein synthesis of cells was inhibited, thus, the ment and inside the host organism. phenomenon was not due to new protein synthesis. Surprisingly, uptake of radiolabeled glucose was not COMPARATIVE GENOMICS OF MICROSPO detectable, therefore, it was considered that the RIDIA acceleration by glucose occurred via glucose sensing and P.J. Keeling signaling pathway. The response to glucoserelated Botany Department, University of British Columbia, Vancouver, compounds was measured, and the results revealed an Canada. Email: [email protected] importance of stereochemistry at carbon 1, 2 and 5, Genomic compaction has occurred many times in and hydroxyl group on carbon 3 and 6 for the response. evolution, but we generally do not know why. Nuclear Glucose recovered the inhibition of Ser uptake by Ca2+, genomes are typically quite spacious, but in several thus, amino acid flux is easily controlled by these lineages compaction has occurred, the most spectacular compounds, implying a possibility of regulatory system being microsporidian (intracelluar parasites) and in Paramecium symbiosis. (endosymbionts). We have used a comparative approach to investigate the distribution of compaction in microsporidia, and variations in its OPHRYOGLENA SP., A PARASITE OF DREISSENA effects on genome function. The best studied microspo POLYMORPHA (PALLAS) IN POLAND, AND RE ridian genomes (Encephalitozoon and Antonospora) are MARKS ON ITS POSSIBLE REPRODUCTION IN all relatively small and fit the expectations for a com THE HOST pacted genome. We have carried out genome sequence S.L. Kazubski1, V.I. Yuryshynets2 1 Museum and Institute of Zoology Polish Academy of Sciences, surveys (GSS) on species with larger genomes, and these Warsaw, Poland, 2 Institute of Hydrobiology National Academy of paint a very different picture. In Brachiola algerae and Sciences of Ukraine, Kiev, Ukraine. Email: [email protected] Edhazardia aedis gene density is four to fivefold lower The interest in parasites of Dreissena polymorpha than observed in Encephalitozoon and Antonospora, and (Pallas) expanded when it became known that this transposons abundant. In both surveys, all genes invasive mollusc has successfully spread within Europe identified are also found in Encephalitozoon, suggesting and North America. The first ciliates included in the the three species contain similar proteomes despite genus Ophryoglena were found by the senior author in genome size differences. One of the interesting effects the lakes Lichenskie and Goslawskie near Konin of compaction described in Antonospora is the high (Poland) on November 9, 1970. Over the next 5 years, frequency of overlapping transcription. We have now more than 2200 zebra mussels from that region were determined that Encephalitozoon transcripts overlap at investigated, and many of them were infected with a similar frequency, but in different ways. Antonospora Ophryoglena. During that time many slide preparations transcription tends to initiate in the upstream intergenic from tissue smears were stained and silverimpregnated, region and terminate beyond the downstream intergenic and morphology of more than 600 specimens of region, but Encephalitozoon transcriptions more often Ophryoglena sp. was investigated in details. The results initiate within the upstream gene and terminate within of these investigations will be published in a joint paper the downstream intergenic region, suggesting compac with Dr. V.I. Yuryshynets. In the present communica tion affected the two genomes differently. We also show tion, the authors want to draw attention to a group of there is little conservation of transcript types between small Ophryoglena ciliates found in 15 specimens (about Antonospora and Encephalitozoon, suggesting the 2.5% of silver impregnated specimens examined). These process is very fluid over time, and that transcription ciliates differ by the form of the body and the pattern of overlap has not stabilized the genome. the argentophilic system at the posterior end of the cell. The Ophryoglena sp. ciliates normally have a regular NOVEL COMPARTMENTALIZATION OF PYRU course of kineties in this region, but in these specimens, VATE: NADP+ OXIDOREDUCTASE IN SPOROZO the kineties were curved and formed "a scar", which may ITES OF CRYPTOSPORIDIUM PARVUM have arisen at the place of splitting the cell during J.S. Keithly1, V. Ctrnacta2, F. Stejskal2, C.A. Mannella1 Protistology · 41

1 Wadsworth Center, New York State Department of Health, New shape within the Favella. In this laboratory culturing 2 York, USA, Charles University, Tropical Medicine, Prague, CZ. E study, the morphology and morphogenesis of F. ehren mail: [email protected] Using pyruvate dehydrogenase, most eukaryotes oxi bergii were observed using the protargol staining method, and small subunit rDNA sequences were also analyzed. datively decarboxylate pyruvate within mitochondria. Our morphological data showed 1618 of oral kinetids Anaerobic parasitic protists use instead the O sensitive 2 and almost 100 of somatic kineties. Compared with enzyme pyruvate:ferredoxin oxidoreductase (PFO), other species, oral kinetids and somatic kineties of F. which is localized within the cytosol, , ehrenbergii certainly differed from those of F. pana or . In contrast, both Cryptosporidium parvum mensis (22 of oral kinetids and 74 of somatic kineties) and encode and express a unique O 2 and F. novaeangliae (24 of oral kinetids and 35 of somatic sensitive fusion enzyme, pyruvate: NADP+ oxidoreduc kineties). tase (PNO), the Nterminal PFO domain of which is fused to a Cterminal NADPHcytochrome P450 CYST MORPHOLOGY AND ENCYSTMENT OF reductase. Unlike E. gracilis, the PNO of C. parvum lacks THE PLANKTONIC OLIGOTRICH CILIATE a mitochondrial targeting peptide. C. parvum does STROMBIDIUM CAPITATUM possess a small sandwiched between the Y. O. Kim1, A. Taniguchi2 nucleus and crystalloid body (CB). Transmission 1 Korea Ocean Research & Development Institute, Geoje 656830, electron microscopy (TEM) and tomographic recon Republic of Korea, 2 Tokyo University of Agriculture Okhotsk, structions reveal a complex arrangement of membranes Hokkaido 0992493, Japan. Email: [email protected] outside and within this organelle. The inner mitosomal Cysts of an oligotrich ciliate were isolated from natural membrane lacks tubular "crista junctions" typical of sediment samples collected in Onagawa Bay of Japan metazoan, fungal, and protist mitochondria. This is and in Masan Bay of Korea, and incubated under labo congruent with the loss through reductive evolution of ratory culture conditions. Excysted vegetative cells were the entire mitosomal genome and the capacity for observed after protargol staining and were identified as oxidative phosphorylation. Nevertheless, the C. parvum Strombidium capitatum. Cysts of S. capitatum have a mitosome shares with other anaerobic protists the spherical shape, with a papula and cyst wall ornamented machinery to assemble [FeS] clusters, an essential with spines. Size varied from 6062 µm in total length function of eukaryotic mitochondria. Western blot and 5055 µm in width. Seasonal changes in the analysis shows that sporozoites of C. parvum express vegetative population and sedimentation of newly the entire fusion protein, and both immunofluorescent formed cysts were investigated in situ. Planktonic and immunogold electron microscopy confirm that vegetative cells were abundant during the cold season CpPNO is primarily cytosolic. Unexpectedly, however, from December to April, when the water temperature CpPNO was also distributed within the crystalloid body, was lower than 15°C. Mass encystment occurred in an enigmatic organelle whose function is unknown. March, when the vegetative population flourished. Because CpPNO is compartmentalized in a novel way, Living cyst abundance in the sediments increased in there is an intriguing possibility that C. parvum has an January and April and empty cysts largely increased unusual type of energy metabolism that it might be from October to March. These results indicate that S. exploited for drug development against human crypto capitatum is well adapted to cold water and aestivates sporidiosis. during the warm period in summer.

MORPHOLOGY, MORPHOGENESIS AND PHY FOOD SELECTION AND FEEDING STRATEGIES LOGENETIC ANALYSIS OF MARINE CILIATE OF PROTOZOA IN PLANKTON OF RIVER FAVELLA EHRENBERGII JORGENSEN, 1924 DANUBE (CILIOPHORA: CHOREOTRICHIA) A.K. Kiss, K.T. Kiss, E. Acs 1 1 2 3 Hungarian Danube Research Station, Institute of Ecology and Botany, S.Y. Kim , J.K. Choi , E.J. Yang , J. Gong Hungarian Academy of Sciences, God, Hungary. 1 Department of Oceanography, Inha University, Incheon, 402751, Email: [email protected] Korea, 2 Marine Environment Research Department, KORDI, Ansan P.O. Box 29, 425600, Korea, 3 Regional Research Center for Coastal Food consumption and feeding strategies of protozoa Environments of Yellow Sea, CCEYS, Inha University, Incheon 402 were investigated in plankton of River Danube during 751, Korea. Email: [email protected] one year. Feeding behaviour and food vacuole contents F. ehrenbergii was collected from Incheon coastal water, were studied in living and preserved samples. Two Korea and cultured in the laboratory. Although this hundred protozoan species heterotrophic flagellates, species is widely distributed in marine water, its infra naked amoebae, heliozoans and ciliates were included ciliature is still unknown. The taxonomical description into study. Half of species were mainly bacterivorous, of this species has been based on lorica shape. However, one quarter algivorous, and smaller proportions several studies have been reported the variation of lorica flagellativorous, mixotrophic and omnivorous. Annual 42 · Abstracts of V European Congress of Protistology · 2007 distribution of protozoan biomasses constituting these sing the species specific trophic relations. Growth rates feeding categories showed different patterns. Bacteri were assumed from literature. In most parts of the year vores and algivores comprised 80% of total biomass, the microbial loop proved to be dominant, nanoflagel 20% were mainly flagellativory. Algivory was most lates were the most productive protozoan group. At high important in growing seasons, while in winter bacteri phytoplankton abundances the classical food web based vory and flagellativory dominated. The most common on phytoplankton was likewise important, sometimes feeding strategy among heterotrophic flagellates was channelling larger fluxes than the microbial loop. Most suspension feeding. Greater part of the flagellates was important herbivores were ciliates and large heterotro free swimming, smaller sedentary. One third of them phic flagellates. The main proportion of primary pro were substrate associated raptors, 15% free swimming duction was usually not utilized directly in the microbial raptors, and 10% osmotrophic. Regarding the annual food web, although at high algivor protozoan biomasses distribution of flagellate biomasses, suspension feeders the half of the primary production might have been dominated, with high peaks of free swimming raptors consumed. in growing seasons. Substrate associated raptors were sparse. Among ciliates half of species were raptors and DIFFERENT MODES OF STOP CODON RES another half suspension feeders, few were diffusion TRICTION BY THE AND PARA feeders. Half of suspension feeders were free swimming, MECIUM ERF1 TRANSLATION TERMINATION and half sedentary, with few loricated free swimming FACTORS tintinnids. Regarding to ciliate biomasses, suspension L. Kisselev1, S. Lekomtsev1, P. Kolosov1, L. Bidou2, feeders were dominant, with growing season peaks of L. Frolova1, J.P. Rousset2 1 raptors, and small summer peak of diffusion feeders. Engelhardt Institute of Molecular Biology RAS, Laboratory of structurefunctional genomics, Moscow, Russia, 2 CNRS, Orsay, F Many feeding niches can be distinguished according to 91405, France. Email: [email protected] food type, food size and feeding strategies of protozo In universalcode eukaryotes, a single translation ans, but overlapping is obvious and resource utilisation termination factor eRF1 decodes three stop codons: may be low some times during seasonal succession. UAA, UAG and UGA. In some ciliates, like Stylonychia and Paramecium, eRF1s exhibit UGAonly decoding PROTOZOAN COMMUNITY IN PLANKTON OF specificity, while UAG and UAA are reassigned as sense RIVER DANUBE: TEMPORAL DYNAMICS, TRO codons. Since variantcode ciliates may have evolved PHIC INTERACTIONS AND ROLE IN MICROBIAL from (a) universalcode ancestor(s), structural features FOOD WEB should exist in ciliate eRF1s that restrict their stop A.K. Kiss1, J.K. Torok2, K.T. Kiss1, E. Acs1 codon recognition. In omnipotent eRF1s, stop codon 1 Hungarian Danube Research Station, Institute of Ecology and recognition is associated with the amino terminal Botany, Hungarian Academy of Sciences, God, Hungary, 2 Department of Systematic Zoology and Ecology, Institute of Biology, domain of the protein. Using both in vitro and in vivo Eotvos Lorand University, Budapest, Hungary. assays we showed that chimeric molecules composed Email: [email protected] of the Nterminal domain of Stylonychia eRF1 fused The planktonic protozoan community of River Danube to the core domain (MC domain) of human eRF1, (Hungary, 1669 river km) involving all major groups, retained specificity towards UGA; this unambiguously was investigated during one year. We examined temporal associates eRF1 stop codon specificity to the nature of dynamics, trophic interactions between protozoa and its Nterminal domain. Functional analysis of eRF1 their food organisms, and structure of the microbial chimeras constructed by swapping the ciliate N food web. Protozoan groups with the largest biomass terminal domain sequences with the matching ones were heterotrophic flagellates, ciliates and naked from the human protein, highlighted the crucial role of amoebae. We determined the type and size of consumed the tripeptide QFM in restricting Stylonychia eRF1 algae for many protozoan species using in situ food specificity towards UGA. Using the sitedirected vacuole content analysis. Examination of the coupling mutagenesis, we showed that Paramecium eRF1 of temporal dynamics of investigated groups may reveal specificity towards UGA resides within the NIKS indirect trophic relations between many protozoa and (amino acids 6164) and YxCxxxF (amino acids 124 their food organisms. We found no coupling between 131) motifs. Thus, we established that eRF1 from two bacteria and heterotrophic nanoflagellates, edible algae different ciliates relies on different molecular mecha and Paraphysomonas vestita, but significant coupling nisms to achieve specificity towards the UGA stop was detected between edible algae and codon. This suggests that eRF1 restriction of specificity triciliatum, many edible algae and algivor ciliate groups, to only UGA might have been an early event occurring and between prey ciliates and predatory ciliates. We in independent instances in ciliate evolutionary history, revealed the structure of microbial food web and the possibly facilitating the reassignment of UAG and UAA quantity of intercompartmental carbon flow summari to sense codons. Protistology · 43

PHOSPHATIDYLINOSITOL SPECIFIC PHOS polymerisation, and blocking Raclike protein aboli PHOLIPASES OF PARAMECIUM TETRAURELIA shed this effect. The Rhorelated signalling pathway INVOLVED IN ANTIGENIC VARIATION exerted its effect on migration and endocytosis by T. Kloeppel, H.J. Schmidt, M.C. Simon myosin inactivation and probably by inhibition of University of Kaiserslautern, Department of Biology, Kaiserslautern, cofilinlike protein. We also observed that Arp2/3like Germany. Email: [email protected]kl.de protein complex played a role in actin filaments bran Surface antigens of several protist species are Glycosyl ching in different areas of migrating amoeba (cortical Phosphatidyl(GPI)anchored and Phosphatidyl layer, perinuclear cytoskeleton, adhesion structures), Inositol specific Phospholipases (PIPLCs) are strongly but not in the fronts of advancing and the assumed to be involved in antigen release in the uroid. This might suggest that in A. proteus Arp2/3 medium, especially during antigenic switching. We dependent actin polymerisation was not engaged in the report here the analysis of six different PIPLCs present frontal membrane proggression. Cofilinlike protein in Paramecium tetraurelia. Transcriptional analysis was involved in actin depolymerisation within the shows that all six genes, including two paralogs, are up middleanterior region of the cell but not in the proces regulated during antigenic switching. Additionally to ses of the cortical network disorganisation occurring in the typical X, Y and C2domains of previously the uroid. In migrating amoebae, the course of changes described PIPLCs of other organisms, two PIPLCs in filamentous (F)/total (T)actin ratio corresponded exhibit calcium binding motifs. Interestingly, these two to the distribution of the tension in the cell cortex, as PIPLCs were those which are shown to be not involved the areas of maximal isometric tension were located in in antigenic variation, as silencing of these genes did the middleposterior region behind the adhesion area, not alter antigenic switching. On the contrary, silencing and in the distal part of the uroid and retracting of the other PIPLCs was characterized by a decelerated pseudopodia. These results aided in elucidation of antigen shift: the old antigen was present much longer mechanisms of actin cytoskeleton dynamics in A. on the cell surface, compared to control cells. Moreover, proteus. our data suggests that PIPLCs in Paramecium are not involved in the normal antigen turnover in stable TIMING OF DIFFERENTIATION OF PERIALGAL serotypes. Therefore, their role in cutting GPI anchors VACUOLE MEMBRANE FROM DIGESTIVE VA seems to be a special mode during antigenic switching. CUOLE MEMBRANE OF THE CILIATE PARAME Apart from the discrimination in function of PIPLCs CIUM BURSARIA DURING INFECTION WITH we have shown that not a single PIPLC is responsible SYMBIOTIC ALGAE CHLORELLA VULGARIS for GPIcleavage during saltalcohol extraction of Y. Kodama1, M. Fujishima2 surface proteins, and therefore that all PIPLCs in 1 Department of Natural Science and Symbiosis, Graduate School of general are able to cut GPIanchors. With this PIPLC Science and Engineering, Yamaguchi University, Yoshida 16771, variety, Paramecium seems to become the organism of Yamaguchi 7538512, Japan, 2 Department of Environmental Science choice to study PIPLC activity, its influence on GPI and Engineering, Graduate School of Science and Engineering Yamaguchi University, Yoshida 16771, Yamaguchi 7538512, Japan. anchored proteins and role in the signal transduction Email: [email protected] pathway. Each symbiotic Chlorella cell of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from SIGNALLING PATHWAYS AND ACTIN DYNAMICS the host digestive vacuole to be protected from lyso IN AMOEBA PROTEUS somal fusion. To know timing of differentiation of the W. Klopocka1, A. Wasik2, P. Pomorski2, D. Suplat2, perialgal vacuole from the host digestive vacuole, algae M.J. Redowicz1 free P. bursaria cells were fed with the symbiotic C. 1 Department of Biochemistry, Nencki Institute of Experimental vulgaris cells for 1.5 min, washed, chased, fixed at Biology, 3 Pasteur St., 02093 Warsaw, Poland, 2 Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur St., 02 various times after mixing, and an acid phosphatase 093 Warsaw, Poland. Email: [email protected] activity in the vacuoles enclosing the algae was detected The results of our recent studies indicated that Rho by Gomori's staining. This activity appeared in 3min familybased regulation played a key role in motility of old vacuoles, and all vacuoles with algae showed activity freeliving Amoeba proteus. Blocking Rac or Rho in 30 min. Algal escape from the digestive vacuoles related signalling pathways with C3 transferase, anti began at 30 min by budding off the digestive vacuole bodies against human RhoA and Rac1, and Y27632 membrane into the cytoplasm. In the budded membra (a specific inhibitor of Rhoassociated kinase) caused ne, the algal cell was surrounded by a Gomori's staining distinct and irreversible changes in amoebae morphology positive thin layer. The vacuoles with a single algal cell and significant inhibition of their migration. We soon moved to and attached at just behind the host cell revealed that protein(s) from the amoeba cytosolic surface. Such vacuoles were Gomori's stainingnegative. fraction facilitated the nucleation step of actin These results indicate that the perialgal vacuole mem 44 · Abstracts of V European Congress of Protistology · 2007 brane differentiates soon after the algal escape from the organelles was thoroughly examined. Twenty year old host digestive vacuole, and differentiation completes samples of buetschliids were washed with water and prior to the algal localization just beneath the host cell prepared for osmicating with or without prefixation in surface. This is the first report to show the timing of 2.5% buffered glutaraldehyde. In both cases, the results differentiation of the perialgal vacuole membrane of TEM were equally satisfactory. All other formalin during infection of P. bursaria cell with the algal cell. samples of ciliates were postosmicated directly after washing in water. SQUARE NUMBER OF FOOD VACUOLES AS A NUTRITION INTENSITY INDEX IN PERITRICHS LEPTOMONAS JACULUM (LEGER 1902) WOOD (CILIOPHORA, PERITRICHIA) OF THE ACTIVA COCK 1914: A LEPTOMONAS OR A BLASTO TED SLUDGE IN THE SEWAGE TREATMENT CRITHIDIA? PLANT A.Yu. Kostygov1, A.O. Frolov2 1 L.A. Konstantynenko Zoological Institute RAS, laboratory of molecular systematics, St. Petersburg, Russia, 2 Zoological Institute RAS, laboratory of Zhytomir State University, Zhytomir, Ukraine. protozoology, St.Petersburg, Russia. Email: [email protected] Email: [email protected] The square number of food vacuoles is a sensitive index The genus Leptomonas Kent, 1880 contains homoxe of the nourishment intensity of peritrichiasedimenta nous trypanosomatids with promastigotes as the main morphotype in their life cycle. Systematics of the genus tors. We studied the possibility of the square number of needs to be revised, since its type species (L. butschlii) food vacuoles as the index of nutrition intensity in five is seemingly not a trypanosomatid, and molecular species of peritrichs (Vorticella striata Dujardin, 1841; phylogenetic studies showed the genus to be polyphyletic. Epistylis plicatilis Ehrenberg, 1831; E. bimarginata One of the first steps of this work should be the choice Nenninger, 1948; Opercularia phryganeae Kahl, 1935. of the type species in the substituting taxon. Since the V. convallaria (Linnaeus, 1758)) and modified this most of leptomonads parasitize insects, Leptomonas method with taking into consideration the peculiarities jaculum, as a first species described from this host group, of Peritrichia biology and the conditions of activated could be considered as a namebearing type. Meanwhile sludge tank in the sewage treatment plant. The square this species has not been used so far in molecular number of food vacuoles depends on the temperature phylogenetic studies due to failure to cultivate it. We determined with the help of the oneway ANOVA. For determined sequences of the 18S rRNA gene of L. all species the authentic connection between the jaculum, amplified directly from the infected bugs, and temperature and the square number of food vacuoles revealed the phylogenetic proximity of this species to was fixed. The square number of food vacuoles is blastocrithidias. The cells of this species resemble maximal under optimal conditions of the hydroche promastigote, nevertheless they possess a cryptic undu mical parameters of the activated sludge in the sewage lating membrane (multiple desmosomes form an exten treatment plant. The usage of the square number of food sive contact between the and the cell body vacuoles permits to check up the effectiveness of sewage inside the flagellar pocket), making them similar to cleaning, to react effectively on the technological epimastigotes, which have such contact extended to the disbalance in the process of cleaning, and to determine outer cell surface. Moreover, presence of the cystlike the optimal technological regimes during the exploita amastigotes in the life cycle, and absolute absence of tion of the sewage treatment plant. the cytostome also bring L. jaculum and blastocrithidias together. L. jaculum should obviously be removed from NEW LIFE FOR OLD COLLECTIONS OF CILIA the polyphyletic group named so far the genus Lepto TES monas. However, the displacement of this species into O.A. Kornilova the genus Blastocrithidia Laird, 1959 is premature until Herzen State Pedagogical University, Dept. Zoology, St. Petersburg, the inclusion of its type species (B. gerridis) in the phylo Russia. Email: [email protected] genetic analysis. Ciliates collected from the intestine and caecum of Equus hemionus kulan in 1987, from Yakut horse in COMMUNITY OF PLANKTON CILIATES OF THE 2001, and from faces of Elephas maximus in 2003, all AZOV SEA fixed in 4% neutral formalin, were postfixed in osmium K.V. Kreneva tetroxide and studied in transmission electron AB MMBI KSC RAS, Plankton, RostovonDon, Russia. microscope (TEM) 2006 2007. Observation of thin Email: [email protected] sections of some species of Buetschliidae, Cycloposthi Ciliates are one of the most widespread and numerous idae and Ditoxidae revealed good preservation of cell groups, though very poorly known. Nevertheless, they structures. Fine morfpology of the concretionvacuoles, are one of the most perspective groups as bioindicators. kinetids, skeletal plates, hydrogenosomes and other Data on the ciliates community of the Azov Sea have Protistology · 45 been fragmentary so far. Some vast regions were not Microsporidia, worldwidely distributed opportunistic investigated at all. Largescale, regular research of the parasites described predominantly from immunocom Azov Sea ciliate plankton was held for the first time promised patients, have been in the focus of attention during the period of 19992006. In total, 91 ciliate taxa of physicians and researchers since the onset of HIV/ were identified during this period in the water body, AIDS pandemics in the 1970s. Nothing is known so far including 72 taxa being reported from the Azov Sea for about occurrence of microsporidiosis in Russia. the first time. Reliable data on distribution of the quan Recently a project funded by CRDF, has been initiated titative characteristics of the ciliate plankton were ob to survey prevalence of microsporidiosis and cryptospo tained. The structure of the community of plankton ridiosis among HIV/AIDS patients in Russia. As a part ciliates, and ecological peculiarities of the most abun of this survey we evaluated sera from 28 HIV/AIDS dant species were examined for the first time. Dynamics patients of City Municipal Hospital St. Petersburg, of the ciliate community structure and quantitative Russia suffering from lasting diarrhea, for reactivity to characteristics of the ciliate plankton under the influence major microsporidian species infecting humans, i.e. of different biotic and abiotic factors have been studied. Encephalitozoon cuniculi, E. intestinalis, E. hellem and Enterocytozoon bieneusi by enzymelinked immunosor THE SPHAGNUM PONDS OF SIMMELRIED IN bent assay (ELISA) and by indirect immunofluo GERMANY: A BIODIVERSITY HOTSPOT FOR rescence assay test (IFAT). We found one stool sample MICROSCOPIC ORGANISMS positive for E. bieneusi detected by E. bieneusimono M. Kreutz1, W. Foissner2 clonal antibodies (mabs) and by rabbit E. bieneusi 1 Private Laboratory, Magdeburger Strasse, Konstanz, Germany, 2 polyclonal sera. Three sera samples were positive for E. Universitaet Salzburg, FB Organismische Biologie, Salzburg, Austria. Email: [email protected] cuniculi in ELISA, and three for E. bieneusi and E. This study describes 656 species of bacteria, protists, hellem, both by ElISA and IFAT. Our study is in accord and micrometazoa occurring in the Simmelried, a three with the results of PCR identification of E. bienusi by hectaresized moorland in southern Germany. Each specific primers. Testing of the samples by other species is shown by an average of two colour micro available methods, including PCR identification with graphs. Further, the surface organization of most main other specific and universal primers and staining groups is demonstrated by scanning electron micro techniques, as well as examination of new samples is graphs. The Simmelried formed after the last iceage, underway. Supported by grant No. RUB2002707SP that is, about 15,000 years ago. The investigations 05 from the U.S. Civilian Research and Development indicate that the 656 species documented represent only Foundation. two thirds of the taxa actually present. Thus, a conside rable diversity was accumulated over 15,000 years, A HIGHER SPECIES DIVERSITY IN THE FAMILY emphasizing the great distribution capacity of micro MICROCHLAMYIIDAE OGDEN, 1985 (AMOEBO organisms. On the other hand, some common species ZOA, TESTACEALOBOSIA): TWO NEW SPECIES are lacking (e.g., the ciliate Colpidium , the OF THE GENUS SPUMOCHLAMYS KUDRYAVT euglenid pleuronectes, and rotifers of the genera SEV AND HAUSMANN, 2007 Proales and Floscularia), and many undescribed species A. Kudryavtsev were discovered. While a mass of undescribed species Department of Invertebrate Zoology, Faculty of Biology and soil Science, SaintPetersburg State University, Universitetskaja nab., 7/9 199034 is comprehensible in the poorly studied , flagel St. Petersburg Russia. Email: [email protected] lates and ciliates, this is surprising in wellknown groups, Two species of testate amoebae isolated from the such as euglenids and chrysophytes. Thus, some of the epiphytic moss have flexible plateshaped tests. Dorsal undescribed species might be regional or local endemics. parts of these tests have spongious structure. They are The book has been published by Shaker, Aachen and is thicker in the center and become thinner at the margin available in printed and electronic form: http://www. to the end in a delicate membrane forming a flexible shaker.de/OnlineGesamtkatalog/Details. asp? ID= aperture. The cell body attaches directly to the dorsal 0&ISBN=3832225447&Reihe=0. part of the test. Amoebae move by gliding on a flattened hyaline sheet, expanding from the aperture. When the SURVEY FOR MICROSPORIDIOSIS IN HIV/AIDS cells are suspended in the medium and turned upside PATIENTS IN ST. PETERSBURG, RUSSIA: SERO down, they twist their test margins towards the ventral LOGICAL IDENTIFICATION surface, and produce several long lobopodia that turn Z. Kucerova1, O.I. Sokolova2, A.V. Demyanov3 the tests back to their natural orientation by dragging 1 Division of Parasitic Diseases, Centers for Disease Control and them to the substratum. The species differ from each Prevention, Atlanta GA, USA, 2 Medical Department of St. Petersburg State University, St. Petersburg, Russia, 3 Institute of Pure other in the test size and structure of its dorsal part. With Biopreparations, St. Petersburg, Russia. Email: [email protected] the ultrastructural data showing the test structure and 46 · Abstracts of V European Congress of Protistology · 2007 relations between the test and cell body, both amoebae relations within Amoebozoa. Supported by DAAD can be unambiguously identified as new species of the fellowship A/05/00103 and RFBR grant 060449387. genus Spumochlamys (family Microchlamyiidae), so far comprising only one species S. iliensis. By contrast, AN UNUSUAL COMBINATION OF LIGHT AND when the largest of the two species was observed only ELECTRONMICROSCOPICAL FEATURES IN by light microscopy, it ideally matched the existing "THECAMOEBA" VERRUCOSA (EHRENBERG, descriptions of another microchlamyiid amoeba, 1838) (AMOEBOZOA) Microchlamys patella, however, the structure of the A. Kudryavtsev1, K. Hausmann2 dorsal part of the test wall resembled Microchlamys 1 Department of Invertebrate Zoology, Faculty of Biology and soil sylvatica Golemansky et al., 1987. The presented results Science, SaintPetersburg State University, Universitetskaja nab., 7/9 confirm previous suggestion that the family Microchla 199034 SaintPetersburg, Russia and Research Group Protozoology, myiidae contains more species, which can be reliably Institute for Biology/Zoology, Free University of Berlin, KoeniginLuise Str., 13, 14195 Berlin, Germany, 2 Research Group Protozoology, distinguished only by combination of light and electron Institute for Biology/Zoology, Free University of Berlin, KoeniginLuise microscopy. Supported by the RFBR grant 0604 Str., 13, 14195 Berlin, Germany. Email: [email protected] 49387. Thecamoeba verrucosa (Ehrenberg, 1838) is often mentioned, when the genus Thecamoeba is discussed LOCOMOTION AND FACTIN DISTRIBUTION IN in the literature, but its ultrastructure is unknown and FLABELLINEAN AMOEBAE OF COCHLIOPODI this species does not exist in culture. We isolated and IDAE, FLAMELLA AND PELLITA (AMOEBOZOA) studied an unusual amoeboid protist that has an A. Kudryavtsev1, K. Hausmann2 elongated triangular shape during locomotion, with the 1 Department of Invertebrate Zoology, Faculty of Biology and soil flattened anterior part, a tapering uroid raised over the Science, SaintPetersburg State University, Universitetskaja nab., 7/9 199034 SaintPetersburg, Russia and Research Group Protozoology, substratum and irregular dorsal longitudinal wrinkles. Institute for Biology/Zoology, Free University of Berlin, KoeniginLuise A single nucleus contains a central coarsely granular Str., 13, 14195 Berlin, Germany, 2 Research Group Protozoology, endosome and an additional smaller homogeneous Institute for Biology/Zoology, Free University of Berlin, KoeniginLuise body located near the endosome. An ultrastructural Str., 13, 14195 Berlin, Germany. Email: [email protected] study shows that this amoeba has a prominent We performed a comparative study of locomotion in four species of , Cochliopodiidae n. gen., microtubular cytoskeleton with irregular bundles of Flamella n. sp. and Pellita digitata, and revealed the microtubules in the cytoplasm. Microtubules are most distribution of Factin in the moving cells by TRITC numerous near the nucleus and converge towards a phalloidin staining. Although Cochliopodiidae and dumbbellshaped MTOC located near the invagination Flamella show a very similar pattern of movement of the nuclear membrane. The central part of the accompanied by an expansion of a broad peripheral nucleus is occupied by the coarsely granular electron hyaloplasmic sheet, while the rest of the cell is dragged dense body surrounded by a less dense layer of homo forward, the spatial organization of their actin geneous material, sometimes forming a smooth sphe cytoskeleton is different. They both have a fine actin rical body. Amoebae have mitochondria with tubular network in the leading hyaloplasm, but in addition cristae, numerous dictyosomes and a typical plasma cochliopodiids possess pronounced bundles of actin, membrane without any visible glycocalyx. All light oriented transversally or obliquely to the direction of microscopical features of this amoeba perfectly movement and resembling stress fibers of motile correspond to the earlier descriptions of Th. verrucosa metazoan cells. Pellita digitata shows a different pattern and allow its inclusion in ; by contrast, of movement and actin distribution. It forms a smooth its ultrastructure shows that its assignment to the family anterior hyaloplasm and a posterior spherical granulo Thecamoebidae is not correct. The revealed combina plasm. The ventral surface of hyaloplasm produces short tion of lightmicroscopical and ultrastructural features fine subpseudopodia; they penetrate a thick cell coat of does not allow a certain inclusion of this species in any this amoeba and adhere to the substratum. In contrast of the amoebozoan taxa; gene sequence data are to Cochliopodiidae and Flamella, the cell surface of P. necessary to clarify the taxonomic position of this digitata demonstrates a typical rolling movement. These amoeba. Supported by DAAD fellowship A/05/00103 amoebae have a thin actin cortex in the granuloplasm, and RFBR grant 060449387. from which numerous actin bundles originate. They are directed forward into the hyaloplasm and end in the SALINITY TOLERANCE OF LOBOSE AMOEBAE ventral subpseudopodia. The revealed differences in the AND CILIATES distribution of Factin confirm an earlier idea of a high A.V. Kudryavtseva1, A.O. Smurov2, A.V. Goodkov3 diversity in the patterns of movement among Amoebo 1 Engelhard Institute of Molecular Biology RAS, 2 Zoological Institute zoa and indicate a possible correlation between the RAS, St. Petersburg, 3 Institute of Cytology RAS, St. Petersburg, spatial structure of the cytoskeleton and phylogenetic Russia. Email: @mail.ru Protistology · 47

Salinity tolerance zone of 13 lobose amoebae and 16 complete. Here are some possible ways of its augmen ciliate species were estimated. Potential salinity tation: special faunal studies in the above mentioned tolerance zone was also evaluated using stepwise areas; due to new benthic and fouling species, which acclimation method. It was either identical to the zone may compose from 5 up to 60% of the total number of of salinity tolerance, or certain differences were species in the sample; due to alien species, with regard observed. For two amoebae and two ciliate species, to increasing level of "mediterranization" of the Black several isolates were investigated. Several ecological Sea fauna and transmission via ballast waters; due to groups of species differing by their relation to salinity description of new species. Such information will allow were distinguished in amoebae and ciliates. The first to add data on biodiversity and to conduct comparative group comprises the absolutely freshwater species, analysis for ecological zoning of the Black Sea area. which have shown growth only in the range from freshwater medium to 2.5 ppt. The second group INTRONS IN 18S RNA GENE AS A TAXONOMIC consists of species, which grow in the range from CHARACTER OF CHLORELLA ENDOSYMBI freshwater medium to 68 ppt. The third group is made ONTS OF PARAMECIUM BURSARIA up of the euryhaline species, they have shown successful K.V. Kvitko1, A.V. Migunova1, E.E. Andronov2, growth in the range from freshwater medium to at least K.P. Vorobiev1, D.V. Karelov3 35 ppt, which is the average ocean salinity. The fourth 1 Biological Institute of St. Petersburg State University, Department group consists of the strictly marine species, which of Microbiology, Stariy Petergof, St. Petersburg, Russia, 2 All Russia cannot exist under salinity less than 510ppt. It is Research Institute for Agriculture Microbiology, Department of , Pushkin, St. Petersburg, Russia, 3 Petersburg Institute of Nuclear interesting that in the case of two amoebae and one Physics, Department of Molecular Genetics. Gatchina, St. Petersburg, ciliate species studied, different isolates had their own Russia. Email: [email protected] zone of salinity tolerance. Therefore, this feature is not Since 1996 (X International Congress of ) we a species attribute and may be determined by the discuss the problem of taxonomic difference of two conditions in the local habitats. Combination of these (southern and northern) ecotypes of Chlorella sp., the data with those on occurrence of species in different endosymbionts of P. bursaria. Southern ecotype is habitats leads to conclusion that freshwater and marine characterized by ts phenotype (sensitive to 32oC), which faunas of amoebae and ciliates can partially interfere. differs from the northern one by isozyme spectra of 8 It can be supposed that an evolutionary trend within enzymes, contrasting serological patterns, and different protists’ taxa is occupation of new types of habitats with types of viruses (NC64A& Pbitypes of Chlorovirus, different salinitiy. Phycodnaviruses). Genomic dactyloscopy by UPPCR patterns also allows dividing zoochlorellae strains into A SYNOPSIS OF THE BLACK SEA FAUNA OF the northern and the southern ecotype, corresponding, PLANKTONIC CILIATES probably, to two different species. In our last publi A.V. Kurilov cations (and in papers of Hoshina et al., 2004, 2005) it Odessa Branch Institute of Biology of the Southern Seas, Department was shown that all strains of northern and southern of ecology of marginal communities, Odessa, Ukraine. Email: [email protected] ecotypes could be distinguished from freeliving Chlo Since 1871, more than 500 species of ciliates were rella vulgaris by occurrence of introns in the anterior discovered in the Black Sea. Basic attention was given (5') region of the 18S RNA gene; the southern zoo to studying of benthic ciliates, ectocommensals and chlorellae had two additional introns in the central and parasites. Using plankton nets ciliates were studied only posterior (3') regions of the gene. By sequences of 18S as a component of zooplankton. As a result, the species rDNA exon zoochlorellae from P. bursaria (northern lists of pelagic ciliates in most of the Black Sea areas and southern ecotypes) are closely related to Ch. vulgaris, are incomplete and usually limited to tintinnid species. Ch. sorokinianа and Ch. lobophora. According to "Intro In Georgia only 9 taxons have been found, for Turkey n early" hypothesis, such difference can arise very early, 12, Bulgaria 23, Romania 14, and Russia 27. Special and therefore we suggest that two groups of P. bursaria taxonomic studies nearshore and in the open sea were endosymbionts belong to different species. A small few. The most complete studies of species composition number of cultivable strains of these groups are the only have been made along Ukrainian coasts: 184 taxons fact that prevents us from the final decision. By new (201 if limans and lagoons are included). Overall, 205 methods (see theses by Vorobyev et al.) we hope to get taxons of ciliates (including common species) were information about more northern and southern popula recorded in the Black Sea plankton. Analysis of species tions of P. bursaria endosymbionts, which will allow us composition shows, that this list is still far from being to solve the problem. 48 · Abstracts of V European Congress of Protistology · 2007

REEVALUATION OF DORSAL SILVER LINE analysis. Results: Cryptosporidium DNA was evidenced SYSTEM IN CHARACTERIZATION OF EUPLOTES in anus swabs from all 7 patients. For each patient, the NOVEMCARINATA (WANG, 1930) (CILIOPHORA: same species was obtained in anus swab and in stools HYPOTRICHIDA) (C. hominis in 4 patients, C. parvum in 2, C. felis in 1). C.B. Kwon, M.K. Shin Cryptosporidium DNA was evidenced in one of the 42 Dept. of Biological Science, University of Ulsan, Korea. swabs sampled in patients' rooms; this swab was sampled Email: [email protected] from the toilet button in a patient's bathroom. Genoty A population of Euplotes novemcarinata was isolated ping identified C. parvum, as for the patient who was from the debris of sesame plants collected at Andong, occupying this room. Conclusion: This study showed Korea. Its morphology of each developmental stage was that Cryptosporidium DNA can occasionally be demon studied by observing a live, protargol and silvernitrate strated on surfaces touched by Cryptosporidiuminfected impregnated specimens. It was discovered that the dorsal patients, suggesting a possible spread of oocysts in pati silver line system varied according to the developmental ent's environment. This underlines the need for a strict state of dorsal ridges of this species as follows. When application of hygiene measures for patients with active the dorsal ridges were protruded extremely from the cryptosporidiosis, and possibly isolation of these pati dorsal surface, more than four rows of small polygons ents to prevent nosocomial transmission of cryptospo were observed to make the highly irregular complex type ridiosis. of the silver line system. In contrast, when the dorsal ridges were protruded subtlety from the dorsal surface, LIVING IN THE DEAD ZONE: MICROZOOPLAN 3~4 rows of the small polygons were found to make a KTON COMMUNITIES DURING SEASONAL multiple type of the silver line system. These facts HYPOXIA IN LAKE ERIE AND THE GULF OF demonstrate that Euplotes muscicola is a potential syno MEXICO nym of E. novemcarianta, and it is suggested that dargy P.J. Lavrentyev1, R.J. Duff1, K.M. Moats1, F.J. Jochem2 rome patterns that characterize Euplotes species has to 1 University of Akron, Biology, Akron, OH, USA, 2 Florida be reevaluated especially in the case of the species International University, , Miami, FL, USA. having the distinct dorsal ridges. Email: [email protected] The northern Gulf of Mexico (Louisiana shelf) and the POTENTIAL RISK OF CONTAMINATION OF central basin of Lake Erie experience seasonal hypoxia, HOSPITAL ENVIRONMENT BY CRYPTOSPO covering broad regions in summer and impacting their RIDIUM OOCYSTS FROM PATIENTS WITH living resources. However, little is known about its ACTIVE CRYPTOSPORIDIOSIS effects on microzooplankton, a key component of the L. Larrouy1, C. Sarfati2, H. Belkhiter3, S. Hamane1, pelagic food web in both systems. This study examined J.M. Molina4, F. Derouin2, J. Menotti2 the composition, distribution, and dynamics of ciliates 1 Laboratory of Parasitology, SaintLouis hospital, AP and dinoflagellates and other planktonic microbes HP, Paris, France, 2 Laboratory of ParasitologyMycology, Saint (bacteria, heterotrophic nanoplankton, phytoplankton, Louis hospital, APHP, and EA3520, University of Paris 7, Paris, and rotifers) using microscopy, singlecell and France, 3 EA3520, University of Paris 7, Paris, France, 4 Department of Infectious Diseases, SaintLouis hospital, APHP, Paris, community 18SrDNA analyses, shipboard incubations, France. Email: [email protected]hopparis.fr and realtime flowcytometry. During the early to mid Background: Cryptosporidium causes prolonged and stratification, epilimnetic microzooplankton was severe diarrhea in immunocompromised patients, with dominated by oligotrich ciliates and removed ca. 80% no effective specific treatment available. As Cryptospo of daily primary production and 30% of phytoplankton ridium oocysts in stools are directly infective, there is a biomass in Lake Erie. The hypolimnion was occupied potential risk of person to person transmission and by the remnants of the spring community (e.g. occurrence of nosocomial cryptosporidiosis in the helveticum and Histiobalantium bodami hospital setting. Aim: To study the potential sites of cum). The dinoflagellate herundinella and contamination by Cryptosporidium oocysts in the close rotifers underwent diel vertical migrations extending to hospital environment of patients presenting active the hypolimnion (dissolved oxygen, DO, 23 mg/l). Cryptosporidium infections. Methods: In the rooms of During the late stratification in September 2005, 7 patients with cryptosporidiosis, surface samples were microzooplankton biomass in the hypolimnion (DO < collected using previously humidified swabs. Additio 1 mg/1) was formed by choreotrich ciliates, which were nally, swabbing of patients' anus and fingernails was grazing on nanosized cryptophyte flagellates. Exami performed. A nested PCR assay amplifying a fragment nation of plankton distribution and composition along of Cryptosporidium spp. 18S rRNA gene was then several meridional transects and a 220 nm crossshelf performed, followed by molecular typing by restriction transect in the Gulf of Mexico in August 2006 revealed fragment length polymorphism and/or sequence significant spatial heterogeneity. Heterotrophic micro Protistology · 49 bes and diatoms peaked in a midshelf warm front and Basin (Australia), was investigated. This community of the Mississippi River plume, respectively. Their diel organisms from marine bottom sediments has not vertical patterns were linked to the fluctuating DO previously been studied in Australia. 82 species are concentrations in the bottom layer, which were appa described with uninterpreted records based on light rently influenced by the currents. The hypoxic waters microscopy of living cells in natural communities. The were dominated by choreotrich ciliates and gymnodi records include two new species: Sphenomonas altus nov. niid dinoflagellates. spec. and Platychilomonas planus nov. spec. Of the 82 species, 11 species are new to Australian marine sites, A NEW CILIATE SPECIES OF THE GENUS but the majority of the species encountered here also AMPHISIELLA (CILIOPHORA, HYPOTRICHA) were found at other locations worldwide, including FROM ESTUARY IN KOREA Australian freshwater sites. The new records for Austra J. Lee, C.B. Kwon, M.K. Shin lian marine sites are: Acanthocorbis unguiculata, Cerco Dept. of Biological Science, University of Ulsan, Korea. monas sp.2, Gweamonas unicus, Helkesimastix faecicola, Email: [email protected] Notosolenus sp.1, sp.2, Petalomonas sp.3, The Amphisiella ciliates collected from the estuarine Platychilomonas planus, Salpingoeca amphoridium, littoral water and weeds (salinity 2‰, location 35° Spironema multiciliatum, Sphenomonas altus. The 32'43"N, 129° 20'18"E) in Korea. The description was relative lack of novelty provides little support for the based on the observation of living specimens, protargol existence of endemic biota among this group of orga impregnated specimens and morphometric analysis. nisms which has been shown also for freshwater species. The present species is most similar to A. annulata (Kahl, 1928) Berger (2004) but differs from it as follows. Body size 125175 × 2540 µm in vivo, elongated, DISTRIBUTION OF HETEROTROPHIC PRO elliptical. Two ellipsoidal macronuclear nodules. On TISTS AND FACTORS CONTROLLING THEIR average three spherical or ellipsoidal micronuclei. 43 DISTRIBUTION IN MASAN BAY, KOREA 1 1 2 3 3 adoral membranelles on average. Amphisiellid median W.J. Lee , N.J. Park , J. K. Choi , J.D. Lee , K. Shin 1 Kyungnam University, Department of Environmental Engineering, cirral row extended from right frontal cirrus to near Masan, Korea, 2 Inha University, Department of Oceanography, transverse cirri, slightly to distinctly sigmoidally, Inchon, Korea, 3 KORDI, Division of Coastal Ecological Processes consisting of 30 cirri on average. Six transverse cirri J Research, Geoje, Korea. Email: [email protected] shaped, with two pretransverse cirri. 30 left and 28 right In order to understand the distribution of heterotrophic marginal cirri. Five types of colorless granules: (1) protists and factor controlling their distribution in usually 814 ringshaped or doughnutshaped granules Masan Bay, which is heavily polluted, this study was in cytoplasm, colorless and gray, about 57 µm in conducted during Feb. 2004 Nov. 2006. During the diameter, (2) 1416 small (ca. 11.5 µm in diameter) study, abundance of heterotrophic bacteria and bacterial colorless or vitreous cortical granules were arranged as production averaged at 2.14 × 106 cells/ml and 84 mgC/ a longitudinal band of 2obliquerows on dorsal surface, m2, respectively. Abundances of cyanobacteria and 57 dorsal kineties running along these longitudinal photosynthetic nanoflagellates averaged at 6.17 × 103 bands, (3) small (ca. 0.4 µm in diameter) colorless or cells/ml and 3.18 × 103 cells/ml. Protists consisted of vitreous cortical granules regularly arranged in heterotrophic nanoflagellates (HNF), heterotrophic longitudinal rows between dorsal kineties, (4) small (ca. dinoflagellates (HDNF) and ciliates, and their abun 0.5 µm in diameter) colorless granules irregularly dances averaged 1.22 × 103 cells/ml, 1.20 × 104 cells/l arranged in dorsal and ventral surfaces, (5) spindle and 0.13 × 104 cells/l, respectively. Generally, the chla shaped or ellipsoidal granules (34 µm long and 1.52 concentration and the abundances of heterotrophic µm wide) colorless granules densely arranged beneath bacteria, photosynthetic nanoflagellates and protists the ventral surface. were higher in the inner zone of the bay with high concentration of organic matters, than in the middle FREELIVING HETEROTROPHIC FLAGELLATES and outer zones. The temporal patterns of heterotrophic FROM DEEPSEA SEDIMENTS OF GIPPSLAND bacteria, cyanobacteria and photosynthetic nanoflagel BASIN, SOUTHEASTERN AUSTRALIA lates showed seasonality (i.e., in summer high density W.J. Lee, N.J. Park and in winter low density). Unlike those microbes, Kyungnam University, Department of Environmental Engineering, protists did not show seasonality. Using the grazing rates Masan, Korea. Email: [email protected] of heterotrophic nanoflagellates on bacteria previously In order to contribute to understanding of geographic reported for this area, it was calculated that about 69% distribution of freeliving marine heterotrophic of bacterial production was removed by HNF grazing flagellates, the diversity of heterotrophic flagellates activity. Also about 24% of initial chlorophylla occurring in a number of deepsea sites in Gippsland concentration was removed by microzooplankton 50 · Abstracts of V European Congress of Protistology · 2007 grazing activity. In conclusion, this study suggests that species were found in the plankton of the small river. in Masan Bay heterotrophic protists control the growth The representatives of kinetoplastids and cercomonads of bacteria and phytoplankton, and heterotrophic were dominated in the samples in terms of species protists represent an important link between bacterial richness. Spumella sp., Bicosoeca lacustris, Bodo saliens, & microalgal biomass and higher trophic levels. Spongomonas uvella, Allantion tachyploon, Cercomonas agilis, and Protaspis simplex were the most common COMMUNITY STRUCTURE AND DYNAMICS OF species. Some rare species such as Rhipidodendron CILIATED PROTOZOA ALONG SALINITY GRA splendidum, Dimastigella trypaniformis, Protaspis DIENTS AT SALTPANS OF THE YELLOW SEA verrucosa, Apusomonas proboscidea, Aurigamonas solis, Y. Lei1, J. Choi2, K. Xu1, H. Hong2 americana have been identified. The 1 Institute of Oceanology, Chinese Academy of Sciences, 266071 majority of observed flagellates were bacterivores, but Qingdao, PR China, 2 Department of Oceanography, Inha University, Rhynchobodo sp., Allantion tachyploon, Protaspis Inchon 402751, Korea. Email: [email protected] verrucosa, Aurigomonas solis were predators. The bogs The community structure and dynamics of ciliates along and river biodiversities considerably differed from each salinity gradients ranging from 28 psu to 311 psu at eight other. saltpans of the Yellow Sea, were investigated in April, June, August and September 2001. A total of 98 species IDENTIFICATION OF TWO NOSEMA SPP. ISO of ciliates were identified using live observation and LATED FROM PIERIS RAPAE AND HEMERO protargol staining techniques. The highest number of PHILA ATRILINETA species (19) was found from the sample collected in J.P. Liu1, J. Hao1, S.T. Liao2 April with the salinity up to 50 psu. With the salinity 1 South China Agriculture University, China, 2 Guangdong Provincial increasing beyond 265 psu, only one species, viz., Agricultural Academy Institute, China. Email: [email protected] Fabrea salina, could be observed. The ciliate abundance Microsporidia are a group of obligate intracellular varied from 44 to 210,929 inds. l1 and the maximum parasites. Some microsporidia play an important role value was mainly contributed by Strombidium styliferum in biological control of the silkworm Pebrine disease. in August with the salinity of 36 psu. The biomass ranged This paper is focused on life cycle studies, ultrastructural between 2.39 and 9,866.63 µg C l1, and the maximum identification, and genetic variations (in SSUrDNA occurred in June due to the dominance of Fabrea salina and ITS region sequences) of two microsporidia, when the salinity reached 148 psu. Both abundance and isolated from Pieris rapae (CFD) and Hemerophila biomass decreased to the minimum with the salinity atrilineta (SCH). Following results were obtained. (1) increasing to 311 psu in June. Statistical analyses show Life cycles of CFD and SCH, were similar, except for that neither ciliate abundance nor biomass was duration of life cycles, which were obviously shorter in correlated to salinity, while species richness and diversity CFD (72h) than in SCH (96h). (2) The spores collected significantly decreased (p < 0.01) along the increased from original hosts varied in shape. The spore shape salinity gradients. Meanwhile, our data indicate that was tending to become uniform (oval and smooth) after there were shifts of ciliate groups from oligotrichs propagating the isolates in the experimental host towards hypotrichs to heterotrichs dominance with the (silkworm) repeatedly. Both isolates had threelayered increase of salinity. Supported by the National Science spore wall, isofilar polar filament, bipartite polaroplast, Foundation of China (No. 40576072) and the '100 and two nuclei. The number of polar filament coils and Talents Project' of Chinese Academy of Sciences. the size of posterior vacuole varied depending on generation. (3) Phylogenetic analyses of CFD and SCH THE BIODIVERSITY OF HELIOZOANS AND isolated from original hosts, based on partial SSU HETEROTROPHIC FLAGELLATES OF SEVERAL rDNA sequence put these microsporidia into Nosema/ BOGS AND SMALL RIVER Vairimorpha clade. If microsporidia were passed M.M. Leonov through silkworms 4 times, the sequences obtained from Voronezh State University, Voronezh, Russia. Email: [email protected] harvested spores fell into the clade of true N. bombycis Heliozoan and heterotrophic flagellate biodiversity in group. (4) ITS region of two isolate displayed high several bogs and one small river was investigated. Cent variations. Different clones from the same PCR rohelids Raphidiophrys intermedia, Acantocystis penardi, amplication also significanly varied. Multiple alignment А. nichollsi, A. takahashii, Pterocystis pinnata, Raine revealed three highly varied sites in the ITS region of riophrys echinata, desmothoracid Clatrulina elegans, the studied isolates. Our results suggest that both isolates rotosphaerid Rabdiophrys sp., actinophryids Actinophrys belong to the genus Nosema, and that microsporidia sp., and Actinosphaerium eichhornii have been found. might possess a selective adaptability and potential to 31 species of flagellates from 12 taxonomic groups were differentiate in the alternative host. Supported by NSF observed in the samples from acid bogs, but only 4 grant 30671588. Protistology · 51

EVOLUTION OF EUKARYOTIC LIFE: OSCILLA the Epalxella sequence with other representatives of the TIONS, RYTHMS AND CLOCKS ARE A NECES Class PLAGIOPYLEA with 100% support in both SITY maximum likelihood and Bayesian analyses. Epalxella D. Lloyd1, M. Aon2, D.B. Murray3, S. Cortassa2 is the basal lineage with trimyemid and plagiopylid 1 Cardiff University, Biosciences, Cardiff, UK, 2 John Hopkins ciliates forming the two terminal sister clades. While 3 University, Department of Cardiology, Baltimore, USA, Keio this molecular support is strong and unambiguous, there University, Systems Biology Institute, Tokyo, Japan. Email: [email protected] are no obvious morphological features to unite these Experiments with ameboid and ciliate protozoa (eg. three clades. Thus, the Class PLAGIOPYLEA must Acanthamoeba castellanii and Tetrahymena pyrformis) continue to be referred to as a "riboclass." Using the Epalxella sequence as a basal marker, we tentatively indicate that the cytosolic metabolism produces H2S, while the mitochondria oxidise it. This is an echo of identified 20 environmental isolates to the terminal primeval organisation in the first eukaryote, which was plagiopylean clades: eight to the genus Trimyema; four formed when an Aarcheon host engulfed a proteobacte to the genus ; and eight to two new species, rial endosymbiont and initiated a syntrophic interaction one of which might represent a new plagiopylean genus. with mutually beneficial characteristics. Energy MICROAEROPHILIC AND ANOXIC CILIATES generation accompanies the disposal of this toxic DOMINATE A WARMMONOMICTIC HYPO common intermediate. Evidence that this interaction SALINE LAKE occurs even today comes from studies of a spontaneous M. Macek1, D. Pestova2, M.E. MartinezPerez1 ly selfsynchronised culture of yeast, where the two 1 National Autonomous University of Mexico, campus Iztacala, Project processes are separated in time, as phaserelated of Investigation in Tropical Limnology, Tlalnepantla, Mexico, 2 metabolic steps in the cellular network. In yeast this Masaryk University, Faculty of Science, Institute of Botany and temporal organisation is the output of a 40 min ultradian Zoology, Brno, Czech Republic. clock; in Acanthamoeba, it is a 69 min clock, and in Email: [email protected] Fouryeardata on the ciliate assemblage structure in a Tetrahymena it is 50 min. H S is also the synchroniser 2 high altitude, athalassohaline maarcrater lake that enables spontaneous behaviour with respect to Alchichica, Mexico (18o10'N; 93o10' W, altitude 2340 intracellular redox potential. Waves of synchrony spread m) were analysed. Experimentally, growth rates of from individual mitochondria through the entire cell ciliates were evaluated. DAPI staining was employed and then to neighbouring cells, so that the whole to count ciliates while the Quantitative Protargol population acts almost as a tissue. We suggest that these Staining for their identification; a total of 40 taxa was processes are recapitulating early events in the identified. Peritrichs often numerically dominated the establishment of eukaryotic organisation at a cellular ciliate assemblage; a maximum of 54 cells ml1 (mainly and multicellular level. H S is pivotal as an evanescent, 2 Rhabdostyla sp.) was observed in the surface layer at the volatile, highly diffusible, messenger molecule. end of the mixing period, during the development of diatoms (Cyclotella alchichicana), cyanobacterial bloom PHYLOGENETIC POSITION OF THE ODONTO (Nodularia sp.) and its decay. Minute spirotrichs STOMATIDS INFERRED FROM THE SMALL (particularly Halteria grandinella, maximum 7 cells ml SUBUNIT RRNA GENE SEQUENCE OF EPAL 1) and a haptorid, Belonophrya pelagica (2.8 cells ml1) XELLA ANTIQUORUM PENARD, 1922 (PHYLUM occasionally dominated in the epilimnion, while CILIOPHORA; ORDER ODONTOSTOMATIDA) were numerically dominant within the 1 2 3 D.H. Lynn , T. Stoeck , W. Foissner hypolimnetic assemblages (Cyclidium glaucoma, 8 cells 1 University of Guelph, Department of Integrative Biology, Guelph, ml1; nigricans, 18 cells ml1; and an anaerobic Canada, 2 FB Biologie/Abteilung Oekologie, Technische Universitat 1 Kaiserlautern, Kaiserlautern, Germany, 3 Universitat Salzburg, cf. Isocyclidium globossum, 46 cells ml ). Mixotrophic Institut for Zoologie, Salzburg, Austria. Email: [email protected] Euplotes cf. daidaleos (29 cells ml1) and Pelagothrix sp. The odontostomatid ciliates have remained a homoge (6.4 cells ml1) were important around the oxycline, neous order of ciliates since the 1930s when they were along with haptorids, particularly Phialina sp. (19 cells recognized as a monophyletic assemblage. Since that ml1). Strictly anaerobic ciliates of genera Caenomorpha time they have been placed with the ciliates, (1.5 cells ml1), Epalxella and Trimyema (36 cells ml1) and more recently transferred as to the were found in considerable numbers at the end of the new "riboclass" Class ARMPHOREA. We were able to stratification period. Calculating the contribution of the obtain the small subunit rRNA gene sequence of the ciliate biomass from the microaerobic metalimnion odontostomatid Epalxella antiquorum Penard, 1922, (dissolved oxygen < 2 mg l1) and from the anaerobic collected from the meromictic alpine Lake Alatsee in hypolimnion (May/July through December) suggested Germany, in July 2005. An alignment with representa these layers to be much more important than the tives of all 11 classes of ciliates unambiguously places epilimnetic/oxygenated ones; however, ciliate growth 52 · Abstracts of V European Congress of Protistology · 2007 rates were lower there. Apparently, microphilic and Dynamics of microsporidian infection in natural insect anaerobic ciliates were not related to higher bacteria populations is well studied for some lepidopteran pests numbers. like corn borer (Siegel et al., 1988), cabbage butterfly and gypsy moth (Issi, 1991). In populations of phyto EARLY BIOSPHERIC EVOLUTION, THE ORIGIN phagous insects, diverse routes of parasite transmission OF EUKARYOTA AND FUTURE PERSPECTIVES are available. Microsporidia develop intensively and OF LIFE ON THE EARTH may regulate host densities, preventing pest outbreaks V.V. Malakhov to some extent. Fouryear observations over the popu Department of Invertebrate Zoology, Faculty of Biology, Moscow State lation of meadow webworm Pyrausta sticticalis L. during University, Moscow, 119992 Russia. Email: [email protected] its depression phase showed that low pest density is According to contemporary data the Solar System and correlated to the high level of microsporidian infection, the Earth appeared about 4.6 4.5 billion years ago. this dependence being especially prominent when the The first indications of photosynthetic activity were insect host abundance is compared to microsporidian discovered in the most ancient sediments dated at 3.8 prevalence in the previous generation. Deliverance from billion years ago. Apparently water, sediments and life microsporidian infection facilitates insect population appeared on our planet almost at the same time about growth and under favorable hydrothermal conditions, 3.84.0 billion years ago. During the first two billion creates necessary prerequisites for the pest outbreak years there were only prokaryotes in the biosphere. The (Malysh et al., 2006). This parasitic system may be prokaryotes have no ability to , therefore referred to as a "loose". On the other hand, in blood enormous quantity of biomass was buried in sediments. sucking arthropods like ixodid ticks, horizontal trans Oil pools, gasfields, and a lot of minerals are the mission is limited as compared to lepidopteran insects. products of prokaryote activity in the early biosphere. Vertical transmission is more probable way for parasite's Appearance of eukaryotic protists (between 2 and 1 maintenance in populations of hematophagous hosts. billion years ago) led to decrease of biogenic elements Microsporidian infections in ixodid ticks are characte burial in sediments. After origin of Metazoa (about 600 rized either with low prevalence rates (Rehacek, Weiser, million years ago), burial rate of biogenic carbon was 1978) or with extremely low intensity of infection reduced three times in comparison with Archaeozoic (Tokarev, Movila, 2004; Tokarev et al., in press). For a era. Nevertheless organisms can not use the already "tight" parasitic system, consisted of microsporidia and buried carbon. Oil, gas, coal, shale and other sources ixodid ticks, significant role of parasite in host density of biogenic carbon remain almost inaccessible for them regulation is highly unlikely. Supported by RFBR (04 until arising of human civilization. From the biosphere 0349629, 060490814) and by a grant from President standpoint the destination of the human civilization is of Russian Federation for Y.S.T. (МК653.2007.4). the extraction and incineration of oil, gas and coal, production of ores and their dissolution in waters of the ULTRASTRUCTURAL ORGANIZATION AND FOR great oceans. After this predestination will be accom MATION OF CYSTLIKE CELL IN HOMOXENOUS plished the human civilization will die in a natural way, TRYPANOSOMATIDS OF THE GENERA LEPTO but the biosphere will receive new resources and get a MONAS AND BLASTOCRITHIDIA new impulse to develop. So, life will continue to develop M.N. Malysheva after human extinction, but its existence is limited by Zoological Institute RAS, St. Petersburg, Russia. Email: [email protected] the seeping of oxygen from the Earth nucleus in the Cystlike stages of two trypanosomatid species, future. It is estimated that the oxygen seep will start in Leptomonas oncopelti from the bug Oncopeltus fasciatus 600 million years. In 800 million years, oxygennitrogen and Blastocrithidia gerridis from the water strider Gerris atmosphere pressure will reach 5 atmospheres and lacustris, were studied. Formation of cystlike cells of temperature 76 degrees centigrade (that means the L. oncopelti begins with separation a small daughter cell destruction of almost all eukaryotes). In 1000 million from the promastigote. This small cell remains years, oxygennitrogen atmosphere pressure will reach associated with the flagellum of the parental promasti 15 atmospheres and temperature will rise up to 110 gote and divides twice to give rise to rosette of 4 "stra degrees centigrade, which means destruction of all phangers". "Straphangers" contact each other by the prokaryotes. modified flagella and form zonal desmosomes. One of the modified flagella attaches to the parental flagellum DYNAMICS OF MICROSPORIDIAN INFECTION by formation of zonal desmosome. Formation of B. IN POPULATIONS OF HARMFUL ARTHROPODS gerridis "cysts" starts with nuclear division. One of the Yu.M. Malysh, Yu.S. Tokarev daughter nuclei becomes a nucleus of the "bud", which AllRussian Institute for Plant Protection, St. PetersburgPushkin, is formed on the body of the parental epimastigote. This Russia. Email: [email protected] nucleus divides twice. Flagellumfree "bud" with 4 sets Protistology · 53 of organelles is formed. "Bud" separates from the describing here a new genus and species Amoeboradix parental cell and divides to produce 4 cystlike amasti gromovi, named in honor of B.V. Gromov, who made a gotes. Formation of "cysts" is accompanied by increase bulk of this study. The molecular investigation of this in the electron density of cytoplasm and cellular strain is in progress. Supported by RFBR grant № 05 organelles. Nuclear divisions in B. gerridis "bud" are 0449667. associated with progressive condensation of the chromatin, which masks microtubules and kinetochores. TWO SPECIES OF EPIBIONT CILIATES (SUCTO L. oncopelti chromatin is transformed into "labyrinthine RIA) ON HESPEROCORIXA SP. (HEMIPTERA: structure". In mature "cysts" chromatin is not structu CORIXIDAE) FROM A POND IN HIDALGO, ME rized. DNA fibrils of the kinetoplast lack circular confi XICO guration; they are condensed and do not exhibit any R. MarinoPerez, R. MayenEstrada regular organization. Changes in envelopes begin with Laboratorio de Protozoologia, Departamento de Biologia Comparada, thickening of all three layers of plasmalemma. The layer Facultad de Ciencias, Universidad Nacional Autonoma de Mexico, Mexico. Email: [email protected] of submembranous cytoplasm is condensed and masks Epibiont ciliates associated with some Arthropod groups, subpellicular microtubules. Cortical complex consisting such as Crustacea and Insecta, were recorded all over of thickened plasmalemma and dense granular sub the world. Concerning the hexapods, few orders were membranous cytoplasm is formed. In the electrondense studied (Hemiptera, Trichoptera and Diptera). In Me mature "cysts" the following zones could be disting xico the studies of epibiont ciliates were performed on uished: the zone of densely packed ribosomes, the mo crustaceans Cambarellus patzcuarensis and Hyalella dified kinetoplast, and the nucleus. azteca, but there were no data for insects. We collected 158 individuals of Hesperocorixa (Hemiptera: Corixi AMOEBORADIX GROMOVI GEN. ET SP. NOV. dae) in a pond of Hidalgo, Mexico, between February ENIGMATIC PARASITE OF THE ALGA TRIBO and August 2005, with aquatic net. We found two species NEMA GAYANUM of suctorian epibionts, Discophrya cybistri and Acineta K.A. Mamkaeva1, A.V. Pljusch1, M.A. Mamkaeva1, tuberosa attached to the legs of the corixid. Discophrya S.A. Karpov2 cybistri was found on 113 (71.5%) of the basibionts. 1 St. Petersburg State University, Dept. Microbiology, St. Petersburg, Russia, 2 Herzen State Pedagogical University, Zoology, St. Petersburg, From total 7203 individuals recorded, 6057 (84%) were Russia. Email: [email protected] observed attached to the middle legs. Few individuals Small amoebae (2.5 µm) with stiff flagellum (8 µm), of Acineta tuberosa were observed on 12 (7.6%) corixids. composed of 9 microtubular singlets, contain a nucleus, We conclude that preference of D. cybistri for the middle contractile vacuole, and mitochondria with lamellar legs can be due to their architecture. These appendages cristae, but no dictyosomes or phagosomes. Amoebae are covered with numerous setae, which provide a attach to the of the alga and transform into shelter to the ciliated epibionts. oval cysts with spines. Rhizoid appears from the base of the cyst and penetrates into the algae cell through the PATTERNS OF PROTOZOAN COMMUNITY cell wall. A round thinwalled cyst is transformed into STRUCTURE: LINKING PROCESSES AND an oval (16 × 9 µm), thickwalled, sessile, monocentric, SCALES and epibiotic sporangium with granular contents. Cysts Yu.A. Mazei have a thin long rootlet, which penetrates the algal cell. Penza State Pedagogical University, Dept. of Zoology and Ecology, Sporangia produce amoebae. Parasite develops on live Penza, Russia. Email: [email protected] algae Tribonema gayanum only. A. gromovi resembles at Patterns of the species diversity formation at different most the chytrid Phlyctidium anatropum, which was spatial scales were studied using protozoan communities observed on different algae including Tribonema. P. as model systems. Three community types at three anatropum also forms flagellated amoeboid zoospores. spatial scales were investigated. Testate amoebae But it has an asymmetrical, strongly arched sporangi communities and heterotrophic flagellate communities um, and its haustorium is very small and rounded. inhabiting sphagnum bogs were studied at the (i) mega Another similar chytrid, Amoebochytrium rhizidioides, scale in different regions, such as northern tundra in also produces amoeboid spores, but without flagella. Karelia, southern tundra in Yaroslavl region, forest Moreover it has polycentric thalli, its sporangia are steppe in Penza region; (ii) mesoscale in different pyriform, basally apophysate with more or less biotopes within one region, such as bogs at various prolonged discharge tubes. A. rhizidioides differs signifi successional stage, with different vegetation and levels cantly from A. gromovi in morphology and habitat as a of anthropogenic disturbance; and (iii) microscale in saprophyte growing in gelatinous matrix of Chaetophora different microhabitats within one biotope, such as elegans. On the basis of LM and EM data we are hummocks, lawns, hollows, sphagnum species etc. 54 · Abstracts of V European Congress of Protistology · 2007

Marine interstitial ciliate communities were studied at mystery. In order to address this question we have the megascale (in Barents, White, Black seas), searched the Bigellowiella natans mesoscale (in biotopes with various salinity, depth, database for the presence of ribosomal protein cDNA sediment characteristics within one sea), and micro sequences. A set of 61 full and partial ribosomal protein scale (microhabitats within one type of sediments at the sequences of the host cell was extracted from the same depth). We examined the way in which total database and aligned with the homologues sequences (gamma) diversity at different scales is partitioned into from over sixty representatives of other eukaryotic alpha and beta components. Alpha diversity, also groups. All analysis yielded a sister group relationship referred to as withinhabitat diversity, is the component between Bigellowiella natans and another protist group, of total diversity that can be attributed to average Heterokonta, with high statistical support. The disco number of species found within homogenous sampling vered monophyly of a group uniting and Hete units, named habitats. Betadiversity, referred to as rokonta allows us to reconstruct the last common an betweenhabitat diversity, is the component of total cestor of this group as a heterotrophic amoebaflagellate diversity that can be attributed to differences in species with a complex life cycle. Even further, this result may composition among the homogenous units in the lead to a revision of our current views on the subject of landscape. Betadiversity is determined by (i) variation loss and gain as it prompts us to reconsider the in environmental characteristics among local habitats, central hypothesis of rhodophytederived plastid and (ii) the degree of habitat specialization of the biota. evolution, which assumes a single plastid gain event in So, we try to understand, how contributions of alpha the last common ancestor of Heterokonta, Dinophyta, and beta to gamma diversity change as a function of Haptophyta and Cryptophyta. Supported by the Rus a spatial scale. Analysis of contribution of alpha and sian Foundation for Basic Research (grants 0504 beta components into total species richness suggested 49705 and 060449288). that the community at the microscale as well as at the megascale was alphadominant. In other words, at these MAIN EVOLUTIONARY TRENDS IN THE FO scales community is constrained by local factors. At the RAMINIFERAL TEST AND TEST WALL DEVE microscale, first of all biotic interactions (competition, LOPMENT predation, mutualism, etc.), as well as some abiotic V.I. Mikhalevich parameters, affected the size of the niche space (biotope Zoological Institute RAS, Department protozoology, St. Petersburg, heterogeneity, limiting factors, disturbance, etc.). At the Russia. Email: [email protected] The main evolutionary trends in the phylum Foramini macroscale it was history of climate, migration, and fera were transition from the unilocular to the multi evolution. At the mesoscale community seems to be chambered test as a result of the process of polymeri betadominant. Total diversity is affected by differences zation (in the classes Miliolata, Spirillinata, Nodosariata, between habitats along environmental gradients and Rotaliata), and to the supermultichambered test in the among different type ecosystems within the region. more advanced groups (in the classes Miliolata, Rotali Ciliate community displays the most clear crossscaling ata), then the differentiation of the chambers by their pattern. Testate amoebae community is rather alpha size and later by their function in some higher represen dominant, whereas heterotrophic flagellate community tatives (in the classes Miliolata, Spirillinata, Rotaliata). seems to be betadominant at all spatial scales. The polymerization of apertural openings and inner elements of apertural structures also took place in paral RESOLVING THE POSITION OF RHIZARIA ON lel in each of these classes. The next step of the multi THE BASIS OF MULTIGENE ANALYSIS chambered test evolution was development of integra 1 2 K.V. Mikhailov , V.V. Aleoshin tive systems: the inner communication between 1 Moscow State University, Division of Bioingeneering and Bioinformatics, Moscow, Russian Federation, 2 M.V. Lomonosov multiple chambers was going from simple foramens to Moscow State University, A.N.Belozersky Institution of Physico stolones, and then to apertural integrative systems (class Chemical Biology, Moscow, Russia. Email: [email protected] Rotaliata) and systems of canals (classes Spirilinata, Our present knowledge of the first step in the radiation Rotaliata). According to a new concept of foraminiferal of eukaryotes may be better illustrated not by a tree but evolution (Mikhalevich, 19922005) the agglutinated by a multifurcation that spawned around a dozen of test wall developed into different types of the calcareous "supergroups", a condition that is referred to as the secreted wall. This process occurred independently in "Eukaryotic Big Bang". The taxon Rhizaria is one such the different phyletic lines (classes) of Foraminifera: in supergroup. The monophyly of Rhizaria was established Miliolata, Spirillinata, Nodosariata, Rotaliata). on the basis of rRNA gene sequences and the sequences Appearance of the light and strong bilamellar (bifonti of the main components of the cytoskeleton, but the nal) test wall of Rotaliata can be regarded as aromor phylogenetic position of the taxon itself remained a phosis. The most primitive class Astrorhizata includes Protistology · 55 exclusively unilocular representatives with tectinous or by chronic inflammation of the bronchial wall. Many agglutinated test walls. The complexity of the multi studies showed an increase in IL8, MCP1 and TNF chambered foraminiferal test with its unique integrative alpha in induced sputum of these patients. Local and canal system represents strikingly high level of organi systemic responses are likely related to such factors as zation among Protista. genetic and immunological characteristics, smoking, and infectious agents. It is known that COPD patients can be colonized by P. jirovecii. Studies of animal H2 PRODUCTION IN THE FISH PARASITE SPI RONUCLEUS VORTENS models showed that infection with Pneumocystis C. Millet, J. Cable, D. Lloyd induced a rise of proinflamatories cytokines. Therefore, Cardiff University, Biosciences, Cardiff, UK. it is possible to think that P. jirovecii colonization plays Email: [email protected] a role in COPD physiopathology. The aim of the study The fish parasite vortens causes major was to identify changes induced by P. jirovecii in problems in aquaculture of ornamental fish. The systemic inflammatory response in patients with organism studied here was isolated from an angelfish COPD. COPD was diagnosed in 51 patients according (Sarah Poynton, 1995) and grown in bile supplemented to GOLD classification. Identification of P. jirovecii was (1%) TYIS33 medium. A membraneinlet mass performed by nested PCR of DNA extracted from spectrometer was employed to monitor, in a closed respiratory samples. The systemic inflammatory system, m/z peaks at 2, 32 and 44 for H2, O2 and CO2, response was analyzed in serum using a commercial respectively. When introduced in air saturated buffer, ELISA (R&D systems) for IL6, IL8, TNFalpha and Spironucleus vortens consumed O2 at the average rate MCP1. Patients with COPD colonized by P. jirovecii, 7 of 34 ±15 nmoles/min/10 cells. CO2 was produced at showed a higher level of proinflammatory cytokine than 7 25 ± 12 nmoles/min/10 cells. H2 production started noncolonized subjects. under microaerophile conditions (O2 = 60 µM) with a rate of 20 ± 11 nmoles/min/107 cells. KCN (15 mM) inhibited H2 production by 85% and 20 mM by 96%, Cytokine levels COPD without P. COPD with Pvalue indicating that an Feonly hydrogenase is responsible (pg/ml) jirovecii (N=23) P. jirovecii (N=28) (StudentT) IL8 13,89 21,26 0.028 for H2 production. Metronidazole (1mM) inhibited H2 TNFalpha 3,57 8,15 0.047 IL6 5,34 16,95 0.038 production by 50%, while CO2 production was not affected. A higher concentration (1.5 mM) inhibited MCP1 726,99 1012 0.048

H2 production by 87% and CO2 production by 36%, suggesting that metronidazole is reduced by an enzyme No statistically significant differences in age, sex, tobac of the H2 pathway, thus competing for electrons with co consume, respiratory function, lymphocytes and leu + H . The question of the source of H2 requires discrimi cocytes counts were found. The data suggest a relation nation between the various organelles/inclusions ship between P. jirovecii colonization and high level of evident in confocal and Normarski direct images. systemic inflammatory response in patients with COPD. Antibodies raised to Trichomonas vaginalis hydrogeno Supported by ERANET PneumocystisPathoGenoMics. somes and their enzymes have failed to reveal hydro genosomes in this organism. We have used 15 nm diam. LONGTERM SURVIVAL OF THE RESTING quantum dots (streptavidin coated) as markers for CYSTS OF THE HALTERIID CILIATE MESERES ingested materials in phagocytotic/pinocytotic vacuoles CORLISSI, AND IMPLICATIONS FOR ITS MAIN and vesicles. 16s rDNA probes will be used to investigate TENANCE IN LABORATORY CULTURES the possible presence of endosymbionts (archeal/ H. Mueller bacterial). The authors thank Prof. J. Kulda for provi Private Laboratory Konstanz, Germany. ding the organism and his expertise on its culture. Email: helga.mueller.konstanz@tonline.de Meseres corlissi is a rare freshwater ciliate adapted to PNEUMOCYSTIS JIROVECII INCREASES PROIN small, ephemeral aquatic habitats. It forms desiccation FLAMMATORY CYTOKINES LEVEL IN COPD resistant cysts to survive dry periods. Longterm survival PATIENTS of these cysts was studied in a 250 g soil sample from R. Morilla, M. MontesCano, F. MunozLobato, the type locality, an astatic meadow pond. The soil was V. Friaza, L. Rivero, N. Respaldiza, F.J. Medrano, airdried and stored at 18 to 22°C in the dark for 3 years. J.M. Varela, E.J. Calderon, C. De la Horra HHUU Virgen del Rocio, Seville 41013, Spain. At irregular intervals, 4 g subsamples were tested for Email: [email protected] viable cysts of M. corlissi, following a standard Chronic obstructive pulmonary disease (COPD) is procedure. These tests were positive up to 24 months of characterized by a permanent air flow limitation caused storage, but negative later on. Viable cysts were also 56 · Abstracts of V European Congress of Protistology · 2007 detected in a subsample which for 2 weeks had been THE DIVERSITY OF MITOCHONDRIONRE stored at 25°C. Based on these findings, a technique LATED PROTIST ORGANELLES was developed to cultivate clonal strains of M. corlissi M. Muller in alternating phases of active populations and The Rockefeller University, New York, USA and Collegium Budapest, desiccated cysts, thus imitating the life history of field Budapest, Hungary. Email: [email protected] Trichomonad flagellates were shown in the 1970s to populations. Commercial garden soil was used as a contain double membranebounded hydrogen produ substrate. Cysts embedded in dry soil remained viable cing organelles, the hydrogenosomes, instead of for up to 7 months at 18 to 22°C; they also tolerated mitochondria. Since then all currently recognized storage at 25°C. Cysts formed in the absence of soil, in major clades of eukaryotes except the Rhizaria were contrast, did not survive desiccation periods longer than found to include some groups that do not harbor typical one week. These results indicate that the M. corlissi aerobic mitochondria but contain other forms of double type population is well adapted to the ranges of membranebounded organelles. These organisms were temperature and humidity occurring in its natural sometimes called amitochondriates and regarded to be habitat. They also suggest that dispersal of cysts by wind ancestral eukaryotes. Such notions are no longer in is unlikely, due to their low tolerance to desiccation favor. The morphology and biochemistry of these when not embedded in soil. This may to a certain extent organelles differs dramatically from group to group. The explain the rare and disjunct occurrence of this ciliate. so far recognized major types are the hydrogen producing hydrogenosomes and the smaller mitosomes ERVIN BAUER, A HUNGARIANSOVIET PIO with no major role in metabolism. Their major diffe NEER OF THEORETICAL BIOLOGY AND PRO rences notwithstanding these organelles share a number TISTOLOGY of biological characteristics with each other and with M. Muller typical mitochondria. In contrast to earlier opinions, The Rockefeller University, New York, USA and Collegium Budapest, Budapest, Hungary. Email: [email protected] today they are all regarded as members of a monophy Ervin Bauer (18901938) is a legendary figure of Soviet letic family of organelles, derived from the unique biology of the 1930s. He developed deductively a mitochondrial endosymbiotic event. The observed consistent theoretical biology starting from his principle diversity reveals the extreme evolutionary plasticity of of "permanent disequilibrium of living matter." Work these characteristic organelles of the eukaryotic cell. in his laboratories in Moscow (Obukh Institiute of Professional Diseases and Timiryazev Biological PNEUMOCYSTIS JIROVECII COLONIZATION Institute) and Leningrad (AllUnion Institute of Expe ALTERS PULMONARY SURFACTANTASSOCIA rimental Medicine) was directed to provide expe TED PROTEINS IN SUBJECTS WITH IDIOPA rimental evidence of this disequilibrium. Among many THIC INTERSTITIAL PNEUMONIA biological objects this work included studies on protists. F. MunozLobato, V. Friaza, C. De la Horra, With A.M. Granovskaya (19031958?) he explored the N. Respaldiza, M.A. MontesCano, R. Morilla, effects of injury on ciliates in various stages L. Rivero, J. MartinJuan, J. Varela, E. Rodriguez between cell divisions, focusing on nuclear processes Becerra, E.J. Calderon and respiration. These were among the first measu CIBER de Epidemiologнa y Salud Pъblica. Virgen del Rocio, University Hospital, Seville, Spain. Email: [email protected] rements of respiration in single protist cells. With A.M. Data obtained on in vitro and animal models with Lunts (19001977), who was a student of Max Pneumocystis pneumonia showed that this patogen Hartmann in Berlin and returned to the USSR in 1932, bound to surfactant proteins and could alter their he explored tactic phenomena in green algae. With V.S. expression and distribution. Pulmonary surfactant Brandgendler (19011941), who joined Bauer briefly associated proteins, SPA and SPD, play an important after work in mammalian , he investigated role in lung host defence and in surfactant homeosta serum toxicity in Paramecium. These efforts made sis.It was demonstrated that SPA and SPD regulated significant contributions to protozoology of the time NFkB and, by this pathway, were able to enhance or and, while seemingly disparate, they all formed integral suppress inflammatory mediators production. Idiopathic part of Bauer's theoretical constructs. After Bauer's Interstitial Pneumonia (IIP) is a heterogeneous group repression in 1937, Granovskaya was also repressed, of poorly understood diseases. All of them include an Lunts became head of the Biology Department of the initial inflammatory response that could be triggered Saratov Medical University, Brandgendler returned by an infectious agent. The objective of the study was briefly to mammalian physiology and was killed in the to identify possible alterations in pulmonary surfactant defense of Leningrad. associated proteins in patients with IIP colonized with Protistology · 57

Pneumocystis. Bronchoalveolar lavage fluid (BALF) implies extensive RNA editing. Although the highly from 40 IIP patients was collected, and identification reduced mitochondrial gene content supports the of P. jirovecii was performed by nestedPCR of mt LSU relationship of the dinoflagellates and Apicomplexa as RNAr gene. SPA levels were determined by densito sister groups, the absence of large regions of noncoding metry, ELISA (BioVendor) and westernblotting. All mtDNA in Apicomplexa suggests that this feature arose data were normalized with total protein concentration after the two lineages diverged. (quickstart Bradford dye reagent, Biorad) for each sample. P. jirovecii was identified in 14 out of 40 (35%) MINIATURE CHROMOSOMES OF MICROSPO IIP patients. The results obtained for surfactantassoci RIDIA: STRUCTURE, SEGREGATION AND COM ated proteins are shown in the table. Conclusions: (1) PACTION high rate of P. jirovecii colonization was found in IIP E.S. Nassonova patients; (2) statistically significant SPD decrease was Institute of Cytology RAS, St. Petersburg, Russia. observed in the P. jiroveccicolonized IIP patients; (3) Email: [email protected] P. jirovecci could play a role in the physiopathology of Genomes of microsporidia consist of a small number this disease through interaction with pulmonary of miniature chromosomes, ranging from 120 to 2700 surfactantassociated proteins. Supported by ERA kbp and showing extensive length polymorphism of NET PneumocystisPathoGenoMics. homologs. It is conceived that microsporidian chromo somes, similarly to many other parasitic protists,

IIP colonized IIP no colonized p contain a conserve core and repeatrich variable extre SPA 11.77 15.25 0.295 mities mediating frequent interchromosomal recombi (CN*/mg prot) SPD nations. Conserved restriction patterns of homologous 406.24 904.86 0.026 (ng/mg prot) chromosomes identified in monomorphic diplokaryotic microsporidium Paranosema grylli, evidence for the * Normalized relative concentration existence of internuclear genome homogenization. The ORGANISATION AND EXPRESSION OF THE meiosis was recently found in this species and is long DINOFLAGELLATE MITOCHONDRIAL GENOME known in Nosema rivulogammari, but most of mono E.A. Nash, A.C. Barbrook, R.K. EdwardsStuart, morphic diplokaryotic microsporidia still are believed K. Bernhardt, C.J. Howe, R.E.R. Nisbet to be agamous. It is unknown if any kind of internuclear Department of Biochemistry, University of Cambridge, U.K. exchange leading to genome homogenization, takes Email: [email protected] place in these species. Microsporidia divide by closed Protist mitochondrial genomes are extremely diverse intranuclear pleuromitosis, which involve neither the in size, gene content and organisation. Although the disintegration of the nuclear envelope, nor the mitochondrion of all aerobic protozoans studied chromosome arrangement in a metaphase plate. contains a genome, the number of genes present is Chromosomes remain weakly condensed; probably highly variable. This situation is thought to have arisen such a simple and fast mitosis does not require high by the differing extents of gene transfer to the host degree of compaction of small chromosomes. Spreading nucleus between lineages over the course of evolution. of interphase chromatin in a low ionic strength solution Within the , a group comprising the ciliates, revealed 10nm nucleosomal and 20nm chromatin Apicomplexa and dinoflagellates, the ciliate and fibers, but none of the higher order structures. In silico apicomplexan mitochondrial genomes have been well analysis of core histone genes in Encephalitozoon cuni studied, and several complete sequences from each are culi and Antonospora locustae demonstrate that they are now available. However, dinoflagellate mitochondrial the most divergent eukaryotic histone sequences of all DNA (mtDNA) has hitherto defied characterisation known to date. However, positions of aliphatic amino due to its unusually complex structure. We have recently acids critical for histone folding are conserved, and shown that the dinoflagellate mitochondrial genome histone fold domain can be recognized unambiguously. encodes the three respiratory complex subunits cox1, Most of potential sites of posttranslational modifica cox3 and cob and we have found small rDNA fragments tions can be also tentatively identified. Hence, despite similar to those observed in Apicomplexa. Unlike the high degree of histone sequence divergence, the Apicomplexa, however, the genome appears to consist overall structure of nucleosome in microsporidia is mostly of noncoding DNA containing pseudogenes, similar to that in other eukaryotes. Data used were in short coding region fragments, and closelypacked part obtained in cooperation with the team of Prof. C.P. stemloop structures. Several differentlysized trans Vivares, Universite Blaise Pascal, ClermontFerrand, cripts are produced for each gene, and comparison of France. Supported by RFBR grants 050449222 and sequences from EST projects and genomic clones 070400662. 58 · Abstracts of V European Congress of Protistology · 2007

MACRONUCLEAR GENOME ORGANIZATION bactericidal cationic peptides (lysozyme, histones). CHANGES AT THE INITIAL STAGES OF SPE Cationic peptides and their inhibitors were estimated CIATION: COMPLEX AS to form functional systems "lysozymeantilysozyme", A MODEL "histoneantihistone". We used the model of interaction I.V. Nekrasova1, A.A. Potekhin1, E. Przybos2, between the ciliate Tetrachymena pyriformis and M.S. Rautian1 isogenic strains of bacteria Escherichia coli, that differed 1 Biological Research Institute of St. Petersburg State University, St. by the antilysozyme activity. The antilysozyme activity 2 Petersburg, Russia, Institute of Systematics and Evolution of Animals, of bacteria was shown to protect them from protozoa Krakow, Poland. Email: ne[email protected] Pulsedfield gel electrophoresis (PFGE) was applied to and thus to contribute to their survival in protist' cells. analyze and compare macronuclear genome organiza At the same time, protists take part in formation of tion of all fifteen species of the Paramecium aurelia heterogeneity of antilysozyme sign expression in complex. The "P. aurelia" PFGE profile was a continuous bacterial population. The same data were obtained using spectrum of differentsized (502000 kb) DNA the other model: bacteria Pseudomonas putida and molecules; specific pattern of banding allowed direct cyanobacteria Microcystis sp. Electron microscopy comparison of different profiles. Each strain of any study showed that the strains with antilysozyme activity species, despite stage of its life cycle, was characterized induced incomplete phagocytosis in protists accompa by constant PFGE profile. Different strains of a certain nied by better bacterial survival in associations. Incom species mostly shared speciesspecific banding pattern. plete phagocytosis of the Escherichia coli strain with Still, for some species noticeable intraspecific differen antihistone activity occurred also during its interaction ces were registered. Sonneborn (1974) had observed with Tetrachymena pyriformis. Moreover, there was interspecific mating reaction for some species of the P. incompact chromatin in cilates' macronucleus. The aurelia complex (P. primaurelia, P. triaureliа, P. tetrau proportion of protist cells differed by contents of histo relia, P. pentaurelia, P. septaurelia, P. octaurelia, P. dec nes was changed. The share of cells with a little amount aurelia, P. dodecaurelia), though no livable progeny was of histones was increased and one with normal contents obtained in such cases. We found that PFGE profiles of histones was decreased. These results are important of strains of all these species were relatively similar, while for investigation of the mechanisms of symbiotic rela for the species which are not able to interspecific mating tions between bacteria and protists at initial stage of (P. sexaurelia, P. novaurelia, P. undecaurelia, P. tredec . Supported by RFBR grant 050449870. aurelia, P. quadecaurelia, P. sonneborni) each species was characterized by the highly distinct PFGE profile. DIVERSITY OF FREELIVING BAIKALIAN CILIA The only exception was P. biaurelia, which belongs to TES WITH SOME ECOLOGICAL COMMENTS the second group but did not have highly individual L.A. Obolkina, N.V. Potapskaya PFGE profile. Interestingly, our data well correspond Limnological Institute SB RAS, Irkutsk, Russia. to the recent of P. aurelia complex Email: [email protected] The freeliving Baikalian ciliate fauna (300 species) (Hori et al., 2006): on this tree all species (again, with forms several specialized ecological complexes, two of the exception of P. biaurelia) in which interspecific which are of a particular interest. The spring complex mating reaction has not been observed, and which are develops during the underice plankton maximum; it characterized by wellrecognizable PFGE profiles, are consists of both rare and widespread coldwater and diverged from other species of the complex. Thus, eurythermic ciliates. The temperature optimum for this changes in macronuclear genome organization pattern complex is within the range 04оС, and some species coincide with initial stages of speciation in Paramecium. Supported by RFBR grant 060449504. are rather associated with the ice algae. The occurrence of endemic ciliates in the Baikal plankton is under THE SYMBIOTIC SYSTEMS OF PROTISTS AND discussion. If Marituja and Liliimorpha may be regarded BACTERIA, INHIBITED PROTECTIVE FACTORS as postglacial relicts preserved in some waterbodies OF EUKARYOTIC CELL: MECHANISMS OF of Europe and Asia (Foissner et al., 1999), then the FORMATION Bursellopsis group, consisting of 45 closely related N.V. Nemtseva, A.O. Plotnikov species, may be a result of adaptive radiation typical for Institute for cellular and intracellular symbiosis, UrB RAS, Orenburg, the Baikal flora and fauna. The psammophilic complex Russia. Email: [email protected] is the most interesting among benthic ciliates. The widespread ability to inhibit lysozyme and histones Pronounced morphological specific for (antilysozyme and antihistone activities), 97 and 76% mesopsammon, as well as the diversity (4 genera and respectively, was revealed among bacteria associated 15 species of Colepidae, 67 Frontonia species, and with protists in water bodies. It demonstrates the others) indicate the intensive speciation in this commu mechanism providing the bacterial resistance to protists' nity. Moreover, many eurytopic ciliates, living in fresh Protistology · 59 water sands of Lake Baikal, show adaptive trends to functional genomics, Moscow, Russia. Email: [email protected] inhabit this biotope only. In spite of predominance of In many ciliates, the universal genetic code is substituted widespread species in the freeliving ciliate fauna of this by variant codes in which one or two of the stop codons ancient lake, it also includes autochthonous endemic are reassigned to sense codons. In some Spirotrichea species and even genera. ciliates, UAA and UAG are translated as Gln (for example, in Oxytrichidae), whilst in Euplotida UGA is EXPERIMENTAL MURINE GRANULOMATOUS translated as Cys. These two types of variant codes are AMOEBIC ENCEPHALITIS: IMMUNOHISTO also found in phylogenetically distant ciliates, like CHEMICAL CHARACTERIZATION OF EARLY (UAA and UAG encode Gln) and STAGES OF THE INFECTION (UGA encodes Cys). We examined M. OmanaMolina1, L. VerdinTeran2, D. Hernandez ciliate species with >100 published proteincoding Martinez1, R. OrtizOrtega1, L. MorenoFierros2, DNA sequences. Surprisingly, in several cases discre P. BonillaLemus1 pancies were revealed between our estimation of the 1 National Autonomous University Mexico, FESIztacala, genetic code deviation and the type of variant code 2 Environmental Microbiology Project, Mexico, UNAM Biomedicine established earlier. We compared frequencies of in Unit, Immunology Lab, Mexico, Mexico. Email: [email protected] frame "stop" codons translated as Gln or Cys with fre Early stages of experimental murine granulomatous quencies of Gln or Cys encoded by convention codons. amoebic encephalitis with Acanthamoeba castellanii, We consider the codes characterized by low frequency were immunohistochemically characterized after 48 of inframe "stop" codons as "young" variant codes, and 72h post intranasal instillation of 1 × 106 / 20 µl whereas the codes with many accumulated mutations trophozoites. Mice were sacrificed. Lungs, kidneys, leading to high frequency of inframe "stop" codons are spleens, liver and heads were fixed and processed for classified as "old" variant codes. Some closely related their inclusion in paraffin, and sectioned to obtain 5 ciliate groups exhibit different types of codes. Variant µmthick slides and then processed immunohistoche codes of these organisms were in most cases "young". mically using IgG of rabbit antiAcanthamoeba and goat These data allowed us to propose that the initial muta anti IgG of rabbit conjugated to peroxidase, revealed tion(s) in stop codondecoding translation termination with H2O2 diaminobenzidine and counterstained with factor eRF1 happened in universal code organism Harris hematoxylin. Analysis of all organs in study, leading to lower efficiency of recognition of one or two revealed the invasion of trophozoites of Acanthamoeba stop codons. This mutation(s) could be compensated castellanii. Heads: some trophozoites were observed in by secondary mutation(s) reversing this eRF1 back to contact with the surface of the layer of mucus of the omnipotent type. Alternatively, this codon or codons olfactory epithelium; in some zones, the trophozoites could be decoded by tRNA which acquired an ability moved through the mucus with disruption of a number to recognize stop codons after mutation(s). We suppose of areas; invasion of the olfactory epithelium was that even in ciliate protein sequences where stop codons observed; cystlike forms were observed in the brain. are not reassigned to sense codons a variant code could Lung, spleen, kidney and liver: trophozoites, as well as operate due to restriction of stop codon recognition by cyst, were observed in all the organs; in some cases eRF1. disorganization but not inflammation of the tissues was evident; amoebae were located near of vessels, THE EFFECT OF DRYING ON COLONIZATION frequently migrating through the different tissues. It was OF PERIPHYTON COMMUNITIES believed that Acanthamoeba spp. reach the brain by A. Ostojic1, R. Matonickin Kepcija1, M. Milisa1, hematogenous dissemination, but we proved that the B. PrimcHabdija1, I. Habdija1, K. Tockner2 parasites were able to adhere, migrate and penetrate 1 University of Zagreb, Faculty of Science, Division of Biology, Zagreb, through olfactory epithelium. In contrast to Naegleria Croatia, 2 Eawag, Department of Aquatic Ecology, Duebendorf, fowleri, A. castellanii migrates and penetrates slowly, Switzerland. Email: [email protected] thus explaining the chronic course of the infection. Surface drying is an important disturbance that shapes Immunohistochemistry is a useful technique to identify biological communities. We tested the effect of drying freeliving amoeba since they are difficult to be observed on the colonization patterns of protozoa and micro with conventional staining techniques. metazoa. A total of 192 glass slides were exposed in replicated 1.5 m experimental flumes that were fed by BIOINFORMATIC ANALYSIS OF VARIANT GENE water from a nearby stream. After an initial colonization TIC CODES IN DIFFERENT PHYLOGENETIC period of nine days one third of the slides were airdried GROUPS OF CILIATES for eight days, rewetted, and the recolonization of N. Oparina, Yu. Mazur, L. Kisselev periphyton was compared with community on the Engelhardt Institute of Molecular Biology, Laboratory of structure permanently wet slides. During the initial period (i.e. 60 · Abstracts of V European Congress of Protistology · 2007 nine days), we observed distinct colonization pattern have been found earlier only in PontoCaspian region, starting with singlecelled algae, mostly diatoms, and evidently have been transported to Central Europe followed by flagellates and ciliates. In contrast to the with the invasive PontoCaspian gammarids. Interest initially slow development of periphyton, recoloniza ingly, no microparasites were found in an invasive tion after the dry period was very fast. At the beginning NorthAmerican Gammarus tigrinus, and in Ponto of the rewetting experiment only desiccated periphyton Caspian Obesogammarus crassus. Also, no transfer of was detected on glass slides. However, in less than 24 microparasites between native and alien gammarids was hours flagellates and ciliates appeared in great numbers. observed. Further, drying resulted in a distinctly altered commu nity composition (i.e. first colonizers and dominant FIRST RECORD OF MICROSPORIDEAN INFEC species), compared to the composition on the perma TION OF MYXOBOLUS PARVUS (MYXOZOA) nently wet slides. The much faster recolonization rate FROM JAPAN SEA GREY MULLET MUGIL SOIUY after drying can be explained by the remaining matrix M. Ovcharenko1, V. Yurakhno2, L. Shvetzova3 on the glass slides and the encysted forms of micro 1 W. Stefanski Institute of Parasitology, Polish Academy of Sciences, organisms. In the initial colonization and recolo Warsaw, Poland, 2 Іnstitute of Biology of the Southern Seas NAS of 3 nization experiments species richness increased Ukraine, Sevastopol, Ukraine, Pacific Research Fisheries Centre, Vladivostok, Russia. Email: [email protected] logarithmically, while ash free dry mass increased An unidentified microsporidium is recorded from exponentially in time. Total biomass and the density of myxozoan parasite Myxobolus parvus infecting grey organisms were much higher in the recolonisation mullet, Mugil soiuy in Russian coastal part of Japan Sea experiment. near Razdol'naja river (46°35’N, 32°16’E). Parasitologi cal material was collected from 63 specimens of grey MICROSPORIDIA AND GREGARINES OF ALIEN mullets in period from July to December 2004. AND NATIVE GAMMARIDS (AMPHIPODA) OC Pseudocysts of M. parvus were found in gills, gill organ, CURRING IN POLAND mesentery, pyloric caeca and intestine of M. soiuy. The 1 1 2 M. Ovcharenko , I. Wita , D. CodreanuBalcescu , infected myxosporeans were assembled in intestine of 3 3 M. Grabowski , A. Konopacka 2 from 33 fish specimens (prevalence: 6 %) catched in 1 Institut of Parasitology, Polish Academy of Sciences, Warsaw, Poland, 2 Research Institute of Biology Romanian Academy, October. Total length of hyperinfected hosts was 34,5 Bucharest, Romania, 3 University of Lodz,Department of Invertebrate 35,0 cm, and weigth 415 435 g. Intensity of myxozoan Zoology & Hydrobiology, Lodz, Poland. Email: [email protected] infection was 1 12 plasmodia per intestine. The The goal of our research was to identify microparasites microsporidean spores were elongated, sometimes (gregarines and microsporidia) of alien gammarids slightly bent, measuring 6.4 ± 0.5 (5.1 7.5) × 2.9 ± colonizing recently Polish inland, and coastal waters, 0.3 (2.2 3.6) µm on glycerin gelatin slides. Most of from those infecting native species occurring in the same the spores were glued to the spore surface of myxozoans. areas. Altogether, over 4000 individuals of 12 gammarid Non infected spores of M. parvus measured 7.3 + 0.7 × species (5 aliens and 7 natives) were collected from the 6.3 + 0.6 µm, their polar capsules were 3.9 + 0.3 × 2.1 deltaic system of the Vistula River, Vistula Lagoon, + 0.3 µm in size. Myxosporean spores from infected littoral of the Baltic Sea near Debki, and from small plasmodia were 7.4 + 0.8 × 6.7 + 0.4 µm in size with rivers draining directly to the sea in this area. All polar capsules measuring 3.9 + 0.3 × 2.2 + 0.4 µm. gammarids were identified to the species level, and Species we found, differs from all documented sectioned under a stereomicroscope. Microparasites microsporidians infecting Myxozoa (Nosema cerato were identified with light and electron microscopy. myxae, N. notabilis, N. marionis, Microsporidium sp. and Gregarines (Apicomplexa, Gregarinidae) were recorded two Microsporidia gen. sp.) by shape and dimensions in the digestive tracks of invasive Pontogammarus of spores, and by host infected. robustoides (Uradiophora ramosa BalcescuCodreanu, 1974 & Cephaloidophora mucronata Codreanu TRANSITION BETWEEN EXTRACELLULARITY Balcescu, 1995) from the Vistula deltaic system, and in AND INTRACELLULARITY IN LOWER SPORO the digestive system of native Gammarus pulex (Cephalo ZOANS: A CASE OF ELEUTHEROSCHIZON DU idophora gammari (Franzius, 1848)) from Stradanka BOSQUI BRASIL, 1906 (COCCIDEA, PROTO river, an affluent of the Vistula Lagoon. Microsporidia COCCIDIIDA) FROM POLYCHAETES SCOLO were found only in two gammarid species: Pleistophora PLOS ARMIGER AND NAINEREIS QUADRICUS muelleri (Pfeiffer, 1895) in G. pulex from Piasnica river, PIDA and Nosema pontogammari Ovcharenko & Kurandina, G.G. Paskerova 1987) from the Vistula deltaic system. All the above SaintPetersburg State University, Department of Invertebrate Zoology, microparasites are new to Poland. Pleistophora muelleri St. Petersburg, Russia. Email: [email protected] is a widespread species in Europe. Other microparasites The order Protococcidiida is a small group of parasites Protistology · 61 that develop, extracellularly or intracellularly without tizing in a certain hostal biotope. Gregarines inhabiting schizogony, in marine invertebrates. Protococcidiida the intestine usually exhibit bending or gliding. These are so much different from other coccidia that they need motility types provoke currents of hostal medium to be investigated in details to infer the early evoluti around a parasite cell. Metaboly occurs in protists para onary history of sporozoans as a whole. We have studied sitizing in a medium with increased viscosity and/or in the morphology of Eleutheroschizon dubosqui Brasil, a limited space. In cavities with mobile liquid the 1906. The scanning electron microscopical investiga motionless gregarines occur. They need no movement tion has demonstrated that these parasites develop on because the cavity medium moves by itself around the intestinal epithelium of the White Sea polychaetes parasites. Motionless gregarines likewise occur in host Scoloplos armiger and Nainereis quadricuspida. They are tissues where tissue pressure prevents them from oval in shape and attach to the epithelium layer by a moving. complicated attachment apparatus. Transmission electron microscopy showed that parasitic cells were THE TWILIGHT OF SARCODINA enclosed in a membranous sac of unclear origin. There J. Pawlowski are two morphs of E. dubosqui: with a large nucleus and University of Geneva, Department of Zoology, Geneva, Switzerland. with several small nuclei. These morphs are respectively Email: [email protected] Traditional morphologybased system of protozoa female and male gamonts reasoning from the life cycle grouped all taxa that form socalled pseudopodia in the description by Chatton and Villeneuve (1936). Life subphylum of Sarcodina, subdivided into superclass of time observations showed that macrogamonts and Rhizopodea and Actinopodea. This system was recently microgamonts were both capable of changing their body challenged by phylogenetic analysis of DNA sequences, shape by alternate shortening and stretching. E. dubos which suggested the polyphyletic origin of amoeboid qui is a parasite sharing characters of coccidia and gre protists. Based mainly on ribosomal RNA and actin garines: body polarity, extracytoplasmic intracelullar genes, the majority of pseudopodiabearing protists localization (similar to that of Cryptosporidium and were placed in two "supergroups": Amoebozoa and Ditrypanocystis) in the host intestinal epithelium, cell Rhizaria. The Amoebozoa include naked and testate motility and life cycle without merogony and syzygy. It lobose amoebae, the mycetozoan slime moulds, suggests an evolutionary transition between extracel amitochondriate entamoebids and pelobionts, as well lularity of gregarines and intracellularity of coccidia. as some flagellated protists (, ). The Rhizaria comprise protists with filopodia, reticulo EVOLUTIONARY TRENDS OF THE LOWER GRE podia and actinopodia, as well as some amoeboflagel GARINES: ULTRASTRUCTURAL AND BIOLO lates. Some amoeboid protists branch within other GICAL DATA "supergroups", for example Opisthokonta (Nuclearia, G.G. Paskerova Ministeria) or (Actinophryidae), while SaintPetersburg State University, Department of Invertebrate Zoology, others (Centrohelida) remained incertae sedis. Phylo St. Petersburg, Russia. Email: [email protected] genomic studies confirmed the monophyly of two The aseptate gregarines are the most primitive groups amoeboid "supergroups", but their position in euka within Gregarinea because they have nonseptated cell ryotic tree remains unclear. Lack of genomic data for body and parasitize in marine invertebrates. In the majority of amoeboid taxa impedes the resolution gregarines, the main evolutionary trend is an improve of phylogenetic relationships within Amoebozoa and ment of the locomotor apparatus and attachment Rhizaria. More extensive taxon sampling is also neces organelle. Here I discuss correlations of the cortical sary for better assessment of the diversity of amoeboid zone ultrastructure with the type of motility and protists. localization in the host of lower gregarines. of archigregarines is formed from the sporozoite apex and PHYLOGENETIC POSITION OF TRICHOSIDAE retains elements of apical complex. Mucron of lecu J. Pawlowski, J.F. Fahrni dinides has a flattened membraneous surface adhering Department of Zoology and Animal Biology, University of Geneva, to host cells as a sucker. In advanced gregarines mucron Switzerland. Email: [email protected] is replaced by epimerite an evolutionary new attach The family Trichosidae comprises testate ment organelle, which has no elements of apical complex marine amoebae with alternation of asexual (schizont) and acts as an anchor.Aseptate gregarines demonstrate and sexual (gamont) generations. The schizont posses three distinct modes of motility: bending, gliding, and ses a supple test covered with mineral spicules ("fuzzy" metaboly (peristalsis), while some of them are motion form) whereas the gamont is devoid of spicules ("smo less. Modes of motility are associated with different oth" form). The family is represented by a single genus structural types of cortical zones of trophozoites. The Trichosphaerium composed of three described species. mode of motility is adaptation of gregarines to parasi In order to establish the phylogenetic position of Tri 62 · Abstracts of V European Congress of Protistology · 2007 chosidae among amoeboid protists, we obtained the Russia, 2 University of Connecticut, Department of Genetics and complete small subunit rDNA sequences from three , Department of Molecular, Microbial and Structural Biology, USA, 3 Zoologisches Institut der Universitat different isolates: Trichosphaerium sieboldi (CCAP Tubingen, Abteilung Zellbiologie, Tubingen, Germany. 1585/2; "smooth" form); Trichosphaerium sp. 1 (Mada Email: [email protected] gascar; "fuzzy" form), and Trichosphaerium sp. 2 In general, DNA in the transcriptionally active macro (Mediterranean Sea; "fuzzy"form). Our data show that nucleus of the hypotrichous ciliates is represented by Trichosidae belong to the "supergroup" of Amoebozoa, short linear molecules minichromosomes, encoding but their exact placement is impeded by extremely rapid a single gene each. Previous studies have not revealed rate of evolution of their ribosomal genes. the presence of conventional eukaryotic consensus sequences involved in regulation of transcription initi PHAGOCYTOSIS BY TRICHOMONAS VAGINALIS: ation and activation in the minichromosomes. In our NEW INSIGHTS work we have redetermined transcription start sites in A. PereiraNeves, M. Benchimol four tubulin genes of Stylonychia lemnae. Then we used Santa Ursula University, Cell Biology Department, Rio de Janeiro, microinjection of artificial a1 tubulin minichromoso Brazil. Email: [email protected] mes into the macronucleus of S. lemnae as a means to The parasitic protozoan Trichomonas vaginalis is the characterize in detail the corresponding promoter. causative agent of trichomoniasis. We have studied the Deletion and block substitution mutations in promoter in vitro capacity of T. vaginalis to phagocytose and region of a1 tubulin gene revealed a TATAlike element, degrade Saccharomyces cerevisiae cells. To analyze the an initiator element, three distinct upstream sequence phagocytic ability and capacity, two isolates of T. elements (USEs), including repressor element, involved vaginalis presenting different virulence grades were in regulation of transcription initiation. Investigation used. Complementary techniques such as fluorescence of a tubulin expression in cells treated with Concana microscopy, computer based fluorescence analysis, valin A revealed a capability of a2 tubulin gene for scanning and transmission electron microscopy and the increasing of transcription level. We revealed a promoter use of drugs that interfere with the actin microfilaments region in a2 tubulin gene involved in regulation of were used in order to follow the actin cytoskeleton transcription activation. Determination of transcription behavior during phagocytosis of yeasts by T. vaginalis. start sites and a sequence alignment indicated that both It was concluded that: (1) T. vaginalis changes its shape TATAlike and initiator elements are conserved compo rapidly and engulfs the yeasts, which are almost as large nents of S. lemnae tubulin minichromosomes, whereas as the parasite; (2) longterm and fresh cultures are able the USEs appear to be specific for the a1 tubulin mini to phagocytose; (3) T016 strain exhibited an amoeboid chromosome. On the other hand, the presence of in morphology during the internalization process; (4) duction promoter element is conserved for the a2 attachment occurred through the whole cell surface, tubulin minichromosome. Supported by grants 0704 including both anterior and recurrent flagella; (5) both 00662 and 030448505a from the Russian Foundation sinking process and the classical phagocytosis where for Basic Research, German Research Foundation pseudopodia are extended toward the target cell; (6) (DFG grants Am 26/31 and Gu371/3) and the German the internalized S. cerevisiae are digested in lysosomes; Academic Exchange Service (DAAD grant 325/lin). (7) competitor sugars Dmannose or Lfucose inhibit the phagocytosis; (8) a thick layer of actin microfila BIODIVERSITY AND ECOLOGY OF PLANKTO ments was present underlying the plasma membrane NIC HETEROTROPHIC FLAGELLATES IN SALI and especially in the pseudopodia and around the NE LAKES phagocytosed particles; (9) a dramatic change in the A.O. Plotnikov1, E.A. Selivanova1, N.V. Nemtseva1, distribution pattern of fibrillar actin occurred during J.D. Silberman2 phagocytosis; (10) Cytochalasin D depressed the pha 1 Institute for cellular and intracellular symbiosis, UrB RAS, Orenburg, gocytosis; (11) a nonspecific recognition and phago Russia, 2 University of Arkansas, Department of Biological Sciences, cytosis is mediated by a mannosereceptor present on Fayetteville, USA. Email: [email protected] the parasite surface; (12) the phagocytic process may Biodiversity and ecology of heterotrophic flagellates was occur during mitosis of the parasite. studied in two hypersaline lakes and one brackish lake (SaltIletsk, Orenburg region, Russia). Seasonal dyna COMPONENTS OF ALPHATUBULIN MINI mics and vertical distribution of species richness and CHROMOSOME PROMOTERS IN THE STICHO occurrence of protists were analyzed. These charac TRICHOUS CILIATE STYLONYCHIA LEMNAE teristics of flagellate communities were related with INVOLVED IN TRANSCRIPTION REGULATION salinity, season, trophic status of lakes and abundance A. Pimenov1, I. Skovorodkin1, I. Raykhel1, of other hydrobionts. The and the Metaki B. Schimanski2, D. Ammermann3, A. Gunzl2 netoplastea were mostly common. Wide spread of Hete 1 Institute of Cytology, Russian Academy of Sciences, St. Petersburg, rolobosea was noted in hypersaline environments, that Protistology · 63 indicated their good adaptation to extreme salinity. Fifty that of freshwater P. jenningsi and T. pyriformis, the latter one species and forms of heterotrophic flagellates have may be characterized as some intermediate type. been found. Dominant and rare, stenohaline and eury Differences in two freshwater ciliates may also be due haline species were distinguished. Cosmopolitan taxa to their different origin: P. jenningsi is a rather new Spumella sp., Cafeteria roenbergensis, Bodo designis, natural isolate and the amicronuclear strain GL of T. Paraphysomonas sp., Monosiga ovata, Rhynchomonas pyriformis has been traditionally culturing under stable nasuta were the most common protists. Two species of laboratory conditions. heterotrophic flagellates with wide ranges of salinity tolerance, Cafeteria roenbergensis and Cafeteria POLAR TUBE PROTEINS IN MICROSPORIDIA marsupialis, were registered in both, hypersaline and V. Polonais, F. Delbac, C.P. Vivares brackish lakes. Halophilic flagellates Pleurostomum Equipe Parasitologie Moleculaire et Cellulaire, LBP, UMR CNRS salinum, Pendulomonas sp., Macropharyngomonas aff. 6023, Universite Blaise Pascal, 63177 Aubiere Cedex, France. Email: christian.vivares@univbpclermont.fr halophila were occured only in hypersaline Razval Lake Microsporidia are obligate intracellular parasites for at salinity varying from 236‰ to saturation. These ming environmentally resistant spores. Members of this protists are poorly known; therefore their ecology, phylum possess an invasive apparatus composed of a salinity toleration, appearance and life cycle were highly specialised structure, the polar tube that is unique studied in a uniprotist culture. The SSU rRNA gene in the eukaryotic world and represents one of the most sequencing allowed to refine the systematic position and sophisticated infection mechanisms. The invasion phylogeny of Macropharyngomonas aff. halophila. These process involves the sudden extrusion of the sporal polar extreme halophilic species appeared to fail growing in tube (also called spore germination) for initiating the low salinity conditions. The salinity tolerance thresholds entry of the parasite into a new host cell. Three polar were individual for each flagellate species. Supported tube proteins the socalled PTP1, PTP2 and PTP3 have by RFBR grants 060496920 and 050449870. been identified by our team in the mammal's microspo ridia Encephalitozoon cuniculi. Tandemlyarranged THE ROLE OF HEAT SHOCK PROTEINS IN THE genes encoding the major PTP1 and PTP2 have been PROCESS OF SALINITY ADAPTATIONS IN EURI also reported in the two other Encephalitozoon species HALINE AND FRESHWATER CILIATES as well as in Antonospora locustae and Paranosema grylli, Yu.I. Podlipaeva1, A.O. Smurov2, A.V. Goodkov1 microsporidian parasites of insects. Surprisingly, several 1 2 Institute of Cytology RAS, Zoological Institute RAS, St. Petersburg, genes coding PTP2like proteins are found in the genome Russia. Email: [email protected] The ecological potencies of unicellular organisms might of A. locustae. We also recently identified two new polar be reflected by the initial level of major inducible heat tube components (PTP4 and PTP5) in E. cuniculi and shock protein of 70kDa family (HSP70), as well as by A. locustae. In both species, the genes encoding these the behaviour mode of their chaperone system in toto. new PTPs are similarly clustered on one chromosome Freshwater ciliates Paramecium jenningsi and Tetrahy and present no homology with proteins from databases. mena pyriformis and euryhaline P. nephridiatum The PTP4 in A. locustae is of peculiar interest, since acclimated to various salinity media were studied in this protein seems to be only localized at the end of the these terms. The constitutive level of HSP70 in P. extruded polar tubes, suggesting a potential role in host nephridiatum, acclimated to fresh water (0‰), was cell adherence. Some preliminary experiments to higher than in P. jenningsi from the same medium. In characterize interactions between PTPs during forma the cells of P. nephridiatum, acclimated to 10‰, the tion and functioning of the polar tube have been under HSP70 level was lower than in 0‰. In T. pyriformis taken. from 0, 2 and 10‰ the contents of HSP70 were more or less comparable, but the specters of constitutive HSPs MOLECULAR BIOLOGICAL IDENTIFICATION differed. In P. nephridiatum salinity shock (transfer from OF CULTURED AND NATIVE PROKARYOTIC ENDOCYTOBIONTS OF ARCELLA SPP. 0 to 10 ‰ for 1 h) did not cause the induction of HSP70 1 1 2 1 synthesis, to the contrary, some decrease of the protein B. Pollak , Zs. Heeger , J.K. Torok , K. Marialigeti 1 Eotvos Lorand University, Department of Microbiology, Budapest, content was observed, whereas the reciprocal transfer Hungary, 2 Eotvos Lorand University, Department of Systematic resulted in an increase of HSP70 level. In T. pyriformis Zoology and Ecology, Budapest, Hungary. Email: [email protected] we could not cause the induction of HSP70 synthesis Although in recent years numerous bacterial endosym after shocks in both directions of salinity changes. bionts of freeliving amoebae have been reported and Euryhaline (P. nephridiatum) ciliates appear to be some characterized by molecular methods and many of them how preadapted to abrupt environmental changes by were identified as possible pathogens, the efforts for the highest extent of constitutive HSP70 level and their cultivation mostly failed. In this study, intracellular reactivity of their chaperone system, to compare with bacteria of Arcella spp. were not only detected by fluo 64 · Abstracts of V European Congress of Protistology · 2007 rescence in situ hybridization (FISH), but also suc became more condensed. Unlike starved D. nasutum, cessfully cultivated. Single Arcella cells were rigorously in fed ciliates numerous holes appeared in the fibrillar washed in sterile water and spread on several types of component located at the periphery of nucleoli. These agar plates, designed to imitate the intracellular holes may presumably serve as channels for transporting composition of the host cells. Based on the analysis of newly synthesized rRNA. their 16S rRNA gene, a phylogenetically diverse group was identified, with members belonging to the α and GROWTH OF EUPLOTES VANNUS VAR. BALTICUS βProteobacteria, Flavobacteria, Verrucomicrobia and KAHL, 1932 ON THE MARINE OIL OXIDIZING Firmicutes. Closest relatives of some isolates were earlier YEASTS identified by other researchers as amoebaeresisting L.A. Popova bacteria, such as Candidatus Chryseobacterium massiliae. Institute of Biology of the Southern Seas NAS of Ukraine, Kiev, Ukraine. Some other isolates represented new taxa or possible Email: [email protected] human pathogens. The strains were also characterized Ciliates participate in selfpurification processes in for ecological tolerance, morphological and basic bio marine environment. Flows of pollutants, such as oil, may occur via alimentary chain: oil → oiloxidizing chemical properties. Besides culturebased methods, → direct PCRbased techniques (molecular cloning) were bacteria infusoria. It is well known that infusoria feed also used to achieve a complete view on the bacterial on bacteria, including oiloxidizing ones. At the same spectrum of the Arcella host. The validity of our findings time, we did not find any reported evidences of ciliates' was proved by FISH using specific oligonucleotide feeding on marine oiloxidizing yeasts. To study probes and by electron microscopy. possibility of using marine yeasts for food, the ciliates E. vannus were isolated from the periphyton of the EM AND 3D RECONSTRUCTION OF NUCLEO Artillery Bay seawall (Black Sea) into laboratory LAR APPARATUS IN THE MACRONUCLEUS OF culture. Ten ciliates (cell size 81 µm) were put into Petri THE CILIATE NASUTUM dishes with marine sterile water, to which 5 drops of V.I. Popenko1, B.P. Karajan2, O.G. Leonova1, the marine yeasts culture with initial concentration of Yu.F. Ivlev3, J.L. Ivanova1 13000 cells/ml were added. Necessary concentrations 1 Engelhardt Institute of Molecular Biology, Russian Academy of were obtained by 10, 100 and 1000 times dilution of Sciences, Vavilov str. 32, 119991 Moscow, Russia, 2 Institute of the yeast culture. Numbers of infusoria and yeast cells Cytology, Russian Academy of Sciences, Tikhoretsky av. 4, 194064 St. were counted with JENAVAL X 500. Active uptake of 3 Petersburg, Russia, Severtsov Institute of Ecology and Evolution, yeast cells by infusoria was recorded; they were observed Russian Academy of Sciences, Leninskij prosp. 33, 119071 Moscow, Russia. Email: [email protected] intact inside infusoria bodies and were photographed. Nucleoli are essential structural components of macro These results suggest the existence of yeasts → infusoria nuclei of ciliates. The aim of this work was to follow chain in the process of transformation of the oil pollu fine structural changes in interphase nucleoli of recently tion flow. fed and starved D. nasutum cells by means of three dimensional reconstruction on the basis of serial ultra SIBLING SPECIES CONCEPT IN CILIATES thin sections. To our knowledge, this is the first attempt PARAMECIUM REVISITED: ZOOGEOGRAPHIC to study the nucleolar apparatus in ciliates using 3D AND GENOMIC ASPECTS OF SYNGEN BIOLOGY reconstruction. In both recently fed and starved cells, A.A. Potekhin1, I.V. Nekrasova1, I.I. Skoblo1, two types of nucleoli were observed. Some of them T. Berendonk2, M.S. Rautian1 1 looked like compact round structures less than 1 µm in Biological Research Institute of St. Petersburg State University, St. Petersburg, Russia, 2 Institute of Biology II, University of Leipzig, size (mainly 0.20.6 µm). Another portion of nucleoli Leipzig, Germany. Email: [email protected] was represented by large structures of irregular shape, Importance of two speciation mechanisms, namely, exceding 1 µm in size. On the individual sections the geographic and reproductive isolation, was examined number of small (0.20.6 µm) nucleoli was about 45% on different levels (genetic species syngens strains) of all nucleoli. 3D models showed that in fact most of within Paramecium. Geographic distribution of Para them were parts of large nucleoli. Only a few of them mecium species in Northern hemisphere was investigated made an exception and the total volume of such "free" basing on longterm collecting of paramecia in nature. nucleoli was less than 1.7 % of large nucleoli. Our data While no defined areas could be attributed to sibling showed that large nucleoli, looking on the single species of P. aurelia complex, some P. caudatum syngens sections like individual separate structures, appeared appeared to be geographically isolated, and true areas to be parts of the large complicated branchy nucleolar for some P. bursaria syngens were confirmed. Molecular networks. A 30 h starvation did not lead to disintegration phylogeny of five P. bursaria syngens inferred from of this network, but stimulated formation of numerous mitochondrial cytochrome c oxidase I (COI) sequences vacuoles in the granular component of nucleoli, which indicated that syngens of this morphospecies already Protistology · 65 have evolved separately far enough to be considered ecological functions and to occupy similar niches. Sup young genetic species. In ciliates, which are characte ported by the Project PGIDT01PXI20001PR (Xunta rized by drastic genome rearrangement in each cell de Galicia). cycle, one kind of reproductive barrier may occur due to genomic incompatibility of two conjugating partners. VARIATION OF THE SAPROBIC POLLUTION So, we checked the level of genomic intra and inter DEGREE IN TWO BRACKISH LAGOONS OF THE specific polymorphism of the strains isolated from geo NORTHWEST OF SPAIN (VIXAN AND SAN PEDRO graphically distant populations of Paramecium species. DE MURO LAGOONS), USING CILIATES (PRO Macronuclear genomes of 15 species belonging to P. TOZOA: CILIOPHORA) AS INDICATORS OF aurelia complex were analyzed by pulsedfield gel WATER QUALITY electrophoresis (PFGE). PFGE profile, similar within P. QuintelaAlonso, S. RuizAlvarez, R. AnadonAlvarez complex, still may serve as an "identity card" for most University of Santiago de Compostela, Faculty of Biology, Department of its species, though intraspecific variation of PFGE of Cell Biology and Ecology, Area of Cell Biology, Santiago de profiles was observed within some species, sometimes Compostela, Spain. Email: [email protected] Ciliate communities of two brackish lagoons of the between geographically isolated strains. P. jenningsi, Galician coast (northwest of Spain) were studied from unlike all other Paramecium morphospecies, appeared March 2000 to March 2002, using the Saprobic System to be very similar by PFGE profile to P. aurelia species. to test saprobity variations in each lagoon throughout According to available in literature and own data, we the studied period. Presence or absence of certain suggest that this species belongs to P. aurelia complex. species is a consequence of environmental water Supported by RFBR grants 060449504 and 0704 conditions, related to decomposition of organic 10073. material. This characteristic differs the Saprobic System from other systems based on the "species deficit", related CHARACTERIZATION OF TAXONOMICAL AND exclusively to the total species number in the commu FUNCTIONAL GROUPS OF THE CILIATE COM nity, without caring of the species composition and MUNITIES FROM TWO BRACKISH COASTAL without classifying them according to different degrees LAGOONS OF THE NORTHWEST OF SPAIN: of pollution. Prevalence of βmesosaprobic and α LAGOON OF VIXAN AND LAGOON OF SAN mesosaprobic species in both lagoons indicated a PEDRO DE MURO moderatehigh level of saprobity, which was quantified P. QuintelaAlonso, R. AnadonAlvarez, S. RuizAlvarez University of Santiago de Compostela, Faculty of Biology, Department by the Pantle and Buck method (1955) to determine of Cell Biology and Ecology, Area of Cell Biology, Santiago de the saprobic index. Although San Pedro de Muro lagoon Compostela, Spain. Email: [email protected] had a higher saprobic index than Vixan lagoon during The structure of the ciliate community that develops in the whole study, differences in the saprobic pollution the superficial water of the brackish lagoons of "Vixan" of both lagoons were small. "S" values of the index were and "San Pedro de Muro" (Galicia, Spain) was analyzed recorded inside the αmesosaprobic area, with water between March 2000 and March 2002. Ciliate com quality of class III during most months, according to munities, which include the main freeliving taxo classification of Friedrich (1990). These results show nomical categories of the phylum Ciliophora, inhabit the progressive eutrophication of the ecosystems periphyton of these lagoons. After we have characterized marked by invasive growth of the common reed specific richness of the lagoons, temporal variations, (Phragmites australis), and necessity of control of this and the "resident component" of both lagoons, it was plant in order to conserve the diversity and richness of possible to sort the ciliated community into few groups these environments. Supported by the Project PGIDT0 and to classify the species in each of them according to 1PXI20001PR (Xunta de Galicia). taxonomic, trophic and size (cellular length) criteria. This simplification of the ciliate community allows us THE FLAGELLATE MIXOTRICHA PA to study its structure in a versatile way, and to obtain a RADOXA: ATTACHMENT AND CYST FORMATION concrete ecological model of the community, that OF ECTOBIOTIC SPIROCHETES permits revealing the role and activity of each species R. Radek, G. Nitsch inside the community, as well as its comparison with Free University of Berlin, Germany. Email: [email protected]berlin.de other communities. Taxonomic, trophic and size The symbiotic flagellate (Paraba structure of ciliate communities of both lagoons had salia) in the gut of the termite darwi similar variety and complexity of groups, and it was niensis. Its association with ectobiotic spirochetes and relatively stable throughout the whole study, suggesting bacterial rods is investigated with light and electron that the succession of species in both communities microscopy. Treatment with different chemicals which always took place among species able to carry out similar disturb molecular interactions and the use of the freeze 66 · Abstracts of V European Congress of Protistology · 2007 fracture and freezeetch techniques show that hydro amoebae belonged to thirteen genera: , phobic interactions and integral membrane proteins Naegleria, , , , Platyamo seem to be involved in the firm attachment at the contact eba, Thecamoeba, , Nuclearia, Saccamoeba, sites. There is a complex and regular system of attach Echinamoeba, Guttillinopsis, Acanthamoeba, Paratetra ment sites for spirochetes and bacterial rods. The adhe mitus and Cochliopodium. The most frequently encoun ring and metachronically rowing spirochetes propel the tered was Hartmannella. The highest numbers of FLA cell. Addition of antibiotics to the food of the termites were in May and June the lowest in October. reduces the number of ectobiots and leads to a disinteg ration of the cortical attachment systems. As a result, MOLECULAR PHYLOGENY OF HOLOSPORA Mixotricha becomes spherical and immotile. Most of INTRANUCLEAR SYMBIOTIC BACTERIA OF the cells die after some days. Antibiotics have a further PARAMECIUM SSP. effect: they lead to a transformation of a part of the M.S. Rautian, N. WackerowKouzova spirochetes into cystic bodies. Cyst formation of ecto Laboratory of Protozoan Karyology, Biological Research Institute of biotic spirochetes is reported here for the first time. Star St. Petersburg State University, 198504 St. Petersburg, Russia. vation has a similar, though less dramatic, influence Email: [email protected] than antibiotics. The cysts are rounded (ca. 1 µm) and Bacterial genus Holospora was described by Hafkin contain protoplasmic cylinders in their periphery and (1890) and redescribed by Gromov and Ossipov (1981). sometimes larger central bodies as well. The ectobiotic At present genus Holospora includes 9 species; each of spirochetes of Deltotrichonympha sp. living in the same them is specific to one or another Paramecium spp., termite form similar cysts when antibiotics are applied. and to one of two nuclear types: macronucleus or The production of dormant cystic forms may be a micronucleus. Application of 16S rDNA molecular mechanism of survival under hostile conditions. phylogeny attributed Holospora to Alphaproteobacteria and revealed close relation of two Holospora spp. studied, PRESENCE AND SEASONAL DISTRIBUTION OF namely H. obtusa and H. elegans (Amann et al., 1991). FREELIVING AMOEBAE IN A GROUNDWATER They both together with distantly related intranuclear SYSTEM bacteria of Paramecium Caedibacter caryophilus, and E. Ramirez, E. Robles, R. Ayala, L. Campos some other endobionts of acantamoeba, constitute an UNAM FES Iztacala, Conservation and Environment Improvement early diverged branch (family Holosporaceae) of the Project, Mexico State, Mexico. Email: [email protected] order Rickettsiales (Emelyanov, 2003 and this volume). The presence of eukaryotic microorganisms in These bacteria possess complicated life cycle with groundwater systems has been studied seriously only infectious and reproductive stages. It means that they since recently. The first microbiological investigations are able to be transmitted both vertically and horizon of shallow water table aquifers indicated that prokaryotes tally. The mode of transmission is supposed to influence were the dominant microorganisms present, and that symbiont evolution and coevolution with the host. eukaryotic might be absent altogether. However, Here we investigated 16S rDNA phylogeny inside genus subsequent studies showed the presence of limited Holospora and compared it with Paramecium phylogeny populations of eukaryotes in groundwater systems. So, (StruderKypke et al., 2002). Supported by grants: protozoa were demonstrably present, but in low num RFBR 070401755 and RNP 2.2.3.1.4148. bers. While the presence of eukaryotic microorganisms is low in pristine aquifer, there is evidence that, in PECULIARITY OF THE CLINICAL ISOLATES OF aquifers contaminated by organic matter, the abun TRICHOMONAS VAGINALIS dance of the eukaryotic may be much higher. This is, N. Razdolskaya1, O. Gavrilova2, A. Ivanov1, probably, a result of higher growth rates of bacteria I. Telichko1, A. Krivoruchko1 which support larger populations of bacterivorous pro 1 Military Medical Academy, St. Petersburg, Russia, 2 St. Petersburg tozoa. The aim of the study was to determine presence State University, Dep. Microbiology, St. Petersburg, Russia. and distribution of freeliving amoebae (FLA) in an Email: [email protected] aquifer of Mexico. Groundwater samples from ten wells Human urogenital parasite Trichomonas vaginalis exists of the aquifer of Cuernavaca, located in the center of in different morphological forms: pearlike motile, Mexico, were collected monthly between May 2005 and amoeboid, and round immotile. Biological significance April 2006. Samples were seeded onto nonnutritive of polymorfism is unclear. Two morphotypes (pearlike agar with Enterobacter aerogenes, and freeliving and round) were observed in clinical speciments from amoebae identification was carried out on the basis of patients with different forms of urogenital trichomo morphological features. Freeliving amoebae were niasis. Strains of parasite were isolated and were main found in all the sampled wells of the aquifer. The isolated tainded in TYM Diamonds medium. Morphotype did Protistology · 67 not change in the course of cultivation. By use of trans mission electron microscopy pecularities of the parasite THE MEMBRA ultrastructure were investigated. Round forms possessed NE OF ENCEPHALITOZOON CUNICULI CON 5 flagella, but were not motile, and preserved basic TAINS PORES WHICH SHOULD ALLOW AN features of cell structure. Nucleus, typical for trichomo EXTENSIVE PARTICIPATION OF THE HOST nads, was located anteriorly; pelta and costa extended CELL METABOLITE POOL from kinetosomes. Unlike pearlike forms, in round K. Roennebaeumer, U. Gross, W. Bohne forms reduction of undulating membrane, and Institute of Medical Microbiology, University of Goettingen, Kreuzbergring 57, 37075 Goettingen, Germany. redistribution of hydrogenosomes were observed. Email:[email protected] Round morphotype of T. vaginalis differed from already Microsporida are intracellular organisms of increasing described cysts of other parabasalids by its ultrastruc importance as pathogens in immunocompromised tural organization. On the basis of data on morphology, patients. Since the reduced genome of the model as well as on abilities to reproduce in humans, to induce organism Encephalitozoon cuniculi displays an extreme inflammatory process and to grow on the selective me loss of biosynthetic pathways, we characterized the dium in laboratory conditions, round form of T. vagina amino acid requirements of this pathogen. For accurate lis may be considered as virulent, but not degenerative quantification of microsporidian replication rate, we form. Fagocytosis of different morphotypes were developed a cell ELISA, which is based on combination examined by Confocal Laser Scanning Microscope of two stage specific monoclonal antibodies, allowing Leica TCS SP 5. The role of phagothrophy in pathoge a simultaneous detection of meronts, sporonts and nesis is discussed. Methods of identification of T. vagi spores in infected host cells. Cultivation in the medium nalis morphotypes in clinical samples are proposed. which is depleted in individual amino acids, resulted for most of the lacking amino acids in a strongly reduced INVITRO COMPARATIVE EVALUATION OF FOUR replication rate. This provides experimental evidence PLANT EXTRACTS (KIWI, MULBERRY, POTATO for the lack of a de novo amino acid synthesis in E. AND MILFOIL) ON HUMAN PARASITIC PRO cuniculi and reveals the extreme dependency of this TOZOA organism on the host cell metabolite pool. Since E. M.R. Rezaimanesh cuniculi resides inside a parasitophorous vacuole (PV), Department of Parasitology, Bagiyatallah Medical Science University, nutrients from the host cell need to cross this barrier Teheran, Iran. Email: [email protected] Human parasitic protozoa affecting the gastrointestinal before entering the pathogen itself. Microinjection of tract are the major cause of morbidity, and contribute fluorescent dyes into the cytosol of infected cells showed to mortality worldwide. Therefore suitable control and that a 0.5 kDa molecule could rapidly enter the vacuole, treatment methods with minimum adverse effects, while a 10 kDa molecule was stably excluded from the which do not provoke development of drug resistance, PV lumen. These experiments indicate that the PV are demanded. We tested possibility of application of membrane possess pores with an exclusion size of 10 natural plant extracts as drugs against intestinal parasitic kDa or less, while smaller molecules, e.g. amino acids protozoa. Specimens were obtained from diagnostic can pass. We recently demonstrated that the parasito medical laboratories and were studied using direct phorous vacuole membrane of E. cuniculi lacked host microscopic observation. Hemogenation, filtration and cell membrane proteins immediately after invasion. The supernatant isolation by centrifugation were used to biogenesis of this membrane is thus still unclear and is prepare natural kiwi, potato, mulberry or milfoil currently under investigation in our laboratory. extracts. In the first series of experiments volumes of parasite suspensions and extracts were added in ratio NEW DATA ON HYPERPARASITIC MICROSPO 1:1, and in the second 1:3. The observed decrease in RIDIANS FROM POLYCHAETES OF THE WHITE parasites numbers registered in 24, 48, and 120 hours SEA 1 2 was respectively: for potato extract 40%, 46%, 46%; for Yu. Rotari , G. Paskerova 1 Institute of Ecology of the Volga river basin, Togliatti, Russia, 2 kiwi 40%, 53%, 54%; for mulberry 57%, 57%, 60%; SaintPetersburg State University, SaintPetersburg, Russia. and for milfoil 60%, 98%, 98%. In the second series Email: [email protected] decrease rates for potato, kiwi, mulberry and milfoil Five hyperparasitic microsporidian species, Metchniko were 77%, 62%, 88% and 98%, respectively. Results of vella hovassei (gregarine Lecudina pellucida from poly this study indicate that natural plant and fruit extracts chaete Nereis virens), M. selenidii ( sp. from are useful for control and treatment of protozoan Ophelia limacina), M. polydorae (Selenidium sp. from infections, and suggest that future studies should be Pygospio elegans), Amphiamblys capitellae (Ancora sagit performed in order to investigate effects of natural plant tata from Capitella capitata) and one unknown species extracts on human intestinal protozoa in vivo. (Lecudina sp. from Pygospio elegans), were revealed 68 · Abstracts of V European Congress of Protistology · 2007 during faunistic investigations of the White Sea. Cysts fected with different GFPfused actin constructs is of the unknown microsporidium had the internal light underway. The analysis of the experimental infection retracting and external layer with spiral buldges. Cysts performed under nocodazole treatment demonstrated resided in the parasitophorous vacuole connected with that disassembly of microtubules blocked the entry of the gregarine pellicle from the inside. These characters bacteria into the nucleus, without affecting their are new to microsporidia and give grounds to establish transportation in the cytoplasm. Taken together our a new genus Vivieria (in honor of E. Vivier) with a data suggest that host actin may play an important role species Vivieria spiralis. Because of lack of data on fine in propelling Holospora to the target nucleus at the early structure of cysts and spores, as well as on the life cycle stages of infection, while host microtubules appear to of this species, the systematic position of this genus be required only for its proper sluicing into the nucleus. remains unknown. We observed dividing plasmodia with nuclei coupled in diplokarya during our studies of MORPHOLOGY, INFRACILIATURE AND PHY A. capitellae life cycle. We suggest these plasmodia to LOGENETIC ANALYSIS OF A NEW TINTINNID be a result of merogonial reproduction, though merogony CILIATE (CILIOPHORA, CHOREOTRICHIA) seems not to be obligatory. The host gregarines are FROM THE COASTAL LAKE FARO (SICILY, ITALY) usually very small, and intensive multiplication of para A. Saccа1, M. StruederKypke2, D.H. Lynn2 sites does not seem feasible within such a limited space. 1 Universitа di Messina, Dipartimento di Biologia Animale ed Ecologia A single cyst with 16 falciform spores is regularly formed Marina, Messina, Italia, 2 University of Guelph, Department of at the end of sporogony. When gregarine is big enough Zoology, Guelph, Ontario, Canada. Email: [email protected] A new planktonic tintinnid ciliate of the genus Tintin to hold numerous meronts, merogony takes place. Up nopsis, found in the meromictic coastal Lake Faro to 80 cysts can be produced in this case. The presence (Sicily, Italy), is described from both living cells and of diplokarya in the life cycle of A. capitellae supports quantitative protargolstained preparations. The the view that the genus Amphiamblys belongs to the sequence of the small subunit rRNA (SSU rRNA) gene family Amphiacanthidae (Larsson, 2000). is also reported. Loricae of this species are composed of a subcylindrical bowl, featuring a fine mesh of HOLOSPORA TRAFFICKING IN PARAMECIUM: hexagonal structures, with sparse biogenic matter THE ROLE OF THE HOST CELL CYTOSKELETON adhering on the chitinous basal layer, and of a branched E.V. Sabaneyeva1, K.A. Benken1, M.E. Derkacheva2, aboral horn, characterized by an alveolated texture. The I.N. Skovorodkin2, S.I. Fokin3 lorica length and oral diameter ranges are respectively 1 SaintPetersburg State University, Department of Cytology and Histology, St. Petersburg, Russia, 2 Institute of Cytology, Russian 152205 µm and 3344 µm. Fully extended living cells Academy of Sciences, St. Petersburg, Russia, 3 Biological Research are elongate obconical, 107 to 144 µm in total length Institute of SaintPetersburg State University, St. Petersburg, Russia. and 28 to 38 µm in maximum width. Two ellipsoidal Email: [email protected] macronuclei are always present. The somatic ciliary Bacteria of the genus Holospora are known to be obligate pattern of this species comprises a ventral, a dorsal, and endonucleobionts of paramecia. They belong to αpro a posterior kinety as well as right, left, and lateral ciliary teobacteria and show species and nuclear specificity. fields. Loricae of Tintinnopsis n. sp. are similar to those Holospora obtusa invades the macronucleus and Holo of Tintinnopsis (Paratintinnopsis) corniger (Hada, 1964) spora undulata the micronucleus of Paramecium cau in their overall shape and in the aboral horn texture. datum. Paramecium/Holospora system has lately become Important differences, however, lay in the presence of a model one for investigating various aspects of sym 1215 spiral turns at the oral end and in the scarce bionthost interactions, the way of bacterial transpor agglomeration of foreign particles in Tintinnopsis n. sp., tation in the host cell cytoplasm to the target nucleus the latter character contrasting with the typical traits being among most crucial for the infection. The of the genus. The phylogenetic analysis of this new bacterium gets into the host cell via phagocytosis but species, based on the SSU rRNA gene, shows it to group soon escapes the digestive vacuole and reaches the target with other Tintinnopsis species, but at a basal level in nucleus. The present study sought to find out whether this clade. Supported by Natural and Engineering Holospora exploits the host cell cytoskeleton to get to the nucleus. For this purpose the early stages of Science Research Council of Canada. experimental infection were either subjected to immu GENOTYPE OF OPPORTUNISTIC PROTOZOA IN nocytochemical study with polyclonal antibodies LATESTAGE HIVINFECTED THAI PATIENTS against paramecium actin 11 or stained with rhoda W. Saksirisampant1, J. Prownebon1, M. Mungthin2, minephalloidin. H. obtusa was shown to cause for S. Leelayoova2 mation of actin "comet tails" in the paramecium cyto 1 Department of Parasitology, Faculty of Medicine, Chulalongkorn plasm at the early stages of infection. To get additional University, Bangkok 10330, Thailand, 2 Department of Parasitology, evidence, experimental infection of paramecia trans Phramongkutklao College of Medicine, Ratchatwithi Road, Ratchathewi, Protistology · 69

Bangkok 10400, Thailand. Email: [email protected] morphological criteria are not sufficient to recognize We performed a crosssectional study to determine phylogenetic species. For allogromiids (benthic opportunistic intestinal parastic infections in HIV/ agglutinated forminifers), the genetic variability is also AIDS patients attending Wat Prabaht Nam Phu, a considerably higher than the observed one. However, hospice AIDS care center in Thailand during February in rotaliids (benthic calcitic foraminifers), the situation 2004 to January 2005. Of 90 Thai HIV/AIDS patients seems to be more complicated. Additionally, the hypo with diarrhea, 59 (65.6%) had fullblown AIDS. Only thesis that planktic organisms evolve faster than benthic 20 patients (22.2%) had a history of antiretroviral ones seems to be confirmed by molecular phylogenetic treatment. The mortality rate was 43.3% during one studies performed on foraminifers. year period. The prevalence of intestinal opportunistic parasitic infections was 42.4 %. By nested PCR, THE ACTIN MULTIGENE FAMILY OF PARA Cryptosporidium was the most common (34.4%). For MECIUM TETRAURELIA species specific diagnosis, RFLP comprised C. hominis I.M. Sehring1, C. Reiner1, J. Mansfeld2, E. Wagner3, (27, 87.1%) and C. meleagridis (4, 12.9%). Enterocyto H. Plattner1, R. Kissmehl1 zoon bieneusi genotype D was identified (5, 5.6%) by 1 University of Konstanz, Germany, 2 ETH, Zurich, Switzerland, 3 specific PCR amplification and sequencing of 243 bp University of Munchen, Germany. ITS region of rRNA gene. Enterocytozoon intestinalis Email: Ivonne.Sehring@unikonstanz.de was not detected. ceyatonensis and Paramecium tetraurelia contains an actin multigene belli were each detected in one patient. Multivariate family with at least 30 members encoding actin, actin analysis showed that patients who had watery and/or related and actinlike proteins. They group into twelve mucous diarrhea and having abnormal stool color were subfamilies; a large subfamily with 10 genes, seven pairs independently associated with opportunistic intestinal and one trio with >82% amino acid identity, as well as protozoa infections. three single genes. Analysis of different features on nucleotide and amino acid level (e.g., the number and TO WHAT EXTENT IS THE DICHOTOMY BET position of introns; the actin consensus regions; amino WEEN DYNAMIC PELAGIC AND STABLE BEN acids of the intermonomer interface in filaments, THIC OCEANIC ENVIRONMENTS REFLECTED binding sites for ATP, myosin and actinspecific drugs) IN THE SPECIES RICHNESS AND LONGEVITY revealed striking differences in isoforms of actin and OF DOMINANT FOSSILIZABLE PROTISTS? actinrelated proteins in P. tetraurelia, both within the M. Schweizer, H.R. Thierstein organism and in comparison to other organisms. This ETHZ, Geological Institute, Zurich, Switzerland. diversification suggests unprecedented specification in Email: [email protected] localization and function within a unicellular eukaryote. live in two clearly separated environ To localize the different actin paralogs and to disclose ments: the water column for the plankton and the sea functional implications, we used overexpression as floor for the benthos. The plankton is subject to rapid GFPfusion proteins and antibody labeling, as well as changes and has to adapt quickly to new conditions, gene silencing. Several isoforms are associated, in whereas the benthos lives in a more stable environment different forms, with food vacuoles of different stages. and is thought to evolve more slowly. Among the Some paralogs occur in cilia. A set of actins are found fossilizable marine protists, (Hapto in the cell cortex where actin outlines the regular surface phyta) and foraminifers (Foraminifera) have left a pattern. Labeling of defined structures of the oral cavity rather complete fossil record and have been extensively is due to still other actins, while others are distributed studied by micropaleontologists. Coccolithophores are in a pattern suggesting association with the numerous planktonic, whereas foraminifers are either planktonic Golgi fields. Silencing of the respective actin genes/gene or benthic. In both groups, the shell is the basis of subfamilies entails inhibitory effects on organelles species recognition. Several questions arise with this compatible with localization studies. Knockdown of morphospecies concept: do these paleontological the actin found in the cleavage furrow abolishes cell species correspond to biological species? Are there division, while silencing of other actin genes alters enough criteria on the shell to distinguish species? Are vitality, cell shape and swimming behavior. such criteria distinguishing true species or just different ecophenotypes? Because both groups have still extant WHICH PROTISTS WERE THE ANCESTORS OF representatives today, it is possible to compare the infor METAZOA? mation given by the morphology with the one given by L.N. Seravin1, A.V. Goodkov2 the genetics. Molecular phylogenetic studies show 1 Biological Research Institute of St. Petersburg State University, frequent cases of cryptic speciation in coccolithophores Laboratory of Invertebrate Zoology, St. Petersburg, Russia, 2 Institute and planktic foraminifers, indicating that the used of Cytology RAS, St.Petersburg, Russia. Email: [email protected] 70 · Abstracts of V European Congress of Protistology · 2007

Some researchers consider that the ancestors of the borrori was sequenced and analyzed, and molecular Metazoa were flagellates, some hold that they were trees were constructed. Meanwhile, the cortical ciliates, others nominate for the role of metazoan development during binary division was observed and ancestors amoeboid organisms. The origin of multi compared with other related taxa. Based on the data cellularity is usually explained by nondisjunction of obtained, evolutionary relationships of P. borrori within individuals in the series of subsequent cellular divisions, the class Spirotrichea were determined. The results which occurs in some protists (colonial hypotheses). indicate that this taxon is likely an outgroup to the At the same time, temporary or permanent multi typical hypotrichs (s. l.), while the morphogenetic cellularity is also known to arise in protists by cytotaxis. features suggest it might be an intermediate form In this case, conspecific individuals are attracted to each between hypotrichs and euplotids, yet closer to the other by special attractants secreted by them. In some former. Hence it might represent an ancestor form for protists, e.g. in representatives of the Dictyostelida, both groups. This understanding disagrees with the metazoanlike individuals formed as the result of suggestion of Lynn and Small (2002), that the suborder contact aggregation of separate mixamoebae (pseudo Discocephalina might be a sister group to other typical plasmodia) can move and orient themselves. Analysis euplotids. Basing on the present work we presume the of the available data shows that in some Metazoa, suborder Discocephalina Wicklow, 1982 should be especially those from the phyla Placozoa, Spongia and regarded as a sister clade to other typical hypotrichs, Cnidaria, oocytes and blastomeres of the cleaving i.e. as an order Discocephalida Wicklow, 1982 nov. grad. embryo are capable of amoeboid movement. However, in the subclass Stichotrichia Small and Lynn, 1985. the cells of the newly formed blastula are flagellated. In the course of gastrulation, some of them may lose MULTIPLE TRANSCRIPTION INITIATION SITES undulipodia and revert to amoeboid movement. After IN THE MAXICIRCLE KINETOPLAST DNA OF experimental dissociation, the cells of lower metazoans MONOGENETIC TRYPANOSOMATID LEPTO (Placozoa, Spongia, Cnidaria) start active movement, MONAS SEYMOURI flagellar or amoeboid. They gradually move close E.I. Sharova1, E.A. Bessolitsyna1, E.M. Merzlyak2, together by means of cytotaxis, and contacts aggrega P.N. Flegontov1, A.A. Kolesnikov1 tion occurs; later cells are reassorted and the original 1 Moscow State University, Department of Molecular Biology, Moscow, 2 multicellular individuals are reconstructed. The above Russia, Evrogen JSC, Moscow, Russia. Email: [email protected] facts testify in favour of the hypothesis that the ancestors Transcription of the maxicircle mitochondrial genome of the Metazoa were some amoeboflagellates, now of Trypanosomatidae remains poorly studied: little is known about promoters and termination signals, about extinct. Choanoflagellata, which are incapable of either the number and composition of transcribed RNAs. We aggregative contact behavior or amoeboid movement, constructed a partial transcription map for the maxi can hardly be considered as possible ancestors of the circle of insect trypanosomatid Leptomonas seymouri Metazoa and even of their close relatives. using hybridization, RTPCR, and RACE methods. We identified four polycistronic transcripts (comprising the PRODISCOCEPHALUS: AN EUPLOTID OR STI following genes: 12SrRNA9SrRNAND8, ND7 CHOTRICHOUS CILIATE? PHYLOGENETIC COIIICybA6, COIIMURF2, RPS12ND5) and three ANALYSIS INFERRED FROM ONTOGENETIC monocistronic primary transcripts (ND1, COI, ND4). AND 18S RRNA GENE INFORMATION (CILIO Mature monocistronic transcripts of the ND8, COII, PHORA: SPIROTRICHEA) and MURF2 genes, apparently resulting from cleavage C. Shao1, W. Song1, Z. Chen1, A. Warren2, L. Li1 of the primary transcripts, were also identified. The 1 Laboratory of Protozoology, KLM, Ocean University of China, Qingdao 266003, China, 2 Department of Zoology, Natural History other two polycistronic RNAs remained intact. Museum, Cromwell Road, London SW7 5BD, UK. According to the constructed map some intergenic Email: [email protected] spacers (before the 12S rRNA, ND7, ND1, COII, COI, The Prodiscocephaluslike ciliates which are known as ND4, and RPS12 genes) must contain transcription cephalized hypotrichs or euplotids, and belong to a initiation sites. Those spacers were shown to promote unique and distinct group discocephaline are confused transcription of an artificial construct in isolated considering their phylogenetic position among mitochondria of Leptomonas seymouri (in organello traditional "hypotrichs" (s. l.). The main reasons thereby transcription system) using the following protocol: a are that these organisms exhibit many intermediate fragment comprising the 5'end region of the primary morphological features and most of them are lacking transcript and the upstream spacer was cloned in a ontogenetic as well as molecular investigations. As the plasmid vector, then a linear construct containing this first one in this group, the complete small subunit rRNA fragment flanked by a vector sequence was obtained by gene of a poorlyknown species, Prodiscocephalus PCR and introduced into isolated mitochondria; Protistology · 71 transcription of this construct was assessed by RTPCR DETECTION OF FREELIVING SOIL PROTOZOA with transcript and vectorspecific primers. It was also PRESERVED IN EASTERN ARCTIC PERMA demonstrated that a short sequence (24 bp) upstream FROST of the 12S rRNA transcription start site was essential A.V. Shatilovich, L.A. Shmakova, S.V. Gubin, for transcription initiation in organello, shorter D.A. Gilichinsky sequences being largely ineffective. Thus, maxicircle Institute of Physicochemical and Biological Problems in Soil Science, fragments involved in transcription initiation were Russian Academy of Sciences, Soil Cryology Laboratory, Pushchino, Russia. Email: [email protected] directly mapped for the first time. Viable ancient microorganisms are known to be present within the permafrost. For the first time the viable ANCIENT SOIL CILIATES (PROTOZOA, CILIO protozoa were discovered in frozen layers, and their PHORA) ISOLATED FROM PERMAFROST: MOR ability to survive within permafrost during dozen PHOLOGICAL AND MOLECULAR STUDY 1 2 thousands years was shown. A total of 200 samples of A.V. Shatilovich , L.E. Petrovskaya Eastern Arctic permafrost and buried soils (including 1 Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Soil Cryology Laboratory, Pushchino, burrows of fossil rodent) were screened for presence of Russia, 2 Shemyakin and Ovchinnikov Institute of Bioorganic viable protists. Samples of permafrost sediments and Chemistry RAS, Moscow, Russia. Email: [email protected] buried soils were sterile obtained by sampling from Permafrost is the unique environment which is capable frozen walls of outcrops or from drilling cores down to of protecting microorganisms for longterm preser 25 m depth. The longevity of protozoans cryoconser vation. The purpose of this study is to detect viable forms vation corresponds to the permafrost age. Only 28 of ciliates in permanently frozen sediments of late samples originated from the late Pleistocene and Holo Pleistocene and Holocene age, isolate and characterize cene layers were inhabited by viable representatives of these organisms. A total of 200 samples of Arctic almost all main groups of protists: naked amoebae permafrost, buried soils and burrows of fossil rodent (Lobosea: , , Leptomyxi ranging from several hundred to 3 Myr were investigated dae, and Heterolobosea: ), heterotrophic for viable ciliates. To avoid contamination of samples flagellates (Choanomonada, , Kineto we developed the protocol in which the necessary plastea), and ciliates (Ciliophora: Colpodidae, Platyo requirements concerning temperature conditions and phryidae, ). The oldest isolated micro sterility, were added. Ciliates' resting cysts were re organisms dated back to the ~30 thousand years. activated using the nonflooded Petri dish method Isolated paleoprotozoans were represented by relatively (Foissner, 1987), which allowed us to isolate several small forms (550 µm), and most of them were species common soil ciliates species of the genus Colpoda from of soil microfauna commonly distributed worldwide. horizons differing in age and origin. Species were The life cycle of protists certainly includes a cryptobiotic identified by various silver impregnation techniques stage that characterized by formation of resting cysts. (Foissner, 1997). Thus, four ancient ciliates cultures The general tendency of increasing the number and derived form 30000yearold paleosol were identify as diversity of protozoa within buried soils and burrows of Colpoda steinii (2 cultures), Colpoda inflata and Colpoda fossil rodent was established. This fact may be explained sp., one strain isolated from permafrost samples of late by more favorable conditions of cryopreservation in rich Pleistocene icy complex (depth 7m) as Colpoda sp. plant debris, and originally rich fauna. Correlations and two isolates obtained from Holocene deposits between viable protozoa populations and physico (depth 1.0 and 1.3 m) as Colpoda steinii. Morpho chemical characteristics of sediments (pH, ice content, logical analysis of four clones of C. steinii isolated from chemical or textural composition) were not observed. sediments aging back to 100 30000 years was carried out. Results of analysis of variances (ANOVA) applied DEPENDENCE OF HETEROTROPHIC FLAGEL to morphometric data showed nonsignificant diffe LATES ABUNDANCE ON THE HYDROCHEMICAL rence between isolates. Besides, morphometric PARAMETERS IN DIFFERENT RESERVOIRS IN characteristics of contemporary species were in close THE CENTRAL PART OF UKRAINIAN POLISSIA correspondence to those of ancient species. Phylo AREA genetic analysis based on partial sequencing of 18S S.Yu. Shevchuk rRNA genes (up to 1200 bp) and using the neighbor Zhytomir State University, Zhytomir, Ukraine. joining method revealed a high similarity between Email: [email protected] strains of C. steinii. All of the ciliates, cultures shared It is well known that heterotrophic flagellates are capable 99% of the 18S rRNA gene sequence identity with of surviving in a broad range of abiotic factors. At the previously described C. steinii from Ribosomal Data same time the highest numbers of these protists were Base. detected in waters, which have a lot of organic sub 72 · Abstracts of V European Congress of Protistology · 2007 stances. We studied dependence of heterotrophic the MEXT, Kakenhi (no. 18760431) (grants to S. flagellates abundance on several hydrochemical Shimano); the Austrian Science Foundation (grants to parameters (pH, dissolved oxygen content and organic W. Foissner). substances) by oneway ANOVA. For species Allantion tachyploon Sandon, 1924, Bodo globosus Stein, 1878, VIABLE ANCIENT ACANTHAMOEBAE FROM truncata (Fresenius) Stein, 1887, Phyllo PERMAFROST OF EASTERN SIBERIA: POSSI mitus apiculatus Skuja, 1948 and Rhynchomonas nasuta BLE REASONS FOR HIGH VIABILITY POTEN (Stokes, 1888) Klebs, 1892 the reliable correlation CIES between flagellates abundance and dissolved oxygen L.A. Shmakova1, Yu.I. Podlipaeva2, D.A. Gilichinski1, content was determined, whereas for Ancyromonas A.V. Goodkov2 sigmoides Kent, 1880, Bodo saltans Ehrenberg, 1832, 1 Institute of Physicochemical and Biological Problems in Soil Science, 2 Parabodo nitrophilus Skuja, 1948 and Protaspis simplex RAS, Soil Cryology Laboratory, Pushchino, Russia, Institute of Cytology, RAS, St. Petersburg, Russia. Email: [email protected] Vors, 1992 the influence of concentration of soluble Heat shock protein of HSP70 family was revealed for organic substances was significant. Furthermore, pH the first time in the cells of Acanthamoeba sp. strain Am8 value appreciably affected the abundance of Parabodo (Acanthamoebidae), which were excysted from cysts, nitrophilus Skuja, 1948. isolated from the permafrost samples (Eastern Siberia) 3000035000 years old and then cultivated in laboratory MORPHOLOGY AND GENE SEQUENCE OF AN at 21 °C. High constitutive level of this protein about 60 ENDEMIC, NEW COLEPID (PROTOZOA, CILIO kDa MW was demonstrated by the method of immu PHORA) FROM THE ANCIENT LAKE BIWA, nnoblotting in the unstressed trophozoites. This level JAPAN was considerably higher, than that in unstressed cells of 1 2 3 S. Shimano , W. Foissner , Y. Kusuoka contemporary freshwater amebae from the genus 1 Miyagi University of Education, Environmental Education Center, Aramaki 149, Aoba, Sendai city, Miyagi, 9800845 Japan, 2 Amoeba (Amoebidae). The comparison of HSP70 levels Universitat Salzburg, FB Organismische Biologie, Hellbrunnerstrasse of those in contemporary representatives of the same 34, A5020 Salzburg, Austria, 3 Lake Biwa Museum, Oroshimo 1091, genus (Acanthamoeba sp., strain 4465, collection of the Kusatsu, Shiga 5250001, Japan. Institute of Parasitology, Czech Republic) was carried Email: [email protected]u.ac.jp out. The treatment of blot with the same antibodies did Endemism is difficult to prove in microorganisms. not result in revealing any zones. The data on tempe However, the ancient freshwater lakes (Lake Baikal, rature preferences of acanthamoebae strains show that Lake Tanganyika, Lake Biwa, Lake Ohrid) provide a strain Am8 is much more termotolerant than strain unique opportunity to look for endemic flagship species. 4465. We failed to cause induction of this protein in Indeed, some unique protists have been described from Am8 cells either by heat (37 °C, 1 h), or by cold ( 8 °C, all the lakes cited, but mostly algae, while ciliates have 1 h) shocks, both treatments resulting in decrease of been poorly researched. We investigated some samples constitutive HSP level. Paleoamoebae under study from Lake Biwa for ciliates and found two undescribed belong presumably to an amphizoic species A. polyphaga. flagship species which are likely endemic to the region Levels of HSP in amphizoic and freeliving acanth or even to the Lake. Here, we report on a new colepid amoebae may reflect the species virulence low initial which belongs to a group of ciliates with highly level and noticeable induction in freeliving species and conspicuous cortical scales. We used live observation, high initial level and weak antistress reaction in patho silver impregnation, scanning electron microscopy, and genic ones. Unusually high constitutive level of HSP in molecular biology (SSU rDNA) to characterize the new the cells of paleoacanthamoeba might indicate the high species. Morphologically, the new colepid differs from survival potential, even in terms of geochronology. most other members of the group by the lack of spines near to the anterior and posterior end of the cell. ULTRASTRUCTURAL FEATURES OF SPORO Genetically, it is far away from the common, likely GONY OF SOME MICROSPORIDIA PARASI cosmopolitan Coleps hirtus (U97109) and two Coleps TIZING BLOODSUCKING MOSQUITOES IN sp. (DQ 487194 and X 76646) contained in GenBank. SIBERIA Thus, our ciliate likely represents not only a new species A.V. Simakova but also a new genus. Interestingly, colepids without Tomsk State University, Tomsk, Russia. spines have been described also from Lake Baikal Email: [email protected] (Obolkina, 1995) and Lake Tanganyika (Dragesco, The developmental and ultrastructural features of DragescoKerneis, 1991). Thus, this group of ciliates sporogenesis of species of Amblyospora (Microsporidia) provides strong support for ciliate endemism. Supported infecting Aedes/Ochlerotatus mosquitoes from Siberia by the Japan Society of Protozoologists, the Lake Biwa were investigated. Sporogony and sporogenesis occur Museum Comprehensive Research Project 0602, and within a sporophorous vesicle. The vesicle contains Protistology · 73 numerous secretory granules of different structure that subjacent layer under pellicle with basal bridges are involved in formation of the mature spore. During separating the inside of the folds from the rest of sporogenesis, the episporontal space is cleared and there cytoplasm, so that there used to be neither cell is a correlation between the structure of the secretory organelles nor inclusions. Some other species had an granules and the morphology of the exospore and additional axial electrondense structure in the fold polaroplast of the spore. A thick complex multilayered tops. At last, some species had not the fibrillar bridges exospore is formed in spores from those species that in the bases of the folds, so that some cytoplasmic possess an episporontal space filled with small tubules organelles and inclusions were penetrating into the arranged in large osmiophil lumps, or the space filled folds. In this case, micropores typically located between with finegrained secretions. The polaroplast is the folds, were shifting on the fold sides. High diversity composed of closely packed lamellae, and the polar of the cortex structure in the lecudinids could be filament coils are smooth and frequently arranged in connected with a low level of their evolution: the final several rows. Thin exospores, by contrast, are formed type of the epicyte organization was not yet formed in in those species that possess secretory granules lower gregarines. The differences among the epicyte composed of an accumulation of small filaments structure also might be used as taxonomic markers (fibers), or "taillike" structures on the surface of (generic or specific) in this problem group. envelopes of divided sporonts, sporoblasts and mature spores. Polaroplasts in these species contain closely ORIGIN OF CHROMOSOMES IN PROTISTS packed lamellae and large chambers filled with a S.O. Skarlato medium electron dense substance. Polar filament coils Institute of Cytology RAS, St. Petersburg, Russia. are arranged in a single row and inner membranes of Email: [email protected] the thin coils are stellate and folded on transverse In spite of the remaining uncertainty in the problem of sections. We consider the structure of the secretory origin of the contemporary taxa, there is a strong belief granules of the sporophorous vesicle to be a useful that just within the "primitive" unicellular organisms taxonomic character in these species. Ultrastructural about 2 billion years ago protochromosomes were examination of mature spores further reveals that formed. In different times, various protists were Amblyospora, Parathelohania and Senoma species attributed to the most probable direct descendants of possess polar filaments, which distal ends lie freely in the first eukaryotes on the Earth. At present two rather the spore cavity closer to the posterior pole and end heterogeneous groups can be distinguished among with a broad clublike membranous structure that is not protists in respect to the condensation levels of their connected with the nucleus or posterior vacuole. chromosomes. Representatives of the first group display condensed chromosomes during the whole life cycle. EPICYTE STRUCTURE DIVERSITY IN THE Among these organisms, the best known are dinofla ASEPTATE GREGARINE FAMILY gellates. A substantial attention was paid to the structure T.G. Simdyanov of their chromosomes and the phylogenetic meaning Moscow State University, Dept. of Invertebrate Zoology, Moscow, was attributed to this cell characteristic. However, the Russia. Email: [email protected] further investigations clearly showed the secondary Structures of epicytic folds in 16 species of the lecudinid simplification of these unicellular organisms during gregarines from White Sea and Far Eastern (Sea of evolution. On the contrary, members of the second Japan) polychetes, turbellarians, nemerteans, and group possess weakly condensed chromosomes even in ascidians were compared with SEM and TEM methods. mitosis. For these "invisible" chromosomes a special The high degree of epicytic fold structure diversity was term was coined "weakly condensed chromosomes" revealed in the species observed. The surface morpho (WCCs, Skarlato, 2003). WCCs are common for logy of the trophozoites varied in the degree of fold bodonids, trypanosomatids, diplomonads, trichomo closeness, and in the way of their undulation: if the folds nads, several groups of other freeliving and parasitic were located closely to each other, undulation was flagellates and amoebae, haemosporidia, microspo vertical; and if these were distributed rather rare, ridia, etc. It is tempting to speculate that such a undulation was horizontal. The different species were characteristic as "weak condensation of chromosomes" also different in the shape of the folds on crosssections has a phylogenetic meaning. Most probably the and in the pattern of the fold internal structures. The chromosome apparatus of protists has developed from most common shape of the folds was ampuliform or a prokaryotetype genetic apparatus, undergone evo fingerlike. Such folds had a typical set of internal lution within the ancient Protista (including the stage structures: rippled dense structures (apical arches) and of weakly condensed protochromosomes), and finally apical filaments in their tops without any supplementary achieved the eukaryotic state in contemporary uni formations. There was a welldeveloped fibrillar cellular and multicellular organisms. It is likely that 74 · Abstracts of V European Congress of Protistology · 2007 some of now existing eukaryotic microbes possess lobose amoebae, isolated from all kinds of environment. WCCs that have a number of protochromosome In traditional view it contained five genera Vannella, characters of their protistan ancestors. It can be , Pessonella, and , assumed that it were the WCCs but not the permanently differing in cell surface structure, locomotive morpho condensed chromosomes of Protista (as accepted logy and floating form. Comprehensive phylogenetic earlier) that have served the starting platform for the analysis performed for 57 Vannellidae sequences evolution of genetic system of all eukaryotes. Supported indicated that species of two key genera of this family by grant No. 070400662 from the Russian Foundation Vannella and Platyamoeba, together encompassing for Basic Research. about 85% of the diversity of vannellid amoebae, are completely mixed and do not form distinct clades. MOLECULAR EVOLUTION OF CHLORARACH Several very closely related species pairs exist, each with NIOPHYTE NUCLEOMORPHS a Vannella and a Platyamoeba differing in only a few C.H. Slamovits, P.J. Keeling nucleotides. Therefore, presence (Vannella) or absence University of British Columbia, Botany Department, Vancouver, (Platyamoeba) of glycostyles in the cell surface coat is Canada. Email: [email protected] an invalid generic distinction; we therefore merged the are a relatively small group of two genera (suppressing Platyamoeba) in a forthcoming cercozoan protists that acquired through paper in Protist. Molecular trees revealed a separate secondary endosymbiosis by engulfing a green alga. clade of vannellids with specific and rather short SSU These amoebae are interesting because they are one of gene (ca 1800 bp instead of the usual ca 2000). We the two known groups of algae that have retained a established a new genus Ripella for the only named vestigial nucleus of the endosymbiont, usually called the species of this clade R. platypodia. Sequencing . The nucleomorph of natans molecular clones obtained from the same DNA sample has been recently sequenced and showed an extremely indicated that vannellid amoebae show unusual compact structure. One of the striking features is a high microheterogeneity of SSU sequences in clonal density of very short spliceosomal introns, with a very cultures. Similar data exist for the genus ; narrow size range: 18 to 21 base pairs. Aiming to shed there are a few more examples of such SSU hetero light on why these introns are retained in spite of their geneity. This finding contradicts the paradigm "species extreme size reduction, and how they are removed, we = rRNA sequence" and makes the "molecular ecology" carried out comparative analyses by generating EST and of these naked amoebae based on 18S rRNA sequences genomic sequences from Gymnochlora stellata, a species from total DNA isolated from the environment very from a lineage that diverged from B. natans at the base difficult, if ever possible, because species cannot be of the chlorarachniophyte tree. Our data from about identified by complete sequence identity. During our 50 nucleomorph genes and 130 introns show that intron study of vannellids that included named species and all size is generally similar to B. natans (18 to 21 bp) with a type cultures plus many unnamed and environmental slightly higher representation of 20 bp introns, sequences we could identify none of the environmental consistent with the larger size of G. stellata nucleomorph sequences and found no identical sequences from (ccc versus 373Kbp in B. natans). We also found that different strains or DNA samples, suggesting under intron loss occurred in the B. natans lineages and that sampling of vannellid diversity and/or intraspecific introns larger than 21 bp can be processed by the heterogeneity. Molecular signatures combined with LM spliceosome. We discuss the significance of these morphology of isolates may help sorting out the species findings in the context of the current hypotheses about problem among gymnamoebae, but a study of single the evolution of eukaryotic introns. copy protein gene sequences not prone to microhetero geneity may be essential to identify better molecular PHYLOGENY OF VANNELLID AMOEBAE SSU markers for amoeba clones. Supported by a NERC HETEROGENEITY IN CLONAL CULTURES AND grant to TCS and RFBR grants (030448718 and 06 THE PROBLEM OF "MOLECULAR ECOLOGY" 0449387) to AS. OF GYMNAMOEBAE A.V. Smirnov1, E.S. Nassonova2, E. Chao3, T. Cavalier THE SPECIES DIVERSITY OF THE FRESH Smith3 WATER TESTATE AMOEBAE OF AZERBAIJAN 1 Faculty of Biology and Soil Sciences, St. Petersburg State University, N.Yu. Snegovaya, I.Kh. Alekperov St. Petersburg, Russia, 2 Laboratory of Cytology of Unicellular Institute of Zoology NAS of Azerbaijan. Email: [email protected] Organisms, Institute of Cytology RAS, St. Petersburg, Russia, 3 Department of Zoology, University of Oxford, Oxford, UK. The current study of testate amoebas from inland waters Email: [email protected] of Azerbaijan started in the 1990s. Prior to our research, The family Vannellidae Bovee 1979 contains 40 species only 41 species were recorded from Azerbaijan inland and is one of the largest and commonest groups of naked waters as a result of general hydrobiological investi Protistology · 75 gations in 19301940. Some areas of inland waters rements of electrical resistance in the contact zone were described in those early investigations do not exist at carried out. Besides, experiments with fluorescent dye the present time. In total, 200 species of testate amoebas were performed. Nor the electrical connection, neither have been identified during our survey covering inland transfer of dye from one cell to another was registered, waters of West and NorthernEast Azerbaijan, as well thus we could not reveal existence of any connection as some freshwaters of Apsheron peninsula. The fauna between primite and satellite. The results obtained in of testate amoebas of these regions appeared to be this work suggest that SJs are not the sites of ion and extremely interesting and contained many taxa, new molecule exchange between both cells. We presume that for science. In our opinion, the study of testate amoebas gregarine SJs play a role in recognition and subsequent of inland waters is still far from being completed. We mechanical maintenance of connection between the believe that regions of Nakhichevan and SouthernEast cells of syzygy in the period of time preceding to their of Azerbaijan (Lenkoran) are most interesting for . Some evolutionary aspects of the further faunistic research, because they are considered presence of SJs in the different groups of animals are as centers of Caucasian formation of species, and are discussed. expected to have many endemic species. AMOEBA PROTEUS MYOSIN VIIMMUNO GREGARINE SYZYGY AS A MODEL FOR ANALOG IS INVOLVED IN CELL MIGRATION, MORPHOFUNCTIONAL STUDY OF SEPTATE PINOCYTOSIS AND PHAGOCYTOSIS JUNCTIONS M. Sobczak1, W. Klopocka1, A. Wasik2, Z. Madeja3, E.S. Snigirevskaya, Ya.Yu. Komissarchik M.J. Redowicz1 Institute of Cytology RAS, Membrane Ultrastructure Research, St. 1 Nencki Institute of Experimental Biology, Department of Petersburg, Russia. Email: [email protected] Biochemistry, Warsaw, Poland, 2 Nencki Institute of Experimental An enormous contribution of specialized intercellular Biology, Department of Cell Biology, Warsaw, Poland, 3 Jagiellonian University, Faculty of Biotechnology, Department of Cell Biology, junctions (SIJs) into intercellular communications of Krakow, Poland. Email: [email protected] Metazoa is now established. It is generally accepted that Actin cytoskeleton plays a crucial role in panoply of septate junctions (SJs) are typical for epithelial tissues key cellular functions, such as maintenance of the cell of invertebrates. Always SJs are accompanied by other shape, intracellular trafficking and migration. Myosin SIJs (desmosomes, tight, intermediate, and gap VI is a molecular motor walking to the "" end of actin junctions). This fact causes difficulties in the interpre filaments (opposite direction to other known myosins) tation of functional role of each contact. During the that is involved in intracellular transport, translocation study of Gregarina polymorpha syzygy from Tenebrio and adhesion. Amoeba proteus has been widely used as molitor by electron microscopy we revealed presence of a model to study cell motility, but molecular mechanisms SJs between syzygy individuals: primite and satellite. underlying its actinbasedonly locomotion and The structure of the SJ was analogous to SJ from intracellular trafficking remain poorly understood. We invertebrate tissues. It should be noticed that the detected a 130kDa protein interacting with several gregarine syzygy is one of rare examples of twocellular antibodies against different regions of myosin VI and stage in the life cycle of Protozoa possessing specialized immunoprecipitating with antimyosin VI antibodies. intercellular contacts. Absence of some other SIJs This protein possessed biochemical features characte makes this system very convenient for morpho ristic for myosins, and peptides derived during mass functional study of SJ. Scanning and transmission spectrometry analysis, revealed significant identity to electron microscopy (SEM and TEM) demonstrated different human and invertebrate myosins VI. In the complicated structure of the gregarine pellicle migrating amoebae, myosin VI immunoanalog (mVIi) forming longitudinal ridges on the cell surface. In the localized to membranous vesicular structures, particu preparations for SEM it was well seen that ridges form larly within the perinuclear and subplasma membrane different regions with various directions along the areas. Moreover, mVIi colocalized with dynamin II in longitudinal cell axis. It seems that during sliding regions enriched in actin filaments; both proteins were movements the cell is being turned around. On ultrathin also detected on the same isolated vesicles. In TEM, sections with application of lanthanum chloride during mVIi was found on actin filaments bundles within the fixation, the typical SJs were observed in sites of contact cell. In pinocytotic cells, mVIi concentrated within of primite and satellite ridges. The gap in this region pinocytotic pseudopodia and strongly colocalized with was measured 1314 nm. It was intersected by periodi dynamin, while in phagocytotic cells mVIi localized to cally located septa. Thickness of each septum was 56 phagosomes. Blocking endogenous mVIi with anti nm, the distance between them 10 nm. On the myosin VI antibodies caused changes in cell morpho tangential sections septa looked as parallel zigzag lines. logy, inhibited the rate of cell locomotion, impaired For understanding a functional role of SJs, measu pinocytosis as fewer pinocytotic channels were formed, 76 · Abstracts of V European Congress of Protistology · 2007 and caused severe defects in phagocytosis. These results pterans, T. solenopsae infects all castes and stages of indicate that this novel myosin VI isoform is involved the host. Each of its four developmental sequences is in cell migration, pinocytosis and phagocytosis. specialized to a certain insect caste or stage and plays a particular role in the T. solenopsae life cycle to promote INTERACTION OF PHOSDUCIN WITH THE G maximum success in parasite multiplication and in PROTEIN BETA SUBUNIT IN THE CILIATE vertical, horizontal, intra, and intercolony transmis PROTOZOAN JAPONICUM UPON sion. Four distinctive spore types are produced: ILLUMINATION diplokaryotic spores, which develop only in brood; K. Sobierajska, H. Fabczak, B. Groszynska, S. Fabczak octets of octospores within sporophorous vesicles, the Department of Cell Biology, Nencki Institute of Experimental Biology, most prominent spore type in adults but never occurring Warsaw, Poland. Email: [email protected] in brood; Nosemalike diplokaryotic spores developing Immunological methods (immunoblotting, coimmu in adults; and megaspores, which occur occasionally noprecipitation, immunocytochemistry, confocal in larvae4 and adults of all castes but predominantly microscopy) and high resolution FRET technique have infect gonads of alates and germinate in inseminated been used to study the in vivo interaction of phosducin ovaries of queens. Nosemalike spores function in β β (Pdc) with the subunit of Gprotein (G ) in the autoinfection of adipocytes. Increased proliferation of ciliate Blepharisma japonicum. The dephosphorylation diplokaryotic meronts in some cells is followed by of Pdc observed in response to cell illumination was karyogamy of diplokaria counterparts and meiosis, found to be followed by translocation of Pdc from the thereby switching the diplokaryotic sequence to octo cell cytoplasm to the vicinity of the plasma membrane, spore or megaspore development. Megaspores transmit β where it colocalized with G . Coimmunoprecipitation the pathogen transovarially to the next generation. β analysis showed that Pdc and G proteins that appear From the egg to larvae4, infection is unapparent and colocalized in an immunocytochemical assay, interact can be detected only by PCR. Juvenile and megaspore β with one another in tested cells. Formation of PdcG sequences are abruptly triggered in larvae4, which is complex in cells exposed to light, was evidenced also the key stage in intracolony food distribution via by FRET between these proteins. The FRET efficiency tropholaxis. Larvae4 lack buccal filters, can consume values for lightstimulated ciliates were several times solid food, and participate in horizontal transmission higher, compared to control darkadapted cells. Similar of spores, presumably via cannibalism and/or meco β Pdc dephosphorylation and its colocalization with G nium utilization. Molecular, morphological and life were observed in darkadapted cells pretreated with H cycle data indicate T. solenopsae must be assigned to a 89 or KT5823, kinase G and kinase A activity new genus, work in progress. inhibitors respectively, whereas no changes in these FRET parameters were estimated in ciliates under the EFFECT OF "MICOBACTOVIR", A NEW BROAD same experimental conditions. The results of these SPECTRUM FUNGICIDE, ON ENCEPHALITO β experiments suggest that the observed PdcG inter ZOON CUNICULI REPLICATION IN VITRO action in the ciliate Blepharisma japonicum is deter Y.Y. Sokolova1, V.V. Tetz 2 mined not only by Pdc dephosphorylation upon cell 1 Institute of Cytology Russian Academy of Sciences, Dept. Unicellular illumination, but it is evident that for this molecular Organisms, St.Petersburg, Russia, 2 St. Petersburg Medical University, process the light activation of cell photoreceptor St. Petersburg, Russia. Email: [email protected] followed by Gα dissociation from Gprotein (Gαβγ) is "Micobactovir" (MBV), a newly synthesized derivate necessary. Supported by grants 2P 04C 014 27 and 2P of arilidenine13pyrimidine, suppressed growth of 04C 013 30 from the Ministry of Science and Higher multicellular fungi and yeasts in vivo and in vitro; was Education. effective against gram+ and gram bacteria; and inhibited reproduction of Herpes virus. MBV is con SOCIAL PARASITISM IN MICROSPORIDIA: sidered as a drug for systemic and topical use, effective THELOHANIA SOLENOPSAE DEVELOPMENT IN against fungal, fungibacterial and fungiviral infec COLONIES OF THE FIRE ANT, SOLENOPSIS tions. The goal of this study was to assess effect of MBV INVICTA on Encephalitozoon cuniculi and E. intestinalis in cultu Y.Y. Sokolova, J.R. Fuxa red rabbit kidney cells (RK13). E. cuniculi or E. intesti Louisiana State University, Dept. Entomology, Baton Rouge, LA, USA. nalis spores were added to confluent monolayers of RK Email: [email protected] 13 cultivated in 6 and 24well plates. Simultaneously Thelohania solenopsae is a unique microsporidium 1 mg/ml water solution of MBV was added to test the whith a life cycle finely tuned to parasitizing fire ant concentrations 500, 250, 125, 61, 30 and 15 µg/ml. colonies. Unlike other microsporidia of social hymeno Statistically significant cytotoxicity against RK13 cells Protistology · 77 was observed at 500 and 250 µg/ml. Visual observation CYTOPLASMIC MICROTUBULES ARE REQUI of monolayers did not show any differences in mor RED FOR EGESTION OF A FOOD VACUOLE, phology of parasitophorous vacuoles (PVs), or timing WHILE ACTIN FILAMENTS ARE INVOLVED IN of spore maturation between treated and untreated MEMBRANE RECYCLING IN A CYTOPROCT IN monolayers. No statistically significant variations were TETRAHYMENA THERMOPHILA registered in number of PVs per cell, or PV volume, or M. Sugita, K. Nakano, O. Numata number of host cells with PVs. Incorporation of BrdU, Biological Sciences, Graduate School of Life and Environmental followed by application of antiBrdU antibodies Sciences, University of Tsukuba, Ibaraki 3058572, Japan. Email: [email protected] conjugated with Alexa488, was observed in treated and Ciliates including Tetrahymena thermophila take untreated parasites, suggesting the parasite replication nutrient through a food vacuole (FV). Undigested was not blocked by the drug. Nontoxic concentrations contents in an FV are egested through membrane fusion of MBV that suppressed the parasites proliferation by in a cytoproct. This cellular event is attractive for 4080%, were 60125 µg/ml. For comparison, Alben studying control of membrane dynamics via cytoskele dazole and Fumagillin caused 100% inhibition at 0.01 tons. We prepared antiserum against one of myosins in and 0.1 µg/ml correspondingly. Microsporidia occurred T. thermophila, Myo13, and found that it specifically to be more resistant to MBV than Candida spp., inhi labeled a cytoproct. Interestingly, it was found that bited by 1 µg/ml. Supported by RFBR grant 0304 localization pattern of Myo13 in a cytoproct was 49629. changed from a linear shape to a ring by a treatment of cells with an inhibitor for actin polymerization, CILIATE DIVERSITY, ADAPTATIONS, AND Latrunculin B. Moreover, SEM observation revealed NICHE OCCUPATION IN TWO CONTRASTING that the cytoproct was dramatically opened, and that LAKES LOCATED AT DIFFERENT ALTITUDES membrane was protruded from there in these cells. B. Sonntag, M. Summerer, B. Steurer, R. Sommaruga Optical microscopic video images showed that the Lab. of Aquatic Photobiology & Plankton Ecology, Institute of Ecology, University of Innsbruck, Austria. Email: [email protected] protrusion was probably derived from egested FVs. In Piburger See (913 m a.s.l.), the planktonic ciliate Inhibition of actin polymerization is likely to suppress assemblage was highly diverse with >100 species found a membrane recycling of an FV in a cytoproct. Immu over the course of the year. The succession of the species nofluorescence microscopy revealed that strong was closely linked to changes in abiotic parameters, e.g., accumulation of actin surrounded an FV when it was oxygen concentration. For example, along the water egested. However, undigested contents in an old FV column, we found common euplanktonic ciliates (e.g., were defecated even in the absence of Factin. prostomatids, oligotrichs) in the upper meters and more Therefore, the actin cytoskeleton seems to be required anaerobic species (e.g., heterotrichs) in the deepest for membrane recycling but not for egestion of an FV. zone. In summer, we found around 20 Chlorellabearing On the other hand, cytoplasmic microtubules localized species that dominated in the upper meters during as dense network surrounding FVs. Moreover, egestion highest levels of ambient solar radiation. In several of of an FV was rarely seen in cells treated with microtu those , we identified UVabsorbing myco bule depolymerizing reagent, nocodazole. It seemed sporinelike amino acids (MAAs). Moreover, we that cytoplasmic microtubules were required for the identified two possible sources of the compounds: the egestion of old FVs in T. thermophila. Chlorellasymbionts and the ciliates' diet. In the highly UVtransparent alpine lake Gossenkollesee (2,417 m CHLORELLABEARING CILIATES AND THEIR a.s.l.), we found by contrast only three predominant UVPROTECTION STRATEGIES species during the icefree period with only one bearing M. Summerer, B. Sonntag, R. Sommaruga Chlorella, i.e., Askenasia chlorelligera. In this species, University of Innsbruck, Institute of Ecology, Laboratory of Aquatic Photobiology and Plankton Ecology, Innsbruck, Austria. Email: we observed higher concentrations of MAAs per cell [email protected] than in the mixotrophs from Piburger See. Thus, we Mutualistic associations between ciliates and unicellular argue that the accumulation of MAAs in Chlorella green algae of the genus Chlorella is a common bearing ciliates represents an additional benefit in this phenomenon. We investigated Chlorellabearing ciliates symbiotic partnership and an adaptation to elevated in terms of their strategies to resist the negative effects UVradiation. In Gossenkollesee, the heterotrophic of UV radiation. Therefore, we carried out UV prostomatid Balanion planctonicum numerically experiments, which demonstrated a much higher UV dominated over the other species and occupied the tolerance of symbiotic P. bursaria compared to their deepest water layers. This distribution pattern was in aposymbiotic counterpart. HPLCanalyses revealed the agreement their sensitivity to UVB radiation. presence of colourless UVabsorbing mycosporinelike 78 · Abstracts of V European Congress of Protistology · 2007 amino acids (MAAs) in several Chlorellabearing ciliate different stages. This may explain why T. vaginalis species, but not in P. bursaria. To test, if Chlorella contain the largest number of genes among all symbionts of the investigated ciliate taxa were unrelated unicellular organisms. The protein expression levels of to each other, we sequenced part of the nuclear 18S all the identified hydrogenosomal proteins were also rDNA, the ITS1 region and the partial chloroplast 16S associated with the gene expression level by using the rDNA of the algae, and detected a pronounced T. vaginalis expressed sequence tag database to study symbiont variability within the investigated host species, the turnover rate of mRNA and proteins. The newly but also indications for species specificity. An ultra identified proteins are being integrated into existing structural characterisation of the P. bursaria symbiosis pathways to decipherate the hydrogenosomal inter and an examination of the distribution of the symbionts actome. within the host cell revealed distinct distribution patterns under different UV and light intensities. Results INTRASPECIFIC DIFFERENTIATION WITHIN from UVexperiments and HPLCanalyses of sun PARAMECIUM NOVAURELIA (CILIOPHORA, screencompounds in combination with the morpho OLIGOHYMENOPHOREA) USING RDNA AND logical analyses suggest at least two different UV MTDNA ANALYSIS protection strategies of the ciliateChlorella symbiosis: S. Tarcz (1) an internal (self)shading of sensitive cell targets (ie. Institute of Systematics and Evolution of Animals, Polish Academy of the nuclei) provided by a specific distribution of the Sciences, Ul. Slawkowska 17, 31016 Krakow, Poland. Email: [email protected] symbionts, depending on the incident radiation as Paramecium novaurelia is one of 15 genetic species of investigated in Paramecium bursaria, and in addition, Paramecium aurelia species complex, characterized by (2) protection through MAAs in other Chlorella moderate inbreeding. Differentiation within Parame bearing ciliate species. cium novaurelia was previously studied by strain crosses and molecular methods like RAPDPCR (Random COMPARATIVE PROTEOMICS OF TRICHOMO Amplified Polymophic DNA). P. novaurelia was NAS VAGINALIS TROPHOZOITE AND PSEUDO regarded to be restricted to Europe, but later the domi CYST nant species was also recorded in Asia (Turkey, Anato P. Tang1, K.Y. Chien2, J.W. Shin3 lian Upland). The first American strain of P. novaurelia 1 Bioinformatics Center, Chang Gung University, Taoyuan, Taiwan, 2 Proteomics Center, Chang Gung University, Taoyuan, Taiwan, 3 was recently identified in a sample collected in Boston, Dept. of Parasitology, College of Medicine, National Cheng Kung USA. We present molecular comparisons of 25 P. University, Tainan, Taiwan. Email: [email protected] novaurelia strains, including the American strain and Hydrogenosomes are mitochondrion organelles which the strain from Asia. The intraspecific differentiation contain hydrogenase and produce hydrogen and ATP of P. novaurelia was studied using sequencing of two by glycolysis. These organelles have been identified in DNA fragments, i.e. a fragment of rDNA: 18SITS1 unicellular eukaryotes such as Trichomonas vaginalis, 5.8SITS228S (1200 bp) and a fragment of mtDNA Tritrichomonas foetus, Neocallimastix and Nyctotherus CO I (660bp). Obtained results were analyzed by NJ, ovalis. The present study elucidated the complete MP, ML and Bayesian analysis. Intraspecific differen hydrogenosome proteome of T. vaginalis by using 2 tiation within P. novaurelia discriminates four groups dimensional gel electrophoresis (2DGE) and strong of the studied strains with different genotypes. P. cation exchange liquidchromatography coupled with caudatum strain was used as an outgroup. Geographical a mass detection system (SCXLC/MS). A total of 121 correlation between genotypes was not observed. The unique proteins were identified by both techniques. distance among P. novaurelia strains in ribosomal However, only 37 of the identified proteins were also fragment was at the level of 2.4%, and COI mtDNA predicted by The Institute of Genome Research fragment revealed much higher divergence equal to (TIGR) from the recently published T. vaginalis 24.8%. genome. The remaining 84 proteins were newly identified hydrogenosomal proteins. We also compared MICROZOOPLANKTON DIVERSITY IN THE the hydrogenosome proteome of the trophozoite and BALTIC COASTAL WATERS pseudocyst stages. Fructose1,6bisphosphate aldolase I.V. Telesh (F1BP), Glyceraldehyde3phosphate dehydrogenase Zoological Institute, Russian Academy of Sciences, St. Petersburg, (G3PD), heat shock protein 70 (HSP70) and thiol Russia. Email: [email protected] peroxidase (Tpx) were expressed at a higher level in Baltic coastal waters are characterized by a variety of pseudocyst. One interesting observation is the expres ecosystems from large exposed shallow gulfs and sion of specific isoforms within a protein family at estuaries to lagoons and fjords. This variability of Protistology · 79 biotopes along with other specific external abiotic STRUCTURE OF FREELIVING HETEROTRO factors provides high biological diversity of mainly PHIC FLAGELLATE COMMUNITIES IN DIFFE freshwater and brackish aquatic flora and fauna. The RENT TYPES OF FRESHWATER AND MARINE ongoing eutrophication of the Baltic coastal ecosys ECOSYSTEMS tems influences the species diversity, population D.V. Tikhonenkov1, Yu.A. Mazei2 structure and of aquatic communities 1 Institute for Biology of Inland Waters RAS, Borok, Russia, 2 Penza which can serve as indicators of ecosystem alterations. State Pedagogical University, Penza, Russia. Email: [email protected] Results of longterm investigations of zooplankton in Species diversity, cell morphology, community structure the Neva Estuary, one of the largest urbanized coastal and distribution of freeliving heterotrophic flagellate ecosystems of the Baltic Sea, DarssZingst Bodden were investigated. Material from different freshwater Chain and other Baltic coastal areas revealed the and marine biotopes was collected during 20022006. necessity of a profound research into biodiversity of Samples from several microbiotopes with different pelagic communities and the need of a regional substratum were taken within each of the studied illustrated zooplankton guide with a revised species ecosystems. 304 species and forms of heterotrophic checklist. The paper provides data on the zooplankton flagellates from all known higher level groups of diversity in the Baltic coastal waters, with special eukaryotes were observed. The main factors of emphasis on planktonic protists and rotifers, in relation community differentiation in different types of to the problems of biodiversity loss, biological invasions, ecosystems were revealed. Heterotrophic flagellate trophic interactions in the pelagic communities, and communities are highly heterogenous systems; com ecosystem functioning. Principles and mechanisms munity differentiation does not depend on geographic regulating the microzooplankton diversity in estuaries remoteness of biotopes; species distribution is caused are discussed. Supported by the Program "Biodiversity" by local factors, such as type of microbiotope, hydro and RFBR grants 040449207, 050490588. chemical and trophic conditions. The most complex communities with specific set of species are formed in CILIATES: A BIOTECHNOLOGICAL APPROACH biotopes rich in organic matter with reduced (or acidic) TO REDUCE ANTIBIOTICRESISTANT BACTE medium, and are characterized by the highest values RIA FROM THE OUTLET OF WASTEWATER of integral community characteristics. Eurybiont TREATMENT PLANTS species with a great tolerance range to environmental A. Tiedtke factors define major patterns of heterotrophic flagellate Institute for General Zoology & Genetics, WWU Munster, Germany. communities. Ratio of alpha vs. beta components of Email: tiedtke@unimuenster.de species diversity depends on the scale of investigation. Sewage treatment plants, especially the biological At the microscale (partial ecosystems elements) species purification steps, are hotspots for antibioticresistant richness of heterotrophic flagellates is determined bacteria, which are released in relatively high numbers mainly by ability of community to differentiation due into the receiving waters. The further entry of antibiotic to intercommunity mechanisms. At the meso and resistances into the environment a major threat to macroscales (ecosystems and regions) species richness human health should be prevented. We developed depends mainly on peculiarities of the ecosystem, the therefore a novel approach to reduce the cellnumbers region type and evolutionary history of taxonomic of suspended bacteria by filterfeeding ciliates, the groups. natural predators of bacteria. The capability of two ciliates Tetrahymena pyriformis wt and Colpidium DIAGNOSIS OF MICROSPORIDIAN INFECTI campylum to decrease three representative bacteria ONS IN ARTHROPOD HOSTS species (Escherichia coli, Bacillus subtilis and Pseudo Yu.S. Tokarev1, Yu.M. Malysh1, N.V. Munteanu2, monas putida) was analysed at five different temperatu Yu.M. Fefelova1, I.V. Senderskii1 res in a 100 mlscale under batch conditions over 8 h. 1 AllRussian Institute for Plant Protection, St. PetersburgPushkin, These results stimulated upscaling of the experiments Russia, 2 Institute of Zoology, Chisinau, Moldova. to volumes of 2 l and 25 l. In addition, the time period Email: [email protected] was increased to 100 h under continuous conditions. Various methods were exploited to reveal micro Both, T. pyriformis wt and C. campylum have the sporidian infection in a broad range of arthropod hosts: potential to reduce efficiently and economically the crickets and locusts (Tokarev et al., 2005, 2007), beet number of suspended bacteria released into the webworm (Malysh et al., 2006); hard ticks (Tokarev, receiving waters. Movila, 2004; Tokarev et al., in press), leafrolling 80 · Abstracts of V European Congress of Protistology · 2007 weevils, cotton bollworm, flour beetles etc. For mass ding to 16S rRNA gene sequence results. Endocyto scale screening of alive and dead material we found biont nature, morphology and localization of symbiotic staining with DAPI the most useful technique. This bacteria were confirmed by transmission electron DNAspecific dye is known also to stain membranes microscopy, which results were compared with the (Favilla et al., 1993) and proteins (Mazzini et al., 1994). published data. In Nosemalike microsporidian spores, DAPI inten sively stains diplokaryon and feebly stains exospore and FIRST EVIDENCE ON BACTERIAL ENDOCYTO cytoplasm (allowing to counterstain the endospore), BIONTS IN THE TESTATE AMOEBAE OF THE extruded polar filament and other intracellular GENUS ARCELLA structures. This "nonspecificity" prominently helped J.K. Torok1, B. Pollak2, Zs. Heeger2, Gy. Csikos3, to discriminate even single spores found on smears, K. Marialigeti2 especially when diplokaryon staining was unclear. In 1 Eotvos Lorand University, Department of Systematic Zoology and lepidopterans, invaded with Thelohanialike spores, Ecology, Budapest, Hungary, 2 Eotvos Lorand University, Department of Microbiology, Budapest, Hungary, 3 Eotvos Lorand University, DAPI did not stain nuclear apparatus of spores and Department of Anatomy, Cell and Developmental Biology, Budapest, bound only to the exospore. The usability of DAPI for Hungary. Email: [email protected] detection of single Nosemalike spores of microsporidia While prokaryotic symbionts in ciliates are extensively on smears makes this method more helpful as compared searched out, medically harmless species of free living to PCR with SSU rDNA primers, as the threshold amoebae have been widely neglected in symbiont sensitivity of the latter is reported to be 100 spores ml studies. The present study gives the first evidence on 1 for human microsporidia (Weiss, Vossbrink, 1999) and bacterial endocytobionts in the genus Arcella. Clonal even 1000 spores ml1 for insect microsporidia cultures of variety of Arcella species were set up and (Sokolova et al., 2004). On the other hand, PCR detec investigated for presence of endocytobionts with bacte tion of microsporidia was a reliable tool to localize latent rial rDNA sequencing, fluorescent in situ hybridization infection in locusts when other methods were not helpful. (FISH), and transmission electron microscopy. Arcella Supported by RFBR (040349629, 060490814) and species were kept axenically for eukaryotes and fed with by a grant from President of Russian Federation for Enterobacter aerogenes. Rich diversity of eubacterial Y.S.T. (МК653.2007.4). sequences, like Acidovorax and Verrucomicrobium among others, have been identified by PCR either by LIGHT AND ELECTRON MICROSCOPY STUDIES direct isolation of rDNA from the testacean cells or by ON MORPHOLOGICAL POLYMORPHISM IN culturing individual Arcella cells on different types of CLONAL CULTURES OF ARCELLA SPECIES media. FISH with eubacterial probes demonstrated WITH SPECIAL REGARD TO THEIR ENDO presence of single bacterial cells scattered throughout CYTOBIONTS the cytoplasm, clearly different from those clumped 1 2 3 3 J.K. Torok , Gy. Csikos , B. Pollak , Zs. Heeger , together in food vacuoles. First transmission electron 3 K. Marialigeti microscopical surveys revealed single rodlike bacteria 1 Eotvos Lorand University, Department of Systematic Zoology and located in different parts of the cytoplasm. In symbiont Ecology, Budapest, Hungary, 2 Eotvos Lorand University, Department of Anatomy, Cell and Developmental Biology, Budapest, Hungary, 3 bearing Arcella clones, lysis of host cells was not Eotvos Lorand University, Department of Microbiology, Budapest, detected; instead cells in older cultures occasionally Hungary. Email: [email protected] started cyst formation. This phenomenon might To date our understanding of the size polymorphism indicate that so far detected endocytobionts are not range in testate amoebae is very limited. Clonal cultures harmful to host cells, but their beneficial role is still to of various Arcella species were established in order to be proved. Bacteria from the same clone appeared to study both inter and intraspecific polymorphism. A belong to phylogenetically distant groups, some were further aim of the study was to find bacterial endo related to symbionts of other eukaryotic organisms, symbionts in this important lobose testate amoeba genus others to human pathogens, suggesting a potential role and investigate their potential impact on the clonal of arcellas as natural reservoirs. growth capacity. We present the morphological and biometric characterization of active and encysted stages PATTERNS OF SPATIAL AND TEMPORAL VARIA of the cultivated Arcella species completed with the BILITY OF TESTATE AMOEBAE COMMUNITY IN growth rates in different culture conditions. We SPHAGNUM BOG demonstrate the first findings on different symbiotic A. Tsyganov1, Y. Mazei2 bacteria in the testate amoeba revealed by fluorescent 1 Moscow State University, Department of Hydrobiology, Moscow, in situ hybridization (FISH) applying general both, Russia, 2 Penza State Pedagogical University, Department of Zoology, eubacteria and more specific probes, designed accor Penza, Russia. Email: andrei[email protected] Protistology · 81

Testate amoebae are common and important compo Features of a structure of nucleus and changes of their nent of Sphagnumdominated ecosystems and are structure are found out during division and formation increasingly used in peatland monitoring and palaeo of myxospore. For a long period of time two factors have ecological research. However data on the spatial and impeded investigations of myxosporidia cytology: the seasonal structure of communities are completely small size of their nuclei and the absence of data about lacking. This is an important aspect since quantitative existence of condensed chromosomes in these orga models used for paleoecological reconstruction and nisms. Significant progress in biological research monitoring are based on species assemblages. We methods provides new approaches to investigations of investigated spatial and temporal distribution patterns Myxozoa chromatin and chromosomal apparatus. In of testate amoebae in sphagnum sward of the raised bog this work for the first time the chromosomes in Bezimyanoe. The study revealed 63 taxa belonging to generative cell nuclei of Zschokkela nova was found out 21 genera. Analysis of horizontal microdistribution and described using confocal scanning microscopy. It patterns in scales from 1 cm to 2 m showed that species was shown that its karyotype consists of six chromo formed feebly marked aggregations with unclear somes (three pairs). Two pairs of chromosomes have bounds. These aggregations probably resembled more the oblong rodshaped form (metacentric chromo or less expressed patches of different sizes smoothly somes). Third pair of chromosomes has the boomerangs passing from one to another, than a distinct spatially form (submetacentric chromosomes). Lengths of chro constrained group. Size of these patches was species mosomes: 5 microns (1 pair), 4.8 microns (2 pair) and specific and in some cases (Assulina muscorum and A. 3 microns (3 pair). seminulum) positively correlated with the amoebae shell sizes. Vertical distribution pattern of testate amoebae A GENOME SURVEY OF ENTEROCYTOZOON assemblages depended on moisture of the biotope. BIENEUSI Communities in drier conditions were the most hetero S. Tzipori geneous. In upper sphagnum layer (03 cm) species Tufts University Cummings School of Veterinary Medicine, North number and species diversity were minimal, whereas Grafton 01536, USA. Email: [email protected] abundance was maximal. The opposite tendencies in Enterocytozoon bieneusi is clinically the most significant distribution of species Hyalosphenia papilioH. elegans pathogen among the Microsporidia that infect humans. were marked, that reflected separation of the ecological Until recently, investigations on E. bieneusi have been niches along the sphagnum stem. From May to Sep hindered by the inability to propagate these organisms tember, it was revealed that species number increased, in the laboratory. Recent developments, which include while species diversity and evenness remained at the methods for spore purification and animal propagation, same level with insignificant fluctuations. During this have made it possible to undertake a genomic survey of period, species abundance in different assemblages of E. bieneusi. E. bieneusi spores were purified and testate amoebae could increase, decrease, or vary concentrated from the stool of a human patient. without welldefined or directed tendencies. Genomic DNA was extracted and a small insert (12 kb) plasmid library was constructed. The first draft CYTOLOGY AND MORPHOLOGY OF MYXO assembly of the genome consists of 1,743 contigs, SPORIDIA (MYXOZOA) ranging in size up to 131,000 bases, covering ~3.8 Mb P.Yu. Tyutyaev of unique sequence. This represents ~55% of the Institute of Cytology RAS, St. Petersburg, Russia. genome, which is estimated to be 68 Mb by pulsed field Email: [email protected] gel electrophoresis. Preliminary analyses of the genomic The analysis of organic and nonorganic substances in sequence suggest a high AT content (>65%) and an the valves of long stored in water Heneguya oviperda absence of introns. Approximately 2,300 open reading (Соhn, 1895) and Myxobolus pseudodispar (Gorbunova, frames (ORFs) have been identified in E. bieneusi. 40% 1936) myxospores has been made using the cytoche of the E. bieneusi ORFs have been assigned a putative mical methods and atomicabsorptional spectroscopy. function based on homology to known genes. Of these, The high content of Si (up to 94%) and the presence of 70% were homologues to genes identified in Encepha Ca, Mg, Fe, Mn cations (Cu, Al, Ni, Cr in fewer litozoon cuniculi. Assignment of identified proteins to amounts) have been detected. The investigation of pathways and syntenic analyses with the genomes of E. plasmodium and spore nuclei, factin, α and γtubulin cuniculi and Antonospora locustae are underway. In of three Myxosporidia species: Zschokkela nova addition, the putative E. bieneusi methionine amino (Klokacewa 1914), Myxidium gasterostei (Noble 1943) peptidase2 and polar tube proteins have been identified and Myxobolus pseudodispar, was performed using and polyclonal antibodies against the polar tube proteins confocal microscopy and indirect cytofluorimetry. have been produced. 82 · Abstracts of V European Congress of Protistology · 2007

APPLICATION OF POLYMERASE CHAIN REAC stains as well as of immunological reactions (direct and TION IN THE DIAGNOSTICS OF AVIAN CRYPTO indirect fluorescence, latex agglutination reaction, the SPORIDIOSIS immune enzymatic method). At the present time, the A.V. Uyezdina1, V.V. Dyomkin2, V.T. Zablotsky Laboratory of Protozoology in cooperation with the 1 Protozoology Laboratory, Russian Research Institute of Experimental Laboratory of Molecular Diagnostics, Institute of 2 Veterinary Science, Moscow, Russia, Laboratory of Molecular Molecular Genetics, RAS, has developed a highly sen Diagnostics, Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia. Email: [email protected] sitive testing system of PCR for detection of cryptospo Studies of the animal disease cryptosporidiosis induced ridia in avian genetic material. The assessment of results by the Cryptosporidiidae family of protozoa and acquired during our examination of an ailing bird's manifested in gastrointestinal, respiratory and immune pathological material by several methods showed that system diseases, have been for long under way all over microscopic testing for kryptospirosis of 363 samples the world. In Russia, the pathogenic agent of crypto taken from 10 to 40 daysold birds revealed 7.4% sporadiosis was for the first time revealed in 25year old positive results, microscopy with flotation produced calves (Nikitin, Pavlasek, 1983), and at present crypto 38% positive cases and the PCR testing system gave sporidia are treated as harmful pathogens for not only 73% positive results. Microscopy methods with or animals but for humans as well. This invasion started without flotation, widely applied at present for the being frequently diagnosed in acquired immune kryptosporidiosis diagnostics, have proved to be deficiency patients. According to the data reported by insufficiently effective. In our opinion, polymerase the WHO, over 500 thousand persons annually caught chain reaction (PCR) seems more promising for the disease in the 1990s, with 2 to 3% of them suffering detection of Cryptosporidium baileyi. from it chronically. Cryptosporidiosis in AIDSaffected patients is manifested by profuse dropsy diarrhea and COMPARATIVE EXTERNAL MORPHOLOGY OF is regarded as a serious complication. Under the disguise DEVELOPMENTAL STAGES OF GASTRIC CRYP of various contagious diseases, cryptosporidiosis makes TOSPORIDIUM SPP. FROM ENDOTHERMIC it difficult to carry out timely diagnostics and adequate AND POIKILOTHERMIC HOSTS therapeutic and preventive treatments. Cryptospori A. Valigurova1, M. Jirku2, B. Koudela2, M. Gelnar1 diosis in birds has been less sufficiently described than 1 Department of Botany and Zoology, Faculty of Science, Masaryk in mammals. It is exhibited in a number of clinical University, Kotlarska 2, 611 37 Brno, Czech Republic, 2 Institute of Parasitology, Biology Centre of the Academy of Science of the Czech manifestations, e.g. diarrhea, suppression, depression Republic, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic. and respiratory conditions such as cough, nasal running Email: [email protected] and huskiness. Clinicall manifestations of the disease Cryptosporidium is a vertebrate pathogen that has gained are dependent on a number of factors, with the bird's much attention in the last years due to its phylogenetic kind, age and immune status being the most important affinities within the phylum Apicomplexa. In Crypto ones. Although the disease in an adult bird proceeds sporidium spp., the invasive stage (zoite) is finally with no symptoms, this one is yet a parasitecarrier. enveloped by parasitophorous vacuole, the inner Cryptosporidiosis is not recurrent in birds that have membrane of which originates from plasma membrane survived it. The amount of isolated oocysts in adult hens of the apical region of the affected gastric cells. Together is considerably smaller as compared with chickens, with the zoite envelopment, a unique structure (feeder owing to the higher immune status of the former ones. organelle) is formed at the zoitehost cell interference Invasion extensiveness of 95% can be observed in 30 zone. Scanning electron microscopic examination of 35 dayold broilers. Poultryraising farms continue the developmental stages of Cryptosporidium muris suffering considerable economic losses. Alabama State Tyzzer, 1910 from the stomach of experimentally (USA), for instance, loses weekly 25 thousand infected multimammate rats (Mastomys natalensis) and cryptospirosis broilers, while the survived birds lose 100 gastric Cryptosporidium sp. from naturally infected toads 150 g of weight. In a Japanese poultrybreeding farm, (Bufo sp.) showed differences in the attachment strategy the high number of morbid cases could not be decreased of the cryptosporidians depending on density of Cryp for over two years and a half. Infected poultry's body tosporidium developmental stages and character of the mass growth decreases 9 to 20% during the disease microvillous border of gastric cells. According to our period even under most beneficial growth conditions. transmission electron microscopic observations on C. Monitoring of the epizootic situation in West Scotland's muris and Cryptosporidium sp., zoites attach to the host poultrybreeding farms showed the infection of the microvillous surface, being apparently epicellular and poultry to be 71.7%. The presence of cryptospiridia is not intracellularextracytoplasmic as it is traditionally supported by complex examination methods. Laborato referred to. The enveloped zoites obviously do not come ries make wide use of the microscopy of CylNilsen into close contact with the host cell cytoplasm, except Protistology · 83 for the region of a feeder organelle. Supported by MSM tory symbiotic system between a monophyletic group 0021622416, grant no. 524/03/H133 of the Grant of Euplotes species and bacteria belonging to the genus Agency of the Czech Republic, and by Research Centre Polynucleobacter. Recent studies revealed the existence "Ichthyoparasitology" (LC522). of obligatory freeliving populations of Polynucleobacter necessariuslike bacteria that are phylogenetically PROTECTION FROM OXIDATIVE STRESS BY closely related to the endosymbiotic ones. Moreover, METHIONINE SULFOXIDE REDUCTASES IN Polynucleobacterharboring Euplotes often harbor also EUPLOTES secondary symbionts. A detailed characterization of A. Vallesi1, N. Dobri2, P. Luporini1 primary and secondary endosymbionts was carried out 1 Dipartimento di Biologia Molecolare Cellulare Animale, University by the 16S rRNA full cycle approach in several strains 2 of Camerino, 62032 Camerino (MC), Italy, Dipartimento di of these Euplotes species. Phylogenetic analyses on the Biologia, University of Pisa, 55100 Pisa, Italy. Email: [email protected] obtained 16S rRNA sequence data were performed to Reactive oxygen species are physiologically synthesized clarify the position of these bacterial symbionts. by every cell and oxidize a vast array of cellular Surprisingly, in two of the Euplotes strains we found, as constituents. If the oxidative damages are not repaired primary endosymbionts, bacteria which do not belong by antioxidative enzymes, oxidized macromolecules to the Polynucleobacter necessarius cluster. These are no longer active and start accumulating within cells, bacteria were representative of a new phylogenetic thus affecting the organism lifespan. Elective targets of lineage branching in a basal position with respect to protein oxidation are methionine residues, in particular genera Polynucleobacter and Ralstonia. Likely they those which are carried exposed on the molecular represent a new genus for which no freeliving relatives surface. Their modification into hydrophilic sulfoxides have been described so far. The characterization of the may result into effective changes in the protein polarity, secondary symbionts showed the presence of many with dramatic effects on its bioactivity and stability. In bacterial species either completely new or formerly E. raikovi, protein oxidation appears to commonly described as endosymbionts of different ciliates. The affect the waterborne signalling pheromones that obtained results suggest that, in the investigated group control growth and mating of this ciliate. Uninterrupted of Euplotes species, a replacement of primary endosym rhythms of cell division lead systematically to the bionts occurred; whether they fulfill the same host production of pheromone molecules carrying methio demand is still under investigation, as well as the analysis nines modified by oxidation and showing mitogenic and of the functional meaning of the secondary symbionts matinginducing activities appreciably lower than non so far characterized. oxidized molecules. In experiments of macronuclear DNA amplification, we identified and cloned two genes MICROSPORIDIA IN GENOMICS AND POST whose nucleotide sequences predict the synthesis of two GENOMICS AGE different methionine sulfoxide reductases (Msr). One C.P. Vivares of these enzymes, responsible for the cell protection Parasitologie Moleculaire et Cellulaire, LBP, UMR CNRS 6023, Universite Blaise Pascal, ClermontFerrand, 63177 Aubiere Cedex, against oxidative damages, is of the type A (MsrA) France. Email: christian.vivares@univbpclermont.fr specific for the reduction of the oxidized methionine The microsporidian world includes more than 1,200 S form; the other one is of the type B (MsrB) specific species of unicellular eukaryotes, all obligate intra for the reduction of the oxidized methionineR form. cellular parasites, that infest some protozoa, many Both MsrA and MsrB are expressed although to invertebrates such as insects and crustaceans, and different extents, and their expression appears to be an vertebrates such as fishes and humans. The microspo inducible phenomenon. ridian genomic era started with the sequencing of the tiny genome of the human opportunistic parasite COMPLEX DISTRIBUTION PATTERN OF Encephalitozoon cuniculi (2.9megabase, 2,000 poten PRIMARY AND SECONDARY SYMBIONTS IN tial protein coding genes on 11 chromosomes). Phylo EUPLOTES genetic analysis of this complete genome sequence 1 1 2 1 C. Vannini , F. Ferrantini , S.I. Fokin , G. Rosati , supported the fungal origin of microsporidia and 1 1 F. Verni , G. Petroni revealed a high frequency of fastevolving genes. 1 University of Pisa, Department of Biology, Pisa, Italy, 2 St. Petersburg State University, Department of Invertebrate Biology, St. Genome compaction was reflected by reduced inter Petersburg, Russia. Email: [email protected] genic spacers related to a high frequency of overlapping Polynucleobacter necessarius (Betaproteobacteria) was gene expression, and by the shortness of most putative first described as endosymbiont of strain E24 of the proteins relative to their eukaryote orthologues. This ciliate Euplotes aediculatus. Further studies showed that protein compaction permitted to analyse the structure the PolynucleobacterEuplotes association is an obliga and mechanism of mRNA cap (GuanineN7) methyl 84 · Abstracts of V European Congress of Protistology · 2007 transferase. Gene shortening may reflect a lowered distributional limits of individual species; (iii) to form diversity of proteinprotein interactions as a result of a baseline from which future effects of anthropogenic the loss of numerous genes during the evolution of disturbances can be estimated. The flora of chryso highly hostdependent parasites. This strong host phytes was documented from 35 sites of oligotrophic dependence was moreover illustrated by the lack of glacial lakes with low content of dissolved salts, and genes for some biosynthetic pathways. Despite being range in pH from 6.5 to 6.8. First investigations of very distantly related, Antonospora locustae partially chrysophytes in the region showed a relatively diverse sequenced genome confirmed these main results as well and abundant flora, including 43 different taxa from as an unexpected degree of synteny which has been the genera Chrysosphaerella (2), Dinobryon (5), Mallo successfully exploited to identify ptp1 and ptp2 genes monas (16), Paraphysomonas (3), Spiniferomonas (6) in two insectinfecting species assigned to the Antono and Synura (8). All taxa are new records for the region spora clade. In E. cuniculi genome, most repeated CDSs and two species are new for Russia: Mallomonas multi are of unknown function and distributed in subterminal setigera and Synura petersenii f. praefracta. The most regions that mark the transitions between subtelomeric frequently distributed species were Chrysosphaerella rDNA units and chromosome cores. In particular, a brevispina, C. longispina, Mallomonas striata, M. alpina, potential multigenic family (InterB family) was M. crassisquama, Paraphysomonas gladiata, Spinifero described. This thirtymember proteinencoding family monas cornutus, S. bilacunosa and Synura spinosa. The is present in Encephalitozoon, Vittaforma and Brachiola majority of taxa are cosmopolites of the temperate genera which differ in their host ranges but are all able rather than arctic zone. Our findings extend the known to invade humans. Genomic studies have also focused geographical distribution of many chrysophycean taxa on chromosomal composition analyses as exemplified into the cold northern region. Issues of occurrence and by the complexity of Paranosema grylli (insect parasite) distribution of the chrysophyte flora in relation to some molecular karyotype. This was related to the pronounced ecological data are discussed. length polymorphism of homologous chromosomes, which may be a consequence of ectopic recombination ULTRASTRUCTURE OF BOTHRIOPSIDES HIS at the chromosome extremities. An important area of TRIO (APICOMPLEXA: EUGREGARINIDA: ACTI proteomics research implies the use of mass spectro NOCEPHALIDAE) FROM ACILIUS SULICATUS metry as a powerful tool to identify small amounts of (INSECTA: COLEOPTERA: DYTISCIDAE) proteins from complex mixtures, frequently after I. Vorob'eva, A. Dyakin separation by 2D gel electrophoresis procedures. So, SaintPetersburg State University, St. Petersburg, Russia. analysis of the E. cuniculi spore proteome has led to the Email: [email protected] identification of about 177 different proteins, one The intestine of the A. sulicatus beetle is infected with quarter of these having no clearly predicted function. attached and nonattached trophozoites of the eugre In a next future, it is necessary to evaluate microspo garine B. histrio. Trophozoites are able to change the ridian protein subsets subjected to major posttrans outline of their bodies from spindlelike to barrellike lational modifications such as phosphorylation and shapes due to contractions. Trophozoites attach to the glycosylation ("phosphoproteome" and "glycopro intestinal epithelium by the highly depressed frontal teome"), as well as to specifically study proteins required edge of the protomerite, which is also kept in non for cell cycle progression or for spore wall and polar attached parasites. The surface of the parasite, except tube organisation. of its attaching site, is covered with numerous epicytic folds of typical structure varying in form and size in CHRYSOPHYTE FLORA FROM GLACIAL LAKES different parts of the cell. On the protomerite folds are IN THE BASIN OF KARA RIVER (POLAR URAL) smaller, than on the deutomerite, and have round L.N. Voloshko profiles. On the deutomerite they have clubshaped Komarov Institute of Botany RAS, Laboratory of Algology, St. profiles. At the tip of the folds there are apical arcs and Petersburg, Russia. Email: [email protected] apical filaments. Internal underlies the pellicle. The Polar Ural region represents one of the last true Structure and location of micropores are typical for wildness areas in Europe (Heal et al., 1998). Future gregarines. Bacteria are accumulated between the effects of global climate changes and local anthropo epicytic folds. When protomerite borders deutomerite, genic disturbances (e.g. coalmining and gas pipeline several annular collars are formed by the ectocyte and building) can be especially significant in the Arctic endocyte. Longitudinal superfolds can also be observed region. The purpose of our study was: (i) to examine by on the deutomerite. They are extended from the anterior means of electron microscopy a wellrecognized group to posterior end of the deutomerite, consist of an ecto of bioindicator organisms from a pristine, but ecologi and endocyte, and bear epicytic folds. There are nume cally sensitive area of the world; (ii) to detect possible rous circular microtubules underlying the surface of Protistology · 85 trophozoites. Presence of attached and nonattached microsporidia of this host group is insufficient. The trophozoites is suggested to represent two different analysis of hosts of 18 microsporidian species showed physiological states of the parasite. Formation of that these parasites are highly hostspecific. The finding superfolds facilitates changes in the trophozoite body of Agglomerata sidae in the cladocerans of two different shape. families (Sididae and Holopediidae) seems to be an exception. Presporal development and a spore structure SYMBIOTIC AND NONSYMBIOTIC ALGAE of many microsporidia of cladoceran hosts have unique DIVERSITY IN SINGLE CELLS OF PARAMECIUM features and on this basis 7 new genera (Agglomerata, BURSARIA Baculea, Berwaldia, Glugoides, Gurleyides, Norlevinea K.P. Vorobyev1, E.E. Andronov2, A.V. Migunova1, and Ordospora) were created. On the other hand the K.V. Kvitko1 taxonomic position of many species is unclear. For 1 St. Petersburg State University, St. Petersburg, Russia, 2 All Russia example, according to the author observations, all life Research Institute for Agriculture Microbiology, Russia. Email: [email protected] cycle stages of Larssonia obtusa have isolated nuclei and It was shown earlier by 18S RNA gene sequence a validity of genus Larssonia must be reexamined. Life analysis, that the algae Chlorella sp., the endosymbionts cycles of microsporidia of these hosts are investigated of P. bursaria of both northern and southern ecotypes, insufficiently. There are 5 positive results of horizontal are closely related to C. vulgaris, C. sorokinianа and C. transmission for Glugoides intestinalis, Ordospora lobophora. The distinctive property of 18S rRNA genes colligate, Microsporidium sp. (Refardt et al., 2002), of symbiotic Chlorella species is the presence of one and Gurleya daphniae (Friedrich et al., 1996) and Gurleya three introns in algae from northern and southern P. sp. (Voronin, Makrushin, 2006). Two species (Flabelli bursaria ecotypes correspondently. Nested PCR with forma magnivora and Gurleya sp. Vavra, 1964) are primers flanking the first intron, was developed for transmitted with nearly 100% fidelity from mother to identification of Chlorella species in the single cell of offspring. The attempts of artificial infection of clado P. bursaria. This method was applied to six P. bursaria cerans with 5 microsporidia species were unsuccessful, cultures of different origin. As a result, at least two which indicates to possible occurrence of complex life fragments were amplified from each cell. According to cycles among microsporidia of Cladocera. the fragment sizes we presumed, that the ~1000 bp amplicon belonged to symbiotic chlorella, bearing the MORPHOLOGY AND DIVERSITY OF MARINE intron, while the ~700 bp fragment corresponded to FREELIVING PERITRICH CILIATES nonsymbiotic chlorella, lacking the intron. For more A. Warren1, D. Ji2, P. Sun2, W. Song2 precise analysis, DNA fragments amplified from two 1 Department of Zoology, Natural History Museum, Cromwell Road, 2 selected paramecia cells were cloned. The RFLP London SW7 5BD, UK, Laboratory of Protozoology, Ocean University of China, 266003 Qingdao, P.R. China. analysis of the cloned DNA inserts was performed. Email: [email protected] Several cloned fragments representing particular RFLP Peritrichs are among the oldestknown groups of ciliates types were sequenced. As expected, specific types of having first been observed by Antony van symbiotic chlorella were detected in ciliates of northern in 1676. Some are freeswimming while others are vs. southern ecotypes. The endosymbionts of both sessile, being attached to a substrate via a stalk, scopula ecotypes possessed one intron of the same size, which or lorica. They have colonised a wide range of habitats, was located at different sites of the gene. Several non marine, freshwater and terrestrial, and may either be symbiotic species similar to C. sorokiniana, C. vulgaris freeliving or ectocommensal. They form one of the and other Chlorella spp., were found in each ciliate. most specious groups of ciliates with around 1,000 species representing 12 families and over 50 genera. MICROSPORIDIA OF CLADOCERANS: DISTRI Although genus identification among peritrichs is BUTION AND LIFE CYCLES relatively straightforward, species identification is often V.N. Voronin difficult mainly due to inadequate species descriptions State Research Institute on Lake and River Fisheries, Department of fish diseases, St. Petersburg, Russia. and morphological variation. Historically, species Email: [email protected] descriptions were based only on in vivo observations. Freshwater microcrustaceans are frequently hosting In recent years, however, modern methods such a microsporidia. More than 40 species of microsporidia silverstaining have been routinely applied revealing have been reported from Cladocera, most of them from taxonomically informative characters such as the pond populations of Daphnia pulex (10 species) and D. pellicular silverlines and oral infraciliature. Using such magna (8 species). The study of microsporidians from techniques, extensive surveys of peritrichs have been Cladocera was performed only in several European carried out in NE coastal regions of China. These have countries and Canada, and therefore investigation of revealed over 100 species which represents more 86 · Abstracts of V European Congress of Protistology · 2007 than10% of the global peritrich species diversity. This pH is a major environmental factor of aquatic talk provides a brief overview of the morphology and ecosystems at the interface of physicochemical and diversity of marine freeliving peritrichs based on studies biological processes. It is regulated by carbonate carried out in coastal waters near Qingdao, China. equilibrium, both in the ocean and in most inland Supported by the Darwin Initiative Programme (project waters, and is impacted by biological processes such as no. 14015) which is funded by the UK Department photosynthesis and respiration. While pH is relatively for Environment, Food and Rural Affairs. constant in the ocean, it varies between <2 to 12 in fresh water. The general reduction of species diversity with EFFECT OF COFILINLIKE PROTEIN ON decreasing pH and the tolerance limits towards low pH MIGRATION AND ENDOCYTOSIS OF AMOEBA are known for major freshwater taxa such as fish, PROTEUS zooplankton and algae, but little is known on pH A. Wasik1, K. Wierzbicka2, P. Pomorski2, W. Klopocka1 tolerance of aquatic protists. We investigated the pH 1 Department of Cell Biology, Nencki Institute of Experimental Biology, reaction norm, i.e. population growth rate (µ) vs. pH 3 Pasteur St., 02093 Warsaw, Poland, 2 Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur St., 02093 and the response of cell size to changing pH, of 6 Warsaw, Poland. Email: [email protected] freshwater ciliate and one cryptophyte species in the Similarly to tissue cells, organization of the contractile laboratory. The protists can be broadly classified into network in amoebae is highly regulated by activity of pH tolerant and pH sensitive species; the former showed actin binding proteins. Capping, severing and other positive µ over more than 4 pH units, the latter over 2 proteins keeping polymerizationdepolymerization pH units or even less under standard laboratory balance, as well as motor proteins, namely myosins, conditions. Using the oligotrich ciliate Meseres corlissi, keep together proper structure and tension of the we also studied intraspecific differences, investigating cytoskeleton. The cofilin homologue from A. proteus 5 clones from geographically distant environments. The has mass of about 19 kDa, similar to the mass of cofilin results obtained suggest local adaptation in the pH from Jurkat cells. The distribution of this protein and response of this species to specific habitat conditions. its colocalization with actin filaments was investigated The significance of pH impact on growth and survival in migrating, pinocytotic and fagocytotic amoebae. In of the protist species was evaluated relative to the effect migrating amoebae cofilinlike protein colocalized with of other environmental factors such as temperature, filamentous actin in the cortical layer of the middle food, and predators. anterior region of the cell, perinuclear cytoskeleton and in the area of cellular adhesion. The examined protein MICROSPORIDIAN DIVERSITY IN LAKE BAIKAL is also randomly distributed in the endoplasmic strea AND SURROUNDING WATERS ming. We suggest that it is involved in actin dynamic T.J. Wilkinson, J.E. Ironside reorganization of the contractile layer and cytoskeleton University of Wales Aberystwyth, Institute of Biological Sciences, network depolymerization of the endoplasm. In the Aberystwyth, United . Email: [email protected] pinocytotic amoebae cofilin homologue and actin Lake Baikal in Siberia hosts a large diversity of endemic filaments colocalized strongly beneath the whole cell amphipod crustaceans. There is virtually no mixing of membrane that may be interpreted as facilitating of the endemic species and the species that inhabit the actin reorganization related to pinocytotic channels surrounding tributaries and other water bodies. formation. In the fagocytotic cells cofilinlikeprotein Microsporidia are a common parasite of amphipods seems to be absent in the newly formed fagocytic cup, and are well studied within European amphipod but colocalized with Factin on the surface of food populations. It should, therefore, be possible to vacuoles. The in vivo role of cofilinlike protein in investigate the distribution of microsporidia throughout amoebae migration and endocytosis was assessed by the populations, and infer whether or not the immisci blocking endogenous cofilin with polyclonal antibody bility of Baikalian amphipod populations has effected against human cofilin. It evoked significant decrease or been affected by microsporidian infection. Amphi of motile activity. We revealed that actin dynamics pods were screened from the lake, its surrounding induced by cofilinlike protein are crucial for normal waters, and from other lakes, for microsporidian morphology and motility of Amoeba proteus. infection. This was done by polymerase chain reaction used to amplify the 16s rRNA gene. Early results have SIGNIFICANCE OF pH AS ENVIRONMENTAL indicated little or no microsporidian infection among FACTOR LIMITING THE DISTRIBUTION OF Baikalian amphipods, an interesting point given that FRESHWATER PROTISTS the majority of European amphipod have screened T. Weisse, U. Scheffel, P. Stadler positive for microsporidian infection. Within Lake Institute for Limnology of the Austrian Academy of Sciences, Austria. Baikal, microsporidia of the genus Dictyocoela have Email: [email protected] been found in a restricted number of amphipod species Protistology · 87 and occur at very low prevalence. In contrast, high the functions similar to those in multicellular species. prevalence of microsporidian infection were found in Dynamin was detected in the coated pits/vesicles during Gammarus lacustris, an amphipod species inhabiting receptormediated endocytosis (RME) where it water bodies surrounding Lake Baikal but absent from colocalized with clathrin (Wiejak et al. Biochem Cell the main lake. Sequencing of 16S rRNA has revealed Biol. 82, 2004; J Exp Biol. 20, 2004) and also in the unusually high genetic variability of Dictyocoela within membrane pool indispensable for phagosome forma these populations when compared with G. lacustris tion (Wiejak et al. Eur. J. Protistol. 39, 2003). Dynamin populations in European fresh water habitats. expression quantitated in immunoblots with a specific antipeptide antibody, was elevated during interna DIVERSITY OF MITOCHONDRIAL PROCESSES lization of transferrin, the marker of RME, and it was IN MICROSPORIDIA diminished when this process was inhibited. Trafficking B. Williams from the early to late endocytic compartment is Department of Botany, University of British Columbia, Vancouver, regulated by Rab7. We cloned two closely related genes Canada. Email: [email protected] encoding this protein in Paramecium displaying 81.6 Microsporidia are important human parasites and are 82.1% homology to human Rab7 (Surmacz et al. Acta one of the most common causes of diarrhoea in people Biochim. Polon. 53, 2006). In Paramecium Rab 7 was with AIDS. They are also important models of extreme detected in endosomes and in phagolysosomal com eukaryotic genome compaction and cellular adaptation, partment (Surmacz et al. Biol. Cell 95, 2003). Expres and appear to be highly reduced and simplified relative sion of two Paramecium Rab7 isotypes increased when to other eukaryotic cells. One such example of reduction cells phagocytosed latex as shown by real time PCR. is that of the mitochondrion or mitosome, which is a Distinct pattern of localization of Rab7a and Rab7b was recently described but poorly, characterised organelle. observed during phagocytosis with the usage of specific An idea of the processes occurring within the organelle antibodies raised against their Ctermini that differ in has been pieced together from proteins encoded in the four amino acids residues out of 206 in total. complete E. cuniculi genome sequence. This suggests as a highly streamlined mitochondrion that appears to MOLECULAR EPIDEMIOLOGIC STUDIES OF have lost both the genome and the electron transport ENTEROCYTOZOON BIENEUSI: THE PERU chain. We have studied the insect infecting microspo EXPERIENCE ridian Antonospora locustae and shown that the function L. Xiao1, V. Cama1, C. Bern1, Y. Ortega2, H.H. Garcia3, of the mitochondrion may differ in this species. The A.E. Gonzalez3, R.H. Gilman4 mitochondrion of A. locustae appears to more complex 1 Division of Parasitic Diseases, Centers for Disease Control and in terms of how it imports proteins, the types of Prevention, Atlanta GA, USA, 2 University of Georgia, Griffin GA, metabolites it can import, and pathways housed within USA, 3 Department of Pathology and Microbiology, University of Peru, 4 the organelle relative to E. cuniculi. We have begun to Lima, Peru, Johns Hopkins School of Public Health, Baltimore, Maryland, USA. Email: [email protected] characterize some of these additional proteins in order Enterocytozoon bieneusi is a common human pathogen, to understand how and why mitochondrial processes responsible for more than 90% of overall human differ form species to species. The data suggest that microsporidian infections. Few epidemiologic studies, mitochondrial function across microsporidia may be however, have been conducted to characterize the quite variable and show different levels of reduction infection patterns, risk factors and transmission routes across the phylum. of human E. bieneusi infections. In recent years, we have characterized the transmission of E. bieneusi in a case ENDOCYTIC ROUTES IN PARAMECIUM: CON control study in AIDS patients and longitudinal cohort SERVATIVE ELEMENTS study in children in Lima, Peru. Seventy five of 2,652 E. Wyroba, J. Wiejak, M. Osinska, H. Bilski, HIV+ patients (3%) studied during September 2000 to K. Krawczyk, E. Wypych Nencki Institute of Experimental Biology, Warsaw, Poland. Email: December 2003 had microsporidiosis detected on stool [email protected] microscopy at the time of enrollment, while 30 We identified conservative elements of endocytic additional patients had microsporidiosis detected on a pathways in Paramecium at the gene, protein and subsequent visit. Of these 105 patients, 89 had cellular level. Cloning, immunoblotting, real time PCR microsporidia amplified and genotyped; all were E. and immunolocalization in confocal and electron bieneusi and 11 genotypes were differentiated. Infec microscopy revealed presence of dynamin and Rab 7, tions with E. bieneusi were associated with chronic the proteins indispensable for early and late stages of diarrhea in logistic regression models adjusted for endocytic pathways, respectively, displaying a very high CD4+ cell count and infections with other enteric homology to mammalian counterparts and performing protozoa. The evaluation of associated clinical mani 88 · Abstracts of V European Congress of Protistology · 2007 festations showed that genotypes Peru3 to Peru11 were cribed and figured in detail, making it unnecessary to significantly associated with a 4fold increased risk in go back to the original literature often difficult to obtain. chronic diarrhea compared to patients without these Two identification keys are provided, viz., one for parasites. The two most common genotypes, Peru1 and taxonomists and another, simple key for users not Peru2, were not associated with significant increases specifically trained in ciliate . The first part in chronic diarrhea. Risk factors for E. bieneusi infection of the monograph that contains the families Protospa segregated by genotype: contact with duck or chicken thidiidae, Arcuospathidiidae, and Apertospathulidae, droppings and lack of running water, flush toilet, or has been published (Foissner, W. & Xu, K., 2006. garbage collection with genotype Peru1 and water Springer, Monographiae Biologicae, 487 pp). The melon consumption with other genotypes. In the second part will come out soon and contain the family longitudinal cohort study of infection in children during Spathidiidae and a new family, Pharyngospathidiidae. March 2002 to March 2006, 71 episodes of microspo This monograph is part of our attempt to revise the free ridian infections were identified in 70 of 656 children living ciliates. Supported by the National Natural studied, of which 51 were genotyped, with 14 E. bieneusi Science Foundation of China (No. 40576072), the '100 genotypes found. The most common genotypes found Talents Project' of CAS, and the FWF, Project P15017. were Peru2 and Peru11, with Peru1 in only one child. Most infections occurred in children under 3 years of A NEW MICROCOSM AND ITS USE IN BIO age, and 29% of infected children had transient diarrhea MONITORING OF MARINE POLLUTION STATUS lasting an average of 2.1 days. Risk factor analysis USING PFU PROTOZOAN COMMUNITIES identified significant association between E. bieneusi K. Xu1, Y. Lei1, J.K. Choi2, E.J. Yang2 infection and poor sanitary and socioeconomic 1 Institute of Oceanology, Chinese Academy of Sciences, Qingdao 2 conditions. Even though a direct transmission of E. 266071, China, Department of Oceanography, Inha University, Inchon 402751, Korea. Email: [email protected] bieneusi (Peru16) was found between a child and A laboratory PFU (polyurethane foam unit) microcosm guinea pigs in a household, contact with animals or was developed to enable the biomonitoring of marine animal feces was not identified as risk factors for E. pollution status using protozoan communities. This bieneusi infection in children. Thus, molecular method consists in soaking several PFU blocks to target epidemiologic tools are very useful in the characte water and incubating in laboratory. The marine rization of microsporidiosis transmission and the pollution status is evaluated through the structural transmission of E. bieneusi might be very different parameters of protozoan communities colonizing the between children and HIV+ persons living in the same PFU. We test the efficiency of the microcosm on two area. occasions by using sea water samples collected from six stations with different pollution levels in the Kyeonggi MONOGRAPH OF THE SPATHIDIIDA (CILIO Bay, Korea. The data obtained suggested that neither PHORA, HAPTORIDA) 1 2 single species richness nor abundance of protozoans K. Xu , W. Foissner could be used as an effective indicator. However, the 1 Institute of Oceanology, Chinese Academy of Sciences, 266071 Qingdao, China, 2 Department of Organismal Biology, University of Margalef diversity index which combines the two Salzburg, A5020 Salzburg, Austria. Email: [email protected] parameters worked well and could clearly distinguish The spathidiids belong to the ciliate subclass Haptoria the different classes of water quality. The highest (Protozoa, Ciliophora), that is, they are predators using diversity index value coincided well with the best water toxicysts to overwhelm the prey. Spathidiid ciliates quality. By contrast, poor water quality corresponded prefer terrestrial and semiterrestrial habitats, but many with the distinctly low index values. Furthermore, the occur also in freshwater, and some are marine. Over diversity index values not only generally correlated with 200 nominal spathidiid species have been described, the temporal marine pollution intensity, as inferred from sometimes based on seemingly minute differences. the physicochemical parameters, but also indicated the Thus, many protozoologists considered them as effects of longterm stress of various pollutants. indeterminable and claimed for a detailed revision. The Practically, the PFU microcosm can be used to carry present monograph carefully revises the taxonomy, out a comprehensive evaluation of marine water nomenclature, and ecology of all nominal species and condition by using both surface and benthos water shows that spathidiid diversity has been greatly under samples. It overcomes the sampling difficulties in estimated. Based on reinvestigation of the described assessing the longterm effects of marine pollution in species with modern methods (silver impregnation, open waters. Supported by the National Natural scanning electron microscopy) and the first description Science Foundation of China (No. 40576072) and the of over 50 new species, the family Spathidiidae is split '100 Talents Project' of CAS. into four families and 20 genera. Each species is des Protistology · 89

COORDINATED DISTRIBUTION OF PARAME their receptors the cell synthesizes. So far, only three CIUM BURSARIA SYNGENS AND VIRUSES OF MTs have been found in the species. These three MTs THEIR ENDOSYMBIOTIC CHLORELLA are stably inherited during vegetative reproduction and V.V. Yashchenko, I.I. Skoblo, A.A. Potekhin, usually demonstrate typical Mendelian behaviour in A.V. Migunova, M.S. Rautian crosses (a single mat locus with three alleles showing Biological Research Institute of St. Petersburg State University, St. peckorder dominance). However, in some crosses we Petersburg, Russia. Email: [email protected] observed MT instability in young, just matured Symbiotic chlorellae from P. bursaria are known to be exconjugant clones. In a clone of this kind, the states of a specific host for the giant viruses Paramecium maturity and immaturity (or adolescence) often alter bursaria Chlorella viruses. Based upon physiology, DNA nate and/or one MT changes to another, sometimes hybridization and virus infection, two types of Chlorella, repeatedly, during the period of several weeks after the called "American" (or "Southern") and "European" (or clone's maturation. On occasion, all three MTs can be "Northern"), have been reported to date. Correspon expressed consecutively. These observations suggest that dingly, two types of viruses: "Southern" and "Northern", the mat locus in dilepti is a compound integral one; it is specific to one or another type of Chlorella, were des inherited as a whole and can specify expression of any cribed. Distribution of two types of Chlorella and viruses one of possible MTs (much as it occurs in Tetrahymena was supposed to depend either on latitude, or altitude, thermophila). Some other mechanisms, supposedly that is why two types of classification were proposed epigenetic ones among them, control what MT will be earlier. We supposed that distribution of different types expressed in a given exconjugant clone in particular. of Chlorella and viruses could be related to the distri Steady functioning of these mechanisms in the bution of P. bursaria syngens. Geographical distribution micronucleus provides stable, unambiguous differen of P. bursaria syngens was examined. 400 strains collec tiation of the compound mat locus to one and only one ted from the distant geographical regions were analyzed, MT and subsequent Mendelian behavior of the charac five syngens were found. Syngens 1 and 2 were found in ter over sexual generations. If, for some still unknown Europe and Asia. Syngen 1 was registered from latitude reasons the control of such differentiation is disturbed, o o 40 to 60 North, whereas syngen 2 was spread from MT expressed in a given exconjugant clone becomes o latitude 40 North up to and across the polar circle unstable and its Mendelian behavior can be violated. (>63o). Syngen 3 was found in Eastern Asia, and in North America. Syngen 4 was found in USA. Syngen 5 MORPHOLOGICAL VARIABILITY OF CILIATES single strains were found in Astrakhan (Russia). A FROM THE GENUS CONCHOPHTHIRUS (OLI hundred of strains belonging to five syngens were GOHYMENOPHOREA: ) examined on the presence of virus, type of virus was V.I. Yuryshynets determined. For each syngen both virusfree and virus Institute of Hydrobiology NAS of Ukraine, Department of Sanitary and containing strains were detected. 45% of examined P. Technical Hydrobiology, Kiev, Ukraine. Email: [email protected] bursaria populations contained viruses. Viruses of the Investigation of abiotic and biotic factors influence on "Northern" type were detected in the strains of syngens organisms, populations and communities of symbiotic 1 and 2, whereas viruses of the "Southern" type in the ciliates is relevant in view of constant anthropogenic strains of syngens 3 and 4. There was only one case of changes of environment. Some peculiarities of diffe "Southern" virus in the strain of syngen 1 from rentlevel response of ciliates could be used as indicative Tajikistan. Such virus distribution indicates that diffe for detection of such environmental changes. Ciliates rent P. bursaria syngens might contain different types of the genus Conchophthirus are common mesobiontes of Chlorella. Supported by RFBR grants 060449504 of different species of freshwater bivalve mollusks. Our and 070410073. investigation included only some species of these habitants of the mantle cavity, living in unionides NUCLEAR DIFFERENTIATION OF MATING (Unionidae) and dreissenes (Dreissenidae): Conchoph TYPES IN THE LOWER CILIATE thirus acuminatus Clap. Lachm., C. unionis Raabe, C. ANSER curtus (Engelmann), C. anodontae (Ehrbg.). Values of A.L. Yudin, Z.I. Uspenskaya 4 meristic characters of ciliate body and 4 indices have Institute of Cytology RAS, St. Petersburg, Russia. been used for statistical analysis. Samples from the Email: [email protected] waters, heated by the power plant (Konin lakes), and Dileptus anser (= D. margaritifer) is one of the ciliates samples from not extremely disturbed biotopes which excrete specific inducers of conjugation (called (Mazurian Lakes, Dnipro River, Dnister River) were mating pheromones or gamones) into the surrounding chosen for comparison. As a result, dependence of some medium. Accordingly, mating type (MT) of a cell is studied parameters upon water temperature has been determined by the kinds of mating pheromones and revealed. Also our data confirmed a significant role of 90 · Abstracts of V European Congress of Protistology · 2007 relative isolation of host individuals for formation of 1 mm in size, well known for their important role as parasitic ciliates variability, in particular, for repre bioindicators of past and present marine environments sentatives of a genus Conchophthirus. Existence of such used in many scientific disciplines, have been dubbed variability of Conchopththirus sp. ciliates dependent on as "Tiny Giants of the Great Seas". Their extremely environmental factors, was tested and confirmed in diversified and exquisite shell morphology are virtually experiments. art forms in Nature. To popularize their scientific and aesthetic application, Zheng Shouyi, Senior Scientist SPECIES DIVERSITY AND ECOLOGY OF of the Institute of Oceanology, Chinese Academy of CILIATES FROM SMALL WATER BODIES IN THE Sciences, personally sculpted the proportionately AREA OF SARATOV RESERVOIR (THE LOWER enlarged 200 original foraminiferal models, which are VOLGA RIVER) used as popular science and educational tools, tourist V.V. Zharikov, S.V. Bykova souvenirs, jewelry, lamps, garment decorative designs, Institute of Ecology of the Volga River Basin, Russian Academy of etc. A Foraminiferal Popularization Base in Qingdao Sciences, Togliatti, Russia. Email: [email protected] City, China, displays foraminifera from different Data on the ciliate fauna of small water bodies (lakes geological ages, as well as foraminifera, indicative of and ponds) adjacent to large reservoirs of the Volga River different marine environments including the entire are lacking. By this study we tested the hypothesis that China seas, Pacific, Atlantic, Indian, Arctic and Ant the "alien" ciliate species recorded occasionally in the arctic oceans. Foraminiferal Sculpture Park consisting Volga reservoirs are native for these small lakes and of more than a hundred foraminiferal sculptures ponds. Survey of the fauna of ciliates in small water enlarged thousands of times their original size, was bodies was carried out in the region of Samarskaya Luka created in Sanxiang Township, Zhongshan City in (the Lower Volga). Ciliate diversity increased in the Guangdong Province, China, being the first of the kind direction: reservoir flood lands super flood lands in the world. A new vista of microscopic foraminifera elevated lakes. In total, 178 and 234 species were integrating science, marine culture, and art, is unfolded identified in Saratov reservoir and the adjacent ponds for the public to appreciate and enjoy the beautiful respectively, among which 76 species were first records artistry of Mother Nature endowed on foraminifera, to for the Volga basin. Twenty five species were observed inspire artistic creations and innovations, and to exclusively in the Saratov reservoir, 72 in elevated lakes promote the cause of popular science. of karstic and nonkarstic origin. As many as 93 species (53% of the total) were common for all investigated PREVALENCE OF ANTI water bodies. Zones of "optimal development" of the BODIES IN SERA OF STRAY CATS FROM SARI ciliate populations varied in different water bodies and CITY, NORTHERN IRAN, 2004 fluctuated from upper to lower threshold tolerance S.P. Ziapour1, M. AlipourSpehkolaei2, M. Sharif3, limits. Ciliates inhabiting only large reservoirs or only B. Esfandiari1, S. Gholami3, H. Ziaei3, M. Assmar4 small drainless lakes displayed the narrowest limits of 1 Amol Research Center, Pasteur Institute of Iran, Department of tolerance and physiological optimum. Changes in Medical Parasitology & Mycology, Amol P.O.Box. 139, Iran, 2 Babol spectrum of dominant species, as well as alterations of medical science university, Shahid Yahyanejad hospital, clinical labratory, Babol, Iran, 3 Mazandaran medical science university, the ciliate community structure and dynamics varied Dept. of medical parasitology and mycology, Sari, Iran, 4 Pasteur in lakes located at different distances from the reservoir. Institute of Iran, Department of Medical Parasitology, Tehran, Iran. Penetration of many "new species" into the reservoir is Email: [email protected] limited by its hydrology. We infer that changes of the Infection by the protozoan parasite Toxoplasma gondii hydrological mode of the reservoir benefits emergence is widely prevalent in humans and animals throughout of nonindigenous species. Thus, ciliate fauna of small the world. The incidence of Toxoplasmosis in urban ponds serves as a potential source for maintenance of areas can be related to environmental contamination ciliate diversity in large reservoirs. Findings of Follicu with oocyst. An interesting alternative for measuring lina boltoni Kent, 1881 in some lakes connected with T. gondii urban spreading is seroprevalence in freeliving large reservoirs of the Central and Lower Volga basin urban animals, such as stray cats. With this aim, we support this hypothesis. tested serum samples from 100 stray cats in five urban regions from Sari city, northern Iran for antibodies to TINY GIANTS OF THE GREAT SEAS: FORA T. gondii by latex agglutination test (LAT). Antibodies MINIFERA to T. gondii were found in 40 (40%) of 100 stray cats, S. Zheng with LAT titers of 1:1 (4 IU/ml) in 4, 1:2 (8 IU/ml) in Institute of Oceanology, Chinese Academy of Sciences, China. 2, 1:4 (16 IU/ml) in 4, 1:8 (32 IU/ml) in 4, 1:16 (64 Email: [email protected] IU/ml) in 6, 1:32 (128 IU/ml) in 4, 1:64 (256 IU/ml) Foraminifera, shelled marine protozoa usually less than in 2, 1:128 (512 IU/ml) in 6, 1:256 (1024 IU/ml) in 7, Protistology · 91

1:512 (2048 IU/ml) in 1. Seropositivity (LAT 1:1 or THE ULTRASTRUCTURE OF MARINE CARNIVO more) was significantly higher in adult (= 26 teeth, ROUS FLAGELLATE METROMONAS SIMPLEX 54.54% of 66) than in nonadult (>26 teeth, 11.76% of (CERCOZOA INCERTAE SEDIS) 34) cats (P = 0.000), in weighting group 1.52.9 kg (56% A.P. Mylnikov of 25) than in weighting group 01.4 kg (11.76% of 34) Institute for Biology of Inland Waters, Russian Academy of Sciences, and weighting group > or = 3 kg (53.66% of 41) cats (P Borok, Russia. Email: [email protected] = 0.000). There were no significant differences among The ultrathin structure of carnivorous marine flagellate female (44.44% of 72) and male (28.57% of 28) cats (P Metromonas simplex has been investigated. This pre > 0.05), or among cats from western (50% of 20), dator captures the whole cell of the prey, normally bodo northern (35% of 20), southern (35% of 20), eastern nids, or chrysomonads. A cytostome as a cell pocket (35% of 20), or central regions (45% of 20) (P > 0.05). has not been found. The cell surface is composed of the These seropositive cats are likely to have already shed plasma membrane covered with two layers of fibrous T. gondii oocysts in the environment around Sari city. material. Long flagellum bears very thin hairs of 0.8 The high seroprevalence of T. gondii antibodies found 1.0 µm in length, short flagellum is naked and reduced in the present study suggested a widespread exposure in length. The transitional zone does not contain the of stray cats to T. gondii. spiral or any other additional elements. The transversal plate is located upper the cell surface. The flagellar root THE ROLE OF PROTOZOANS IN PERIPHYTON system is very simple and has at least one microtubular COMMUNITIES band of two microtubules, which originates near the V.A. Zolotarev kinetosomes. The latter are approximately parallel to Institute for Biology of Inland Waters of the Russian Academy of each other and interconnected with the bridges. The Sciences (IBIW RAS), Borok, Yaroslavl, Russia. Email: [email protected] vesicular nucleus, and endoplasmic Studies of microperiphyton were conducted in the lakes reticulum are of usual structure. Oval mitochondria of of Karelia, the Rybinsk reservoir, Lake Baikal, Lake 0.6 x 2.5 µm contain lamellar cristae. The rodlike Ladoga, Lake Windermere, the acidic lakes of Darwin () 1.01.4 µm in length and 0.12 National Park, the White Sea, the River Amur and in 0.08 µm in diameter derive from the Golgi apparatus. experimental microcosms/mesocosms. Protozoans are Trichocysts have wheelshaped structure with 13 spokes an important component of the periphyton com visible in cross sections. Contractile vacuole is absent. munities at the early stages of colonization. Abundances M. simplex is similar to Metopion fluens and cryotheco of unicellular organisms in most natural and artificial monads. Supported by the Russian Foundation for periphyton assemblages range from 100 to 100 000 cells Basic Research grants 050448180 and 060449288. per sq.cm, and the number of species is usually more than 150. Microbial species are important members of HOW CHOANOFLAGELLATES CONQUERED attached communities in all ecosystems and play major THE WORLD: A SYNTHESIS BASED ON MOR roles in energy flow. The diversity and abundance of PHOLOGY, ECOLOGY AND EVOLUTION protozoan species provide information about bacterial B. Leadbeater activity, hence protozoa are integral members of the School of Biosciences, University of Birmingham, UK. decomposer community. Periphyton communities that Email: [email protected] are formed on artificial substrates, can be defined as How could a group of humble filterfeeding unicells, "periphyton model communities" (PMC). These now considered to be the nearest living protistan rela communities are easily manipulated in the laboratory, tives of the animal lineage, become one of the most suc and member species span the range of sensitivities of cessful assemblages of freeliving protozoa in the world? more familiar species. Periphyton biodiversity and The answer would appear to lie in the most remarkable relative abundance of ciliates and other protozoans can versatility of their cell coverings. This is splendidly be used as indicators of toxic pollution and acidification. illustrated by the evolution of a basketlike lorica which Multivariate statistics were used to design "the scale of surrounds the cells of many marine species. Subtle toxicity" across a gradient of toxicant stress and organic variations in this structure have allowed the choano compounds. A new index of periphyton flagellates (IPF) flagellates to colonise a myriad of microniches within as the indicator of the trophic status of waterbodies the oceans. Recent evidence for the evolutionary was developed. Several advantages of utilizing PMC and closeness of the choanoflagellates to the animals will new indices for pollutional stress assessment are be presented as well as the most detailed illustrated discussed (http://biomonitoring.narod.ru). Since biota information yet available on the evolution and the may differ essentially from one ecoregion to another, a remarkable lifestyle of these flagellates in their natural universal biological monitoring system should use environment. Structural complexity blends seamlessly species with cosmopolitan distribution. with functional significance and ecological importance.