Protistology 5 (1) 9 91 (2007) Protistology
Abstracts of V European Congress of Protistology and XI European Conference on Ciliate Biology
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 non cultivatable human 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, NY 10461, USA; 2 Department of Pathology, Albert Einstein ABUNDANCE, DYNAMICS AND SPECIES SUC College of Medicine, Bronx, NY 10461, USA; 3 Departments of CESSION IN SOIL PROTISTS Pathology and Medicine, Albert Einstein College of Medicine, Bronx, M.S. Adl NY 10461, USA. E mail: [email protected] Department of Biology, Dalhousie University, Canada. Microsporidia are parasites of all classes of vertebrates E mail: [email protected] and most invertebrates. They have recently emerged as Soil protozoa 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 microtubule 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. coli based 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 baculovirus driven expression in Sf9 cells yielded patterns with seasons, and with plant 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, post mining 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 macronucleus 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 micronucleus 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, MICROSCOPY 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. E mail: [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 lorica 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 Foun dation micronuclei. Capsules (probably extrusive organelles) (FWF; projects P17752 B06 and P19699 B17). in striae (beaded strands) on the collar membranelles and tentaculoids (finger like cytoplasmic processes) DIVERSITY OF EPIBIONT CILIATES OF VARIOUS originating from the intermembranellar ridges. Somatic ORGANIC SUBSTRATES IN MEXICO ciliary pattern is complex and comprises the right, left, M.A. Aladro Lubel, R. Mayen Estrada, M. Reyes Santos 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 E mail: [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. P17752 B06). On the leaves of the angiosperms Thalassia testudinum, Phyllospadix sp. (marine), Halodule beaudetti, Rhizo CONJUGATION IN HALTERIA GRANDINELLA: phora mangle roots (brackish) and freshwater plants, 77 THE MYSTERY OF ITS GENEALOGY PERSISTS species of epibiotic ciliates have been identified. On S. Agatha, W. Foissner seven species of brackish algae, 47 ciliate species have University of Salzburg, Department of Organismal Biology, Salzburg, been observed, and on filamentous freshwater algae we Austria. E mail: [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 animals to serve as potential epibiotic ciliates (apostomatids, folliculinids, licnopho reservoirs of opportunistic Microsporidia in HIV infec rid, peritrichs, prostomatids, stichotrich and sucto ted humans, we examined farm animals and rodents in rians), the peritrich ciliated were the most diverse group, semi desert areas of Azerbaijan: in vicinity of Baku City represented by 62 species (58.5%). Acineta tuberosa, megalopolis, and in Gobustan Absheron and Davachi Vorticella 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 FREE LIVING CILIATES IN AZERBAI microscope. Positive samples were double checked with JAN: MODERN STUDIES AND PERSPECTIVES PCR. Microsporidia like objects were found in calco I.Kh. Alekperov fluor stained fecal smears of 22 Meriones libycus and 1 Institute of Zoology NAS of Azerbaijan. E mail: [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 05 113 4445 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 (cold resistant ROENTERITIS (CRYPTOSPORIDIUM PARVUM, or heat loving) species which are distributed in various SPOROZOA, APICOMPLEXA) 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 E mail: [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 cryptosporidiosis in suckling (10 14 during the first years the researchers register ciliate days old) and weaned (18 22 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 long term 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 burst like 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 genomes, 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. E mail: [email protected] substantially raised above control values and were Microsporidia is a group of obligatory intracellular pa similar to those in 1 to 2 3 month old uninfected rats rasitic protozoans of high practical significance. Infec respectively, i.e. rats after transition from fluid (milk) tion of HIV positive 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 life these lakes. Identification and a morphological charac of infected rats. Supported by CRDF Project RUB2 terization of peritrichs were performed by live observa 002707 SP 05 and RFBR. tions in light microscope equipped with Lumenera INFINITY digital camera. Infestation density, load, PHYLOGENETIC RELATIONSHIPS WITHIN mean number of zooids per colony on the host (adults, PENICULINE CILIATES AND THE PARAPHYLY copepodites and nauplii of A. salinus) and morfometric OF FRONTONIA characteristics of Epistylis 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 bell shaped zooids (in the lake Shira). Zoologia, Pisa, Italy. E mail: [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 genus 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 GENOME PLASTICITY IN THE DICYEMID ME the genus Frontonia is paraphyletic, with the clade re SOZOANS presented by genera Apofrontonia and Paramecium 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 920 1192, Japan. associated with Apofrontonia dohrni, despite the E mail: [email protected]