abstracts of the second european congress of protistology and eighth european conference on ciliate...

Europ.]. Protistol. 31,402-476 (1995)December 15, 1995 I Abstracts

Abstracts of the Second European Congress ofProtistology and Eighth European Conference on CiliateBiology, July 21 - 26, 1995, Clermont-Ferrand (France)

1Potentialities and Limits of Soil Protozoa asBioindicators in Field StudiesE. AESCHTBiology Center of the Upper Austrian Museum,J.-W.-Klein-Str. 73, A-4040 Linz, Austria

Research in most groups of soil protozoa is hindered by meth­odological inconveniences and the lack of taxonomic guides.Culture (dilution) techniques can at best estimate the abun­dance of active and cystic cells. Direct counts provide dataon active individuals and species, which are - beside a clearexperimental design - prerequisites for successful ecologicalwork. Naked amoebae cannot be enumerated reliably at pre­sent. Research on flagellate taxocoenoses is restricted by theirvery small size. Standardized and adequately tested quantita­tive methods are available for testate amoebae and ciliates.Ciliates must be counted on the day of sampling due to theirability to encyst rapidly, while testacean samples can be con­served. Both are equivalent indicator groups in less evolvedsoils, namely the litter layer. In evolved natural and culturedsoils ciliates are less suited, because they are suppressed bymicrobial exsudates. Ciliates are better short-time indicatorsthan testaceans due to their rapid division and cystation abil­ities. Testaceans are more important than ciliates concerningthe energy cycle and production studies. Most papers on tax­ocoenoses show that total individual and species numbers arerarely sufficient to estimate treatment effects. In contrast,these parameters often obscure that some species decrease,while others increase. This clearly illustrates the necessityof identifications at species level. Since few species usuallydominate in a particular site, it could be possible to restrictecological studies on relevant species, i.e. dominance > 2%.

2Treatment of Amebiasis due to Entamoeba histolyticaPossibilities. Limitations. UncertaintiesP. AMBROISE-THOMASDeparternent de Parasitologie-Mycologie Medicale etMoleculaire, CNRS EP 78, Faculte de Medecine,Grenoble, 38706 La Tronche, France

Little progress has been achieved recently in the treatment ofamebiasis due to Entamoeba histolytica. The use of imida­zoles (Metronidazole and Ornidazole) still prevails as thesedrugs have replaced the emetine salts, which have recently

0932-4739-95-0031-0402$3.50-0

been withdrawn from the market. In cases of liver abscess,the treatment may be completed by a drainage puncture,guided with the aid of ultra sounds and allowing, if neces­sary, the in situ injection of amebicides. However, cases ofresistance to metronidazole, as well as side effects, have beenreported. Research work is therefore necessary to developnew tissue amebicides, active against the histolytica formsof the parasite. The main problem, on a practical level, isthe treatment of healthy carriers eliminating cysts with fournuclei. Indeed, only in case of the presence of Entamoeba his­tolytica is the treatment justified, but not of carriers of Enta­moeba dispar which cannot, unfortunately, be distinguishedfrom the former with a mere examination under the micro­scope.

3Seasonal evolution of Glugea stephani (Protozoa:Microspora) Parasitic of Flounders Platichthys flessus,from the Western Coastes of DenmarkJ. M. AMIGO, M. P. GRACIA, H. SALVADO, M. Rms andP. A. MAILLOLaboratori de Protozoologia, Departament de BiologiaAnimal, Universitat de Barcelona, 08028 Barcelona

The seasonal evolution of Glugea stephani in the coasts ofwest Denmark has been examined. The fish used for thiswork (the flounder, Plathichtys (lesus) were commerciallyreared and the prevalence of the parasite was examined fromFebruary 1992 to December 1994, however and due to com­mercial reasons the best studied period was from October1992 to December 1993. The origin of these fish was semi­extensive cultures for the beginning of 1992, and wild popu­lations for the rest of the studied period.The prevalence of this microsporidian ranged between 5%and 53% and was found to be the higher in winter than insummer months, which seemed to be in contradiction withdata indicating that G. stephani does not develop at tempera­tures below IS-16°C (McVicar, 1975), and also in contradic­tion with data of seasonal evolutions of this parasites foundby most authors (McVicar, 1975; Olson, 1976; Takvorian andCali, 1986), nevertheless our data were similar those of Bekhti(1984). The reasons for these radically different results areboth the migratory behaviour of flounders, that migrate todeeper waters in winter, and the cold or warm condition ofstreams that do influence the temperature of water at thedepths where flounders usually live in winter.

© 1995 by Gustav Fischer Verlag, Stuttgart

4Study of Sphaeromyxa balbiani,TheI. 1892(Myxozoa, Myxosporea: Bivalvulida) a Parasite ofCepola macrophthalma L. 1758G. C. AMOROS, M. P. GRACIA, J. M. AMIGO, P. A. MAILLOand H. SALVADOLaboratori de Protozoologia, Departament de BiologiaAnimal, Universitat de Barcelona, 08028 Barcelona

S. balbiani was extracted from its host, the red-band fishC. macrophthalma, captured in the Catalan Coastes (NWMediterranean). The prevalence of the parasite was 71 %(63 infected fishes from 90 observed). Despite having ob­served fish of sizes ranging 28-55 em the highest prevalencewas found to be between 31-40 em. The parasites were pre­pared for its study by light microscopy, transmission electronmicroscopy and scanning electron microscopy.The parasite was found in the bile bladder as plasmodia(usually more than one per bladder) and free spores. Plasmo­dia were polynucleated and polysporous, discoid-shaped ofabout 3.7-4 mm in diameter and about 23 11m thick, andshowing an electrondense ectoplasm with villosities, mito­chondria and lipid vesicules. The endoplasm was a vacuo­lated mass.The spores were fusiform with truncate ends, showing twopolar capsules in both extremes of the spores, and a binu­cleated sporoplasm. Spores sized 13.6 (± 4) x 4.5 (± 1.5)11m and the polar capsules 4 x 3 11m (mean of 20 spores, mea­surements made with fixed material). Under the transmissionelectron microscope the spores showed two valves, and thepolar capsules had a folded polar filament.

5Studies on a and ~ tubulins of Euplotes focardii usingsequence and Posttranslational Modification SpecificMonoclonal AntibodiesL. ARREGUI, S. SERRANO, J.M. DE PEREDA ", C. DE INES" ,I. BARASOAIN, J. M. ANDREU* and A. GUINEADept. Microbiologia, Fac. Biologia, U.C.M.; "CentroInvestigaciones Biol6gicas, C.S.I.C, Madrid (Spain)

Eight site-directed monoclonal antibodies reacting with posi­tions 1-13 (PlC lOC 3 ) , 155-168 (P12BlCl), 214-226 (PlFsC3 ),

430-443 (P12Ellel) of the Cl chain of porcine brain tubulin, aswell as to positions 1-13 (Pl4B4Cl), 153-165 (P4D6C4), 241­256 (PlGgC l) and 416-430 (DM1B) of the ~ chain, all ofthem reactive with fixed microtubules of mammalian PtK2cultured cells, were employed to check whether the cellularmicrotubules of the marine antarctic ciliate Euplotes focardiiwere recognized. Each epitope was chosen because of its evo­lutionary conservation and possible functional role in tubulin(Arevalo et aI., 1990; Andreu and de Pereda, 1993). Amongthese, the three anti-B tubulin antibodies decorated typicalmicrotubular patterns of the genus consisting of basalbodies, microtubular derivatives associated with the kineto­somes and superficial microtubular networks. A recent studyon the sequence of the ~-tubulin gene of this ciliate (Miceli etaI., 1994) allowed us to compare the aforementioned syn­thetic sequences with that of the gene, finding homologouszones. The relative accessibility of each of these residues aspredicted by the ProteinPredict computer program was alsochecked. As far as the remaining antibodies are concerned(anti-a tubulin) only two of them (positions 155-168 and

Abstracts . 403

430-443) showed a clear reaction. Additionally, certain dif­ferences in the localization of the tubulin isoforms were ob­served when permeabilized cells of E. focardii were reactedwith monoclonal antibodies directed to well identify post­translational modifications: 6-11B (anti-acetylated tubulin;Piperno, 1985), 1A2 (anti-tyrosine tubulin; Kreis, 1987)and GT335 (anti-polyglutamylated tubulin; Wolff, 1994).

6New Data on MugU cephalus MyxosporidiaS. BAHRI!, A. MARQUES!, F. COSTE I and O. K. BENHASSINE2

lLaboratoire de Parasitologie et Immunologie,Universite Montpellier 11,34095 Montpellier CEDEX,Prance, 2Paculte des Sciences de Tunis, Tunisie

In Tunisia, as they are very prosperous on shores and in la­goons, mugils are fishes of a high economical interest. Theyare often parasitized by myxosporidiosis (32 %), a fact thatsometimes decreases their value.The inventory of the various Myxosporodia, parasite ofMugil cephalus, shows evidence of different infested siteson the host (scales, gills' lamellae, gill arch, mesenteric ves­sels, intestine) and variations on the size and aspect of thecysts developed by the parasite.Although, according to their localization, these Myxosporidiadiffer: vascular forms give numerous isolated cysts of 0.2 to3.8 mm in length and 0.2 to 3.3 mm width liable by theirgrowth to block the lumen of the blood vessel in which theyhave settled (it is the case for the gills' cyst). Tissular forms donot appear as isolated and well defined cysts but as a cysticmass of 6 to 9 mm length by 4 to 6 mm width where each massis formed by the juxtaposition of microcysts: this is the casefor the species appearing on the scales (Siau, 1978; Menezes,1984; Bahri, 1993 and 1995). On the gill arch, the scalopedcysts can reach 2.2 to 3.75 mm in length and 1 to 3 mm inwidth.According to the infected area, these Myxosporidia sporesshow, under light microscopy, variable sizes (length of sporalbody: 8 to 14 11m; width of sporal body: 6 to 14 11m); theirgeneral shape can be round or oval. The pear-shaped polarcapsules measure from 3 to 6 11m in length and 1.5 to4,5 11m in width. Our transmission electron microscopy ob­servations show a different sporogenesis: the scales' speciesin rnonospored when all the other Myxosporidia observedon various sites are dispored or polyspored. Although Siau(1978) connected all Myxosporidia found in mullets to Myx­obolus exiguus.According to our results, we can confirm that there are severalspecies of Myxosporidia belonging to the Myxobolus genusin Mugil cepbalus. The monospored species can be connectedto Myxobolus exiguus, our results are similar to those of Siaufor this species. Concerning the others Myxosporidia, couldwe be facing various species?To differentiate these specie, we are pursuing our researchusing two methods: immunology: with the electrophoreticanalysis of the spores from the various species and the immu­notransfer study of their common antigenic fractions: mo­lecular: PCR restriction enzymes analysis.

404 . Abstracts

7Biotecton Investigations in the Lazberc ReservoirD. OSCAR BALAZSNorth-Hungarian Env ironmental InspectorateMiskolc Hungary

Lazberc reservo ir is a 16 m deep reservoir for drinking watersupply in Northern Hungary. Th e water body of the reservoiris stratified consisting of an anaero bic hypolimnion, metalim­nion and aerobic epilimnion. Th e quality of the wa ter iseutro phic. In 298 9 a deep water aera tion system was in­stalled into the lake to aerate the lower water layers of a re­servoir,Th e investigations of biotecton n glass slides in the 3 differentlevels (epi-meta- and hypolimnion ) of the reservoir were car­ried out dur ing the summ er of 1982 and 1991.The volume of the dry matter, the quality and quantity of themost characteristic organisms (Diatoms and settled Pro tozoa)of the biotecton show differences in the biological of th e bio­tecton show differences in the biological compositio n be­tween the anaerobic and aerobic hypolimnion.

8The Decreasing of Parasitemia of Plasmodium vivax inThick Film of Blood of Ills in M alaria Residual FociA. M . BARANOVA, V. P. SERGIEVand T. P. SABGAIDADepartment of medical protozoology, Mar~sinovskyInstitute of Medical Parasitology and TropicalMedicine , Moscow, 119435, Ru ssia

Th e decreas ing of parasit ic density in blood of vivax malariaills can be connected with properties, inherent to given Plas­modium vivax stra in, with nonsterile immunity of habi tantsof endemic regions, inefficient tr eatm ent or mass chemopro­phylaxis. Low parasitemia is difficul t for microscopic diag­nostics thu s the time of research of blood 's film is increase d.97 th ick films of blood were examined in residual malari a foci(Panj region, Tajikistan ) at low level of endemia af~er epi­demic' eradication. Among manifestat ions of malana withshort incubation (July-October) parasitemia in 50 films hasmad e on the average 4262 parasites per mm ', Subsequentyear was achieved the break of transmission by mosquit~es

after insecticide spray. All manifestati ons were afte r long m­cubatio n. The parasitemia in 47 th ick films of blood , exa m­ined from ills on Ma rch- July, was 1550 parasites per mm-'onthe average. The distinctions between gro ups ills are validity(p < 0.05 ).

9On the Control of Photomovements inChlamydomonasC. BECK, H. HARZ, P. HEGEMANN'f, E. HOLLAND* andR. U HLBotanisches Institut der Uni versitar M iinchen,M enzinger Str. 67, 80638 M imchen,*Insti tut fur Biochem ie der Univers ita t Regensburg,93040 Regensburg, Germany

Th e unicellular green alga Chlamydomonas shows twoclasses of light-induced behavioral responses. Weak flashescause alterations of the swimming direc tio n performed by

the differential act ivat ion of the cis- and trans-flagellum, re­spective ly (phototaxis). Brighter flashes cause the cell to stopand afterwa rds to switch transiently to a slower backwardswimming mod e (photophobic stop response). Th e photore­cept or for both, the phototactic and pho tophobic response, isa rhodopsin. Rhodopsin-mediated ion fluxes across the mem­brane bring up changes in intracellular Ca 2+-concent rationand , thereby, control the flagellar beat ing pattern. Electro phy­siologica l measurements have given new insights into thesignal transduct ion chain for photomovements in Chlamydo­mona s: In response to a light stimulus Ca 2+ ions enter the cellvia ion chan nels in the eyespot region . Thi s primary transientinwa rd current is termed photoreceptor curre nt P. Its ampli­tud e is graded with photon exposure. Provided the concomi­tant cell depolarizat ion exceeds a critica l level voltage­sensitive ion channels in the flagellar membrane open. Thefollowing inward curre nt is termed flagellar cur rent F. Aswe kn ow from single cell analysis, whe re we can record fla­gellar beating and photocurrents simultaneously, the flagellarcurrent F is alwa ys correlated with a switc h from foreward tobackward swimming mode and, thus, tr iggers the photoph o­bic stop respo nse. At lower light levels on ly the photorecepto rcur rent but any flagellar current is apparent. We can measurephotoreceptor currents down to single photon levels with cur­rent amplitudes of 200 - 40 0 fA. It is estimated that about150.0 00 Ca 2+ ions enter the cell as a result of a single photonabsorpt ion. Th e corresponding membrane ~~po!arization of15 mV is not sufficient to open voltage-sensitive Ion channelsin the flagellar membrane. Therefore, we assume the photo­receptor current alone to control E hototactic responses inChlamydomonas, probably via Ca "diffusion from the eye­spot to the flagellar ro ots.

10The Secretory Pathway of Scherffelia dubiaB. BECKER and M . M ELKONIANBotanisches Inst itut, Univer sitat zu Koln,D-5 0923 Kaln, Germany

Th e cells of the flagellat e green alga S. dubia are covered bystru ctures of distinct size and shape, called scales. On the cellbody the scales coalesce to form a rigid cell wall ( thec~),

whereas the flagella bear ind ividual scales. Scales co nsistmainly of acidic polysaccharides. and scale-ass.ociated pro­teins (SAPs). SAPs are glycopro tems and conta in N-g~ycans

of the high-mannose, hybr id and complex type. Th e blO!?en­esis of scales was investigated during flagellar regeneration.Following deflagellati on, cells regenerat e flagella .and ~agel­

lar scales within fou r hours. Scales are synthesized m theGolgi apparatus (GA) and obviously move through t~e ~A

by cisterna l progression, since they are never found mSld~

the numerous transition vesicles wh ich accompany the Golgistack. Using a monospecific polycl~mal antibody t<;> a 123 ~Da

SAP and immunogold electron microscopy the biogenesis ofSAP123 was examined. Non-regeneratin g cells contain a poolof SAPs which is located at the plasma membrane, scale re­ticulu m' and GA. Experiments employing tun icamycin indi­cate that th is glycopro tein pool is ut ilized during flagellarregenerati on. Th e GA was isolated and the proteins we:e ana ­lyzed by various extrac tions and 2D gel elect ro phoresis , Theisolat ed Golgi fraction is enriched in .a r~ b ~ h? molog.ue asrevealed by immunoblo~ting. Four ~aJor m~nnslc G~lgI pr?­teins were chosen for rrucrosequencmg. A fifteen ammo acid

N-terminal sequence was obtained for a 60 kDa protein(GP60). Peptide antibodies were raised and are being usedfor immunogold localization of GP60 in the GA of S. dubia.

11Tetrahymena pyriformis Behaviour in the Presence ofthe Simian Rotavirus SAIlM. BENYAHYA1,2, H. LAVERAN3, J. BOHATIER2, 4 andJ. SENAUD2

IDepartement de Biologie, Faculte des Sciences DharMehraz, B.P. 1796 Atlas, Fes (Marocco); 2URA CNRS1944 "Biologie Comparee des Protistes" UniversiteBlaise Pascal - 63177 Aubiere (France); 3HygieneHospitaliere, Faculte de Medecine, B.P. 38 - 63001Clermont-Ferrand (France); "Laboratoire de BiologieCellulaire, D.ER. de Pharmacie. B.P. 38 - 63001Clermont-Ferrand (France)

Ciliate-virus interactions are not thoroughly studied. We triedin this work to define the behaviour of Tetrahymena pyrifor­mis in presence of a simian rotavirus: SAll, which was pro­pagated on kidney cells of green monkey. Tetrahymenamultiplied normally when SAIl is added to its usual culturemedium PPYS. Its increase is slowed down in the EMEM(kidney cells culture medium) with or without virus.Short time after ciliate-virus contact, some viral particles hadbeen found by electron microscopy in the digestive vacuolesand not in other cell compartment. So, this protozoan is cap­able to engulf the SAIL But using immunoflorescence assaymethod, we noted only a slight decrease of viral titer at 24hours like at 48 hours post infection in the PPYS medium.The decrease of viral antigen quantity showed by the ELISAtest in PPYS, is a medium effect. In EMEM the decrease inpresence of Tetrahymena would be the result either of virusaggregation into clumps or virus adsorption onto the ciliatemembrane. When Tetrahymena cells were lysed after theircontact with the SAll in the EMEM, so viral antigen wasdetected.

12Phylogenetic Analysis of Anaerobic Protozoa from theTermite GutM. BERCHTOLD, J. BRANKE and H. KONIGAngewandte Mikrobiologie, Universitat Ulm,89069 D-Ulm

Termites harbour symbiotic microorganisms in their digestivetract, which are necessary for the digestion of wood. Thishindgut flora consists of eukaryotic and prokaryotic microor­ganisms. Especially the flagellates and spirochetes seem to beunique in nature. Since most of these microorganisms are notcultivable yet, their characterization is difficult and their pre­cise phylogenetic position is unknown.The Australian termite Mastotermes darwiniensis possesses atleast six different flagellates and five different spirochete sym­bionts. After isolation of DNA from the hindgut microorgan­isms of this termite, we amplified the small subunit ribosomalRNA (SSU rRNA) genes using PCR. We have cloned the am­plification products and sequenced several clones. Based on

Abstracts . 405

the obtained SSU rRNA sequences the phylogenetic positionof some pro- and eukaryotic hindgut microorganims was de­termined.

13The Ciliate Atlas, a Unique Guide to 300 Species Usedas Indicators of Water Pollution: 2,000 Pages, 6,000FiguresH. BERGER and W. FOISSNERUniversitat Salzburg, Institut fur Zoologie,Hellbrunnerstrasse 34, A-S020 Salzburg, Austria

This treatise comprises 4 volumes with about 500 pages eachand describes in detail the morphology and ecology of about300 species listed by SLADECEK et al. (1981) as indicators ofwater quality. The morphology is documented by 6,153 fig­ures, 2,000 of them are original light micrographs of livingand silver prepared specimens and original scanning electronmicrographs. Moreover several species are redescribed in de­tail (e.g., Dileptus margaritifer, Trachelius ovum, Loxophyl­lum meleagris, Platynematum sociale). Volume IV containsan easy to use picture key (74 pages) for beginners andnon-specialists to all taxa described in Vo!s. I (Cyrtophori­da, Oligotrichida, Hypotrichia, Colpodea), II (Peritrichia,Heterotrichida, Odontostomatida), III (Hymenostomata,Prostomatida, Nassulida), and IV (Gymnostomatea, Lox­odes, Suctorial. We also reviewed the faunistic and ecologicliterature distributed in many thousands small papers. Thisprovides the saprobic evaluation of individual species witha more reliable basis and shows research needs. Vo!s. I andII, out of print since 1993, were reprinted. Thus the completeseries is available! It can be ordered from the Wasser­wirtschaftsamt Deggendorf, Postfach 2060, D-94460 Deg­gendorf, Germany. Price per Volume about 100 GermanMark. Supported by the Osterreichischen Fonds zur For­derung der wissenschaftlichen Forschung (Projekt P8924­Bio) and the Bayerisches Landesamt fur Wasserwirtschaft.

14Mixotrophic Protists in Planktonic Food WebsC. BERNARD, Marine Biological Laboratory, Universityof Copenhagen, 3000 Helsinger, Denmark

Mixotrophy means two types of nutrition, the most often re­garded as heterotrophy (namely phagotrophy) coupled withautotrophy. Mixotrophy seems to occur in almost all protistgroups encountered in the plankton. This is encounteredeither in primarily autotrophic organisms that can displaya phagotrophic behaviour (e.g., in flagellates and in dinofla­gellates) or primarily heterotrophic organisms that harbourpermanent or temporal, algae or plastids, as symbionts(e.g., mainly in ciliates).Various degrees of mixotrophy exist, but it is often suggestedthat mixotrophy should provide the protists with an ecologi­cal advantage, leading to a better adaptation in patchy or poorenvironments. From an ecological viewpoint, the ability ofthese mixotrophic protists to be predators and at the sametime to perform photosynthesis has complicated our viewof the microbial food web.

406 . Abstracts

15Microaerophilic and Facultative Anaerobic CiliatesC. BERNARD and T. FENCHELM arine Biological Laboratory, University ofCopenhagen, 3000 H elsingar, Denmark

Microaerophilic ciliates are aerobic ciliates which prefer lowoxygen tensions (between 1 and 20 % of atmospheric satura­tion ). They are sensitive to high oxygen tensions, and theirtolerance to anoxia is variable. Some of these ciliates, namelyCyclidium d. flagellatum Kahl, Euplotes sp. and E. aberransDragesco, Paranophrys sp. and Strombidium d . sulcatum,show the ability to survive successfully in anoxic environ­ments, where they can perform cell division: they are faculta­tive anaerobes. Grow th rates obtained under anoxia arecomparable to or slightly lower than those of microaerophi­lic conditions (e.g., in Strombidium d. sulcatum). Mitochon­dria, involved in the oxic and anoxic metabolism, do not showobvious ultrastructural variations when the ciliates are main­tained under anox ic conditions. These ciliates may be re­garded as intermediary stages in the evolution, from anaerob ic metaboli sm towards an anaero bic one.

16Vertical Distribution of Benthic Protists in Relation tothe Geochemical Gradients in Shallow MarineSedimentsU. G. BERNINGERMax-Planck Inst. for M arine Microbiology,Fahrenheitstr. 1, D-28359 Bremen

The distribution and activity of benthic protists inhabit ingshallow marine sediments is dependent on a variety of fac­tors, such as chemical gradients, water flow, the availabilityof food and the presence of predato rs. In turn , the hydrolo­gical and biogeochemical parameters in this type of environ­ment underlie frequent changes due to differences in the lightfield, the flow regime, the characteristics of the wat er column,and others. In laborator y flow through systems, light inten­sity, flow velocity and nutr ient load of the water columnabove sediment cores were varied. These changes affectedthe profiles of oxygen and sulfide in the sediment. The che­mical conditions were determined employing microelec­trod es at a vert ical resolution of 100 urn, Sediment samplesfor the enumeration of protists were collected at a verticalresolution of 1- 3 mm.Higher light intensities increased the oxygen concentrat ionand the penetration depth of O2 in the sediments, whereasincreasing flow velocity and raised nutrient load in the waterhad the opposite effect (decreased oxygen concentration andpenetration depth). The ciliate community could be dividedinto two distinct groups. Ciliates considered epibenth ic re­mained close to the sediment surface at all times and werenever found in anoxic layers. In contrast, the majority ofthe ciliates adapted to an interstitial life style were alwaysfound in anoxic sediment layers and appeared to activelymove away from high oxygen concentrations. Interstitial cili­ates probably migrate between oxygenated and anoxic sedi­ment layers and feed in both environments. This implies thatmediated through protozoan grazing and ensuing nutr ient re­generation the flux of matter between oxic and anoxic sedi­ment layers may be increased.

17Development of a Multispecies Terrestrial ToxicityTest Using Ciliated ProtozoaA. BERTHOLDUniversity of Salzburg, Institute of Zoology,Hellbrunnerstr. 34, A-5020 Salzburg, Austria

Protozoan bioassays using well-known ciliates such as Tetra­hym ena sp. or Dexiostoma campylum (formerly Colpidiumcampylum) are widely used for aquatic science. For soils onlytwo single species toxicity tests were recently introduced usingthe indigenous soil ciliates Colpoda steinii and C. inflata.These tests only take bacterivorous species into account.The soil ciliate community, however, also comprises manyhighly specialized fungi-feeders and predators. Litera turedata and recent own investigations show that fungivorousciliates may be the dominant feeding group in forest littersand disturbed soils. Thus, this diversity in feeding strategiesmust not be neglected. Terricolous ciliates are ideal candi­dates for rapid toxicity testing due to their high reprodu ctionrate, their good cultura bility and their storage in resting cysts.The new screening test consists of three parallel series eachusing one ciliate species of a main trophic group, i.e. a colpo­did (bacteriafeeders), grossglocknerid (fungi-feeders) and hy­potrich (predators). The aim is to develop a terrestrial toxicitytest, which is well standardized, fast, reliable, reproducibleand easy to conduct and shows a higher ecological signifi­cance than single-species tests. Methodological details aswell as first results will be presented.

18Contribution to the Understanding of theDinoflagellate Chromosome Structure and to it sReplication Process: A Conventional and ScanningLaser Fluorescent Microscope (CLSM) StudyY. BHAUD, B. GILLET, M. ALBERT, M .-L. GERAUD andM.-O. SOYER-GOBILLARDDeparternent de Biol ogie Cellula ire et Moleculaire,Observatoire Oceanologique de Banyuls-sur-mer,URA CN R S N° 11 7, Universite Paris 6, BP44,F-66651 Banyuls su r Mer, France

The genomic material of dinoflagellate protists is organized inquasi permanentl y compacted chromosomes devoid of anylongitud inal differenciation 1. The nucleofilaments are orga­nized in a super twisted double helix? which architectur e ismaintained by divalent cations! and structural RNAs4 . In or­der to localize the replication sites of these chromosomes andthe progression of the DNA replicat ion along the S-Phase, wehave incorporated in vivo the thymine precursor, bromode­oxyuridine (BrdUrd), into the DNA during 6 days, takinginto account the course of the complete cell cycle (132 h)of the autotrophic dinoflagellate Prorocentrum micans Ehr.(S-phase: 4 h)5. The newly synthetized DNA was detectedwith an anti-BrdU Antibody" coupled to FITC on squashedor cryosectioned cells and the ancient DNA was contras tedeither with propid ium iodide for CLSM or with DAPI for con­ventional fluorescent light microscopy. We have localized thefirst origins of replicat ion on one tip of several chro mosomesclose to the persistant nuclear envelope. In the same nucleus,the initiation of the DNA synthesis is not synchronous. Bymeans of CLSM we have observed the spatial organizationof the replication domains": the neo-synthetized DNA orga-

nizes in helix around the ancient DNA. These results are con­firmed by T.E.M. In the light of these results, the polytenicorganization of the chromatids is discussed.References:1 Haapala OK, Soyer MO (1974) Hereditas, 78, 141-1452 id, 1973, Nature, 244, 195-1973 Herzog M, Soyer MO (1983) Eur. J. Cell BioI. 30, 33-414 Soyer MO, Herzog M (1985) id 36 (2) 334-3425 Bhaud Y, MO Soyer-Gobillard (1986) Protistologica 23 (1)

23-306 Just, Wet al. (1993) Cytogenet. Cell Genet., 62 60-637 Mazzotti G. et al. (1990) J. Histochem. Cytochem. 38(1)

13-22

19Genome Organization of the MicrosporidiumEncephalitozoon cuniculi: Molecular Karyotype,Chromosome-specific DNA Probes and Gene LocationC. BIDERREt, E. PEYRETAILLADEl, P. PEYRET 1, M. PAGES2

and C. P. VIVARES 1

lLBCP, Protistologie mol. cell. des parasitesopportunistes, URA CNRS 1944, UBP, 63177 AubiereCedex France; 2Lab. genomes des parasites, UMI, rueA. Broussonnet, 34000 Montpellier France

Among unicellular eukaryotes, micro sporidia are obligatelyintracellular amitochondrial parasites and are considered tobe of very ancient origin as deduced from the prokaryoticfeatures of their ribosomes and rRNA phylogenies.In the species Encephalitozoon cuniculi, an AIDS opportunis­tic parasite, DNA separation was performed by pulsed-fieldgel electrophoresis (PFGE). We show that its electrophoretickaryotype is organized in 11 chromosomal bands within anarrow size range (from 217 to 375 kb). This correspondsto the smallest haploid genome (2.9 Mb) known for a mono­nucleate eukaryotic organism.A partial DNA library, prepared from total DNA ofE. cuniculi by shotgun, has allowed us to characterize 64DNA probes (size range of 200-1500 bp, content GC50%) specific for ten of the eleven chromosomes. These chro­mosome-specific DNA probes could then be used as chromo­somal markers for mapping studies or for examiningallocation in other strains of E. cuniculi.In order to gain insight into the molecular organization of therRNA and ~ tubulin genes in this peculiar organism, we haveidentified the chromosomes on which these genes reside. Totalgenomic DNA was subjected to PCR and the amplified frag­ments (1200 pb for rRNA and 400 pb for ~ tubulin) werecloned into plasmid pUC 18 and sequenced. These fragmentswere used as probes for the Southern hybridization and theiranalysis revealed that rRNA genes are on all chromosomeswhile ~ tubulin genes are present on chromosomes II and III.

Abstracts . 407

20Studies on the Whey Degradation by TetrahymenapyrifonnisP. BOGAERTSt, C. A. GROLIEREt, J. BOHATIER1,2 andJ. SENAUDI 1

lLaboratoire de Biologie Comparee des Protistes,U.R.A. C.N.R.S. 1944, Universite Blaise Pascal,63177 Aubiere Cedex (France); 2Laboratoire deBiologie Cellulaire, Faculte de Pharmacie, Universited' Auvergne, 24, Place Henri Dunant,63001 Clermont-Ferrand Cedex (France)

The potentialities of the ciliated protozoan Tetrahymenapyriformis to transforme the whey were studied. The wheywas beforehand filtered on a 0.45 11m mesh in axenic condi­tion.In a first time, 100 ml of undiluted whey were seeded withabout 5 x 105 cells/ml of an axenic culture of ciliates. Theresults show a decrease of 25% of lactose concentrationand of 66% of proteine concentration respectively. The secre­tion of ~-galactosidase, responsible for hydrolysis of lactose,and of a protease (trypsinase) increases a lot of during thetime.In a second experiment, we followed the growth of the ciliatespopulations on various dilutions of whey (1,1/4 and 3/4) andin presence or without Enterobacter aerogenes. We observeda most important growth in undiluted medium and 1/4 di­luted medium with the bacteria during the first week. Simul­taneously, we noticed a stronger fall of protein concentrationin both media in absence of bacteria than in their presence.For lactose concentration, it was the contrary. In all cases,the enzymic activities increased, especially ~-galactosidase ac­tivity, which was most important with Enterobacter aero­genes in undiluted medium and 1/4 diluted medium.By light microscopy, we observed the ingestion of whey ele­ments by the ciliate with the formation of digestive vacuoles.In transmission electron microscopy, we can see endocyticvacuoles formation from plasma membrane.

21Calcium Sites and Calcium Binding Proteins ofToxoplasma gondiiA. BONHOMME, L. PINGRET, P. BONHOMME, G. BALOSSIER,M. PLUOT~- and J.-M. PINONINSERM U.314 CHU Reims "Laboratory ofAnatomo-Pathology, CHU Reims - France

Toxoplasma gondii, the etiological agent of toxoplasmosisrequires an intracellular site for growth and replication. Itsinvading process is active and depends on energy sourcesand cations such as calcium. Ca 2+ sequestering organelles,Ca 2+ binding proteins and ionic pumps like Ca 2 + ATPaseare studied in order to determine the Ca 2+ metabolic eventsof tachyzoites. In the whole freeze-dried tachyzoites, Ca 2+

concentration appears to have a homogeneous repartitionin the anterior, middle and posterior regions of the cell(27 ± 29 mMoles/kg of dry weight) except in the middle pos­terior area (nucleus area) where Ca 2+ concentration is 15 foldhigher (450±310 mMoles/kg dry weight). X-ray microana­lyses of sections of cryofixed, cryosubstitued and cryoem­bedded tachyzoites conclude a variation of the Ca 2+

distribution from 1-45 mMoles to 100 mMoles and evensuperior to 100 mMoles/kg of resin. The nuclear and perinu-

408 . Abstracts

clear area with endoplasmic ret iculum and mitochondriashows the higher Ca concentratio ns associated with higherP concentrations. Calmodulin (CaM), the ubiquitous modu­lator of cellular calcium functions, was detected by immun o­blotting of tachyzoite soluble antigen, and estimated toconstitute 1.8 % of the total protein of the tachyzoite (i.e.:5.3 ug/rng of protein). CaM is distributed in the nucleus,the conoid and in the secreto ry organelles: microneme s, espe­cially in the dense granul es and to a lesser extent, in the rhop­tries. Two major localizaions of Ca z+ ATPases weredetermined; one associated with the membrane complexwhere Caz+ ATPase acts as a Caz+ pump for Caz+ uptakeand efflux pathways and the other is the nucleus area {nu­cleus, and endoplasmic reticulum}. The immunolabeling ofthese two areas was thre e times higher than that of secretoryorganelles. Nucleus area and, to a lesser extent, secretory or­ganelles appear as intracellular Caz+pools with a capacity forCaz+ sequestration and release. Caz+/CaM complex could beinvolved in the main event of the penetration and the inter­nalization of the parasite: the secretion of the secretory orga­nelles. Rhoptries exocytose a Caz+ dependent PLAzand a Caz+stimulated penetration enhancing factor (PEF) and densegranules release a Caz+ binding protein GRA1 (P23). More­over it could also regulate the Cal. ATPase in order to main­tain the intracellular calcium homeostasis.

22Soil Ciliates as Bioindicators: A Study in Urban AreasS. BONI!, F. ERRAt, A. D'ULlV02, L. LAMPUGNANI2 andF. VERNI llDipart imento di Scienze dell'Ambiente e Territorio,Universita di Pisa, 2Istituto di Chimica Strumentale delCNR Italy

The community of soil ciliates have been used as bioind icatorsto monitor five urban zones in the city of Pisa. The ciliatefauna present in the stations were exam ined for a period ofseven months (February-Au gust 1994). The results obtainedin the urban area were compared between them and also witha station localized at 15 km from the city. The index of gen­era l diversity (Shannon , H) has been used to caracterize thecommunity structure. The presence of lead in the moss hasbeen also verified. The comparison between the H and thequantity of lead present in the samples of moss was indicativeof an inverse relationship between these two variabl es. Thepresence of a major quantity of lead (1.500 ppm ) in the mossspecimens was indicative of a high traffic-road. The analysisof the ratio Ciliates/Metazoa (Rotifera-Nematoda-Tardigra­da) indicated that the ciliate community reacted rapidly tothe ambiental stress than the metazoan community.

23Protozoa in the Rhizosphere of Hordelymus europaeusM . BONKOWSKI, J. ALPHEI and S. SCHEUII. Zoologisches Institut, Abt . Okologie, Berliner Str,28, D-37073 Gottingen, Germany

Influence of protozoa, nematodes and earthworms on micro­bial bioma ss, nutri ent cycling and plant growth was studied inmicrocosms with the grass Hordelymus europaeus L. In con­trast to endogenic earthworms and bacteria feeding nema­todes, protozoa had strong influence on each of the

parameters investigated. Protozoa increased shoot biomassof H. europaeus by 22%, root biomass by 58%, while C­and N-content of plant s was significantly reduced. Prot ozoaalso increased the N O.3 content of soil water by a factor of 10,whereas the mineral nitrogen content of the soil was de­creased by 72%. Reduction in microbi al biomass by proto­zoa was more pronounced in nonrhizosphere soil (- 31% )than in rhizosphere soil (- 4% ). On average, protozoa re­duced total microb ial biomass by 13%, but did not affectthe respirato ry activity of microorganisms.

24Effect of Cadmium on the Functioning and theMicroorganisms of Wastewater Lagoon LaboratoryPilotsJ. L. BONNETt, M. SIMONUTTI l, C. A. GROLIEREt,D. PEPIN2 and G. FOURNERET31Laboratoire Biologie Comp aree des Protistes, URACNRS 1944, 63177 Aubiere Cedex. 2LaboratoireHydrologie, Hygiene et Environnement, UFR dePharmacie, BP 38, 63001 Clermont-Ferrand Cedex.3SATESE du Puy de Dome, BP 43, Marmilhat,63370 Lempdes

The laboratory pilots which were previously perfected andvalidated, simulate in a satisfactory way the treatment pro­cess of a sewage treatment lagoon. We have used these pilotsto assess the effects of an heavy metal, the cadmium, on thefunctioning, the distribution and the activities of the micro­orga nims in these ecosystem. Th ree systems receiving thesame effluent were put into experiment simultaneously. Fol­lowing the necessary sta bilization period of about one month ,the first system served as control; the second and the thirdwere contaminated only once by CdClz with final concentra ­tions of cadmium of 0.06 mg/I and 0.30 mg/I in the lagoonwater respectively.The treatment efficiencies decreased progre ssively in the trea­ted systems after adding the cadm ium in the water. We ob­served that the nitrate concentration increased with toxicconcentration. The dissolved oxygen concentration de­creased strongly before a progressive return towards initialvalue. The effect of cadm ium on the total bacterial popu la­tion was relatively weak while we noticed a concentrationdependant decrease on autotrophic and heterotrophic flagel­lates density. For the ciliates, the effect seemed to be less sig­nificant. Global esterasic activity, essentially microb ial,measured in spectrophotometry with fluorescein diacetate,was enhanced very quickly in presence of the toxin in thewater.

25Evidence for an Immunoanalog of TetrahymenaEpiplasmins as a Component of the IntermediateFilament Network in Mammalian Central NervousSystemP. BOUCHARD, B. VIGUES, V. RAVET and A. VELLETLaboratoire de Biologie Comparee des Protistes, URA1944 CNRS, Universite Blaise Pascal, 63177 AubiereCedex, France

The membrane skeleton in ciliated protozoans, called "theepiplasm" is well developed and serves to anchor kineto­somes and other structures at the cell surface. Epiplasmic pro­teins characterized so far in ciliates exhibit a strongheterogeneity in number and apparent molecular weight; asituation that closely resembles that described for intermedi­ate filaments in higher organisms. Thus far only a few studieshave examined the question of the relationship between theepiplasm and intermediate filaments. The purpose of the pre­sent investigation was the characterization of immunoanalogsof epiplasmic proteins among members of the main classes ofIF in mammalian cell lines and tissues. This was accomplishedusing monoclonal antibodies raised against "band C" a majorepiplasmic component in Tetrahymena pyriformis. We reporthere that anti-band C antibodies cross-react with a 51 kDaprotein found in brain, cerebellar, spinal cord and in theC6 neuroglioma cell line. Using double immunolabelling ex­periments and molecular cloning methods, we demonstratethat the 51 kDa antigen is GFAP,the glial fibrillary acidic pro­tein, a class III IF protein expressed in astrocytes and cells ofglial origin. Experiments are underway to characterize theprotein sequence of band C which should provide further in­sights into the relationship between the epiplasm and inter­mediate filaments.

26Graviresponses in CiliatesR. BRAuCKER and H. MACHEMERArbeitsgruppe Zellulare Erregungsphysiologie, Ruhr­Universitat, D-44780 Bochum, Germany

Ciliates, like other organisms, have to orient in their environ­ment. The most constant physical parameter for orientation isthe direction of the gravity vector. Consequently most ciliatesexamined show a gravitactic behaviour. Although geotaxish~s been ~nown especially in Paramecium since the begin­nmg of this century there was some uncertainty about thepossible mechanism.Having analysed the behaviour of large populations of ciliateswe now have evidence, that gravitaxis is not only a physicalphen.o~en.on, but includes a physiological component, calledgravikinesis,By comparison of graviresponses of different ciliate speciesand by modification of the gravity stimulus we got indica­tions that gravisensors may be similar to the mechanorecep­tor channels which are well described by electrophysiologicalmethods.

Abstracts . 409

27The Actin of Trichomonas vaginalis: A MolecularStudyG. BRICHEUXUniversite Blaise Pascal, URA 1944, 63177 AubiereCedex

Actin represents one of the major proteins in Trichomonasvaginalis. eDNA and gDNA libraries of T. vaginalis havebeen screened using a chicken eDNA actin probe. One gDNAclone and 5 cDNA clones have been selected and sequencedby the dideoxynucleotide chain termination method. The nu­cleotide sequence of the gDNA clone has been determined andthe potential protein deduced. The encoded protein is 376amino acids long. Unlike the actin genes from most other or­ganisms and like all known Trichomonas genes, it does notcontain introns. By Northern blot analysis a single speciesof RNA of 1.3 kb was identified. The sequencing of the dif­ferent clones has shown at least 4 different genes, but the ami­no acid sequence of all is nearly identical. It is one of the mostdivergent actin sequenced to date, being only about 75%homologous to many actins. There is a potential TATA box90 bp upstream from the start site, but the A+T-rich natureof the non coding region makes it hard to identify with cer­tainly. A "TCATTTTTCT" motif could represent the con­served DNA sequence supposed to initiate the transcriptionand found upstream of all T. vaginalis protein-coding genesreported to date.

28The Presence of F-actin in the Microsporidian SporeA. M. BRONNVALLDep. of Zoology, University of Lund, Helgonaviigen 3,5-223 62 Lund, Sweden

There are very few cytochemical investigations made concern­ing microsporidia. In the 70's Vavra showed the existence ofpolysaccharides in the polar filament and anchoring discusing periodic acid-Schiff (PAS). Also glycoproteins havebeen found in the polar filament, spore wall and polaroplasta,nd chit~n in the spore wall. I decided to investigate the pos­Sibleexcistence of a cytosceleton using fluorescent probes andconfocal laser scanning microscopy (CLSM).There are probes for three major types of cytoskeleton: tu­bules (tubulin), intermediate fibers (F-actin) and microfila­ments (G-actin). I used a probe for intermediate fiberssince they are most commonly found beneath the plasmamembrane. The microsporidian, Tuzetia lipotropha, where~he spores measure approximately 2 x 4 urn, was fixedin GA for 24 h, washed in buffer, and incubated withBODIPY-phallacidin for two hours.The result was a heavy incorporation of the probe beneath theplasma membrane, but due to resolution-problems the exis­tence of intermediate fibers could not be proven. However,the probe is also incorporated into the polar filament andthe anchoring disc, raising the question if the extrusion ofthe polar filament is purely due to pressure change in thespore or if there is also an active evagination of the filament.

410 . Abstracts

29Phylogenetic Relationships Inferred from SmallSubunit Ribosomal DNA Sequences Demonstrate thatVahlkampfia species do not belong to one GenusS. BROWN and]. F. DE ]ONCKHEERECulture Collection of Algae and Protozoa, Institute ofFreshwater Ecology, Ambleside, U.K. and Institute ofHygiene and Epidemiology, Brussels, Belgium

Molecular biology techniques have been applied to the iden­tification, differentiation and phylogeny of species of free-liv­ing amoebae assigned to the genus Vahlkampfia. The degreeof inter-specific differences in small subunit ribosom al DNA(SSU rONA) sequence, detected using PCR amplification andriboprinting techniques, allows new strains to be assigned tospecies and their phylogenetic relationships to be investigated.The sequence diversity within the genus was confirmed whenthe SSUrONA of described Vahlkampfia spp. was sequencedand compared with published sequences from V. lobospinosaand species representing other genera of the family Vahl­kampfiidae; the number of nucleotide differences betweensome Vahlkampfia spp. significantly exceeds those betweenthe genera. Consequently, Vahlkampfia spp. do not form asingle cluster in the phylogenetic trees generated from the se­quencing data, suggesting that some Vahlkampfia spp. shouldbe considered distinct genera .

30Oxidant Defense System of Entamoeba histolyticaI. BRUCHHAUS and E. TANNICHBernhard Nocht Institute for Tropical Medicine,Bemhard-Nocht-Str, 74, 20359 Hamburg, Germany

All protozoan parasites studies so far have been found to con­tain at least one of the major anti-oxidant enzymes, e.g. super­oxide dismutases, catalases or glutathione peroxidases. Theseenzymes are important for the protection against host-gener­ated oxidants. In Entamoeba histolytica the detoxification ofsuperdoxide radicals is catalyzed by an iron-containing super­oxide dismutase. Cultivation of the amoebae in the presenceof superoxide radical anions or a ferrous iron chelator re­vealed substantial increase of SOD expression. On the otherhand, E. histolytica does not possess any catalase or peroxi­dase activity. Therefore, its ability to inactivate hydrogenper­oxide and organic hydroperoxides remains obscure.We isolated two genes of E. histolytica encoding polypeptidesof 316 and 233 amino acid residues with molecular masses ofabout 34 kDa and 29 kDa, respectively (Eh34 and Eh29).Each polypeptide revealed striking similarities to one of thetwo subunits of the alkylthydroperoxide reductase (AHP)from Salmonella typhimurium. Eh34 was found to be homo­logous to the F52a subunit of AHP (25% identity) and to thethioredoxin reductase from Escherichia coli (27% identity).Highly conserved between the three proteins are a pair of re­dox-active cysteines, two FAD binding domains as well as anNAD(P) binding domain. Eh 29 was revealed to be similar tothe C22 subunit of AHP (23% identity), which is involved insubstrate binding of hydroperoxide.AHP plays an important role in protecting bacteria againsthydroperoxide mutagenesis. Therefore, it is suggested thata similar enzyme takes part in the protection ofE. histolytica against toxic oxygen metabolites.

31Trimastix convexa a Free-living AmitochondriateFlagellate without Close Relationships withPercolozoa, Retortamonad or Trichomonad FlagellatesG. BRUGEROLLEBiologie des Protistes, Universite Blaise Pascal deClermont-Ferrand, 63177 Aubiere Cedex, France

Trimastix convexa is an heterotrophic flagellate living inanaerobic habitats. It has been described by A. Hollande inGrasse 1952 as bearing 4 flagella, one forming an undulatingmembrane with the rim of the ventral groove, possessing aparabasal, an axostyle and a large nucleus and nucleolusand a contractile vacuole. It has been considered to be theancestor of the trichomonads.Microscopic studies showed the basal bodies are arranged intwo pairs, from which arise 3 Mt roots. Two Mts roots arelining the rims of the ventral groove; the right one gives alsoMts under the groove membrane, the left one is associatedwith a microfibrillar/striated structure. The third Mt rootserves as a MTOC for Mrs radiating on the dorsal surface.The right rim of the groove is thinner than the right one.The recurrent flagellum in the groove has 2 unequal fin-likeextensions. The Golgi apparatus situated near the basalbodies is not supported by a para basal fibre. A contractilevacuole-like is positioned backward to the nucleus. The cyto­plasm does not contain mitochondria, but some hydrogeno­some-like bodies with 2 closely apposed membranes whicharc surrounded by rough endoplasmic cisternae.The organization is compared to that of Percolozoa relatedflagellates: Percolomonas , Psalteriomonas, Tetramitus andto other amitochondriate flagellate Trichomonads, Retorta­monads, Metamonads from which it differs.

32Genetic Comparison of Virulent and Non-VirulentLeishmania major StrainsS. A. BULAT and M. V. STRELKOVAMartsinovsky Institute of Medical Parasitology andTropical Medicine, Moscow 119830, Russia

Two groups of Leishmania major strains of different virulencehave been studied by using PCR technique with set of univer­sal primers. The first group was represented by four highlyvirulent strains, the second one consisted of three non-viru­lent strains and one low virulent . To select the proper primerable to differentiate two L. major groups 29 single universalprimers of unique design (Bulat et al., 1990) have been tested.Most of them appeared to be unsuitable for differentiation.These primer combinations failed to yield the desired effecteither. Primer AA2MI was the only exception. By makinguse of the above mentioned primer as the basic one in com­bination with other universal primers another attempt hasbeen made to differentiate highly virulent and non-virulentstrains in the two-primer system. As a result a combinationof AA2MII15119 was obtained that was able to differentiatehighly virulent and non-virulent strains, though it failed todifferentiate highly virulent and low virulent ones. Thus, incase the obtained results were proved on a greater samplingof non-virulent strains it would be possible to suggest a geneti­cally found L. major virulence determinants or loci chainedtogether.

This investigation has received financial support from theUNDPlWorid BankIWHO Special Programme for Researchand Training in Tropical Diseases (TDRIDIF grant ID920645).

33Digestion of 14C-Iabelled Gram( -) and 14C-IabelledGram (+) Bacteria by Living Cells and Cell-extracts ofParamecium multimicronucleatum and Parameciumcf. sonneborni at Different Temperatures and pH­valuesA. BURZLAFF*, K. HAUSMANN~- and J. WECKE''''"Division of Protozoology, Institute of Zoology,Free University of Berlin, Konigin-Luise-Str, 1-3,14195 Berlin. "'~Robert-Koch-Institute of the FederalHealth Office, Nordufer 21, 13353 Berlin

E. coli was labelled by growth in 14C-palmitate for 120 min­utes. 75% of the incorporated 14C-palmitate was incorpo­rated into phospholipids. The labelled bacteria were fed toP. multimicronucleatum. 40 minutes after beginning of theingestion paramecia were separated from not ingestedE. coli by filtration. 95% of the 14C-label of the bacteriawas incorporated by the paramecia. Eight hours after the be­ginning of the ingestion the lipids and phospholipids of thelabelled paramecia were extracted by the method of Blighand Dyer (1959). The phospholipids were separated by thinlayer chromatography and autoradiography. The 14C-Iabelofthe phospholipids of the bacteria was incorporated into otherclasses of phospholipids by the paramecia.The cell wall of Staphylococcus aureus was labelled with 14C_N-acetyl-glucosamin. The labelled living bacteria or labelledheat-inactivated bacteria were added to a cell homogenate ofP. multimicronucleatum and P. cf. sonneborni at differenttemperatures and pH-values. By measuring the release ofthe radioactive marker into the medium it could be shownthat the cell extract was able to digest living and heat-inca­tivated bacteria. The influence of different pH-values wastested at 28°C. The digestive enzymes of the cell extractsof P. multimicronucleatum and P. cf, sonneborni showedthe highest activity at pH 6. Seven hours after the beginningof the digestion 80-90% of the radioactive marker of the bac­teria was released. A temperature of 19°C did not influencethe activity of the digestive enzymes at pH 6. However, at atemperature of 37°C and pH 6 the digestive enzymes of thecell extracts showed an increased activity. Within one hour100% of the radioactive marker of the bacteria was re­leased. The heat-inactivated bacteria were digested fasterthan the living bacteria.

Abstracts . 411

34Use of a Soil Protozoan Bioassay to Assess the Toxicityand Bioavailability of Heavy Metals in a Long TermSewage Sludge Treated SoilC. D. CAMPBELL, A. WARRENZ, C. M. CAMERON! andS. J. HOPE2

lMLURI, Craigiebuckler, Aberdeen, AB9 2QJ, UK;2The Natural History Museum, Cromwell Road,London SW7 SBD, UK

A simple bioassay of the growth and reproduction of the com­mon soil ciliate Colpoda steinii was used to examine the soilsolutions from a long term sewage sludge experiment. Soilsfrom the Lee Valley experiment, which were treated usingsewage sludge rich in either Cu, Ni or Zn, were extractedusing a centrifugation technique to obtain soil solution andcompared to soil from control plots which had receivedeither no sludge or uncontaminated sludge. The soils, whichhad been previously air dried, were re-wetted and sown withclover plants to restore normal biological activity. The chemi­cal composition of the soil solution was measured and therelationships between cell growth, metal concentration, pHand dissolved organic carbon were examined. The bioassayshowed that the highly contaminated soils were significantlyinhibitory to C. steinii growth and were most toxic in the or­der Cu>Ni>Zn. This finding is in contrast to other studies onother soil types indicating the bioavailability may be differentin this soil. The response to individual metals was modelled bycurve fitting and an effective concentration resulting in a 50%reduction (ECso) in relative growth was predicted at 1.44 and4.22 mg/l for Ni and Zn respectively. These values are muchgreater than those obtained with single metal salt solutionswith high free ion activity and suggest that significant quan­tities of the total metals measured were either in an unavail­able form or that non-toxic competitive ionic species anddissolved organic compounds in solution had increased thetolerance of C. steinii to toxic metals.

35Erythrocyte Recognition by PlasmodiaD. CAMUSINSERM, U 42, Villeneuve d' Ascq, France

The entry of Plasmodia inside host cells depends on a specificreceptor-ligand interaction which determine a close contactbetween erythrocyte and merozoite membranes. It has beenclearly demonstrated that glycophorins and particularly thetetrasaccharides of glycophorin A, rich in sialic acids playan essential role for the invasion of human erythrocytes byP. falciparum. Erythrocytes with abnormal forms of tetrasac­charides, such as Tn cells lacking one galactose-sialic acidresidue or Cad cells with an extra N-acetyl galactosamine,are resistant to infection. Moreover, the removal of the 9­O-acetyl group from sialic acids of mouse erythrocytes whichconverts the sialic acid to the human form enhance the bind­ing of the parasite receptor. However, it has been possible toadapt either a P. falciparum strain in vitro to Tn cells or aP. falciparum clone to neuraminidase-treated erythrocytes(lacking sialic acids). It has been postulated that a switchin a receptor-binding protein of the merozoite changed thespecificity of invasion.

412 . Abstracts

The P. [alciparum receptor mainly involved in the attachmentof merozoite s to erythrocytes is an Erythrocyte Binding Anti­gen of 175 kDa (EBA-175). Hom ologou s prote ins have beendescribed for P. vivax and P. knowlesi. This pote ntial targetfor vaccine development does not appear, in fact, as one of thebest candidates for the following reasons: (i) Specific antibo­dies against the binding domain of the EBA-175 were raised inrabbits. The immune sera is able to inhibit the binding of EBAto erythrocytes but must be used at a dilution of less than 1/5to significantly inhibit the invasion of erythrocytes by mero­zoites. (ii ) Different strains of mice were immunized with a 43amino acid pept ide corresponding to the erythro cyte-bindingdomain of EBA-175. In this experiment H-2 b mice were un­able to produce ant ibodies. Moreover, in view of the restora­tion of the immune response using peptides coupl ed to acarrier, EBA-175 was tested after being coupled to either te­tanus toxoid or tri-palmitoil-LYS. Anti-EBA-175 antibodieswere not obtained in H-2 b mice. (iii) Sera from patients in­fected with P. [alciparum were incubated with erythrocytescovered with EBA-175. Under these conditions antibodiesform immune complexes which elute the EBA-175 fromthe erythrocyte surface and are found in the supernatant .Moreover, the cell ligand appears to be accessible for the bind­ing of a new EBA-175 parasite receptor. If this phenomenomoccurs in infected humans , it could be efficient for the parasiteto evade the antibody response and could explain why rabb itsera were only efficient when used at low dilution.

36Molecular Phylogeny of ZooflagellatesT. CAVALIER-SMITH and E. E. CHAODepartment of Botany, University of British Columbia,Vancouver, Canada V6T 1Z4

We shall discuss molecular evidence, including our own un­publ ished 18s rRNA data, on the diversity and relati onshipsof zooflagellates (phagotrophic heterotrophic flagellateswithout plastids). Althou gh most zooflagellates are probablyprimitively without most zooflagellates are probably primi­tively without chloroplasts, our trees suggest that the pedinel ­lids Ciliophrys and Peteridomonas and the colourlesschrysomonad Oikomonas evolved from ochristan algae bythree independent losses of plastids within the Heterokon­tao Thus zooflagellates are polyphyletic. Trepomonas agilisgroups with Hexamita and Giardia, refuting the suggestionthat their early divergence is an art efact caused by parasit­ism. Th e opalozoan Apusumo nas is specifically related to an­imals, fungi, and choanoflagellates, whereas the sareomonadsHeteromita, Cercomonas, and Tbaumatom onas are related totestate filose amoebae, which probably arose polyphyleticallyfrom them, and to chlorarachneans. Sarcomonads, chlorar­achneans and filoseans constitute a revised phylum Rhizop o­da, which excludes the Lobosea as a separate phylumAmoebozoa, which clearly lost cilia independently from Filo­sea. The non-flagellate proti st Corallochytrium is a specificrelative of choanoflagellates and is placed in a separate classCorallochytrea within the phylum Choanozoa.

37Validity of Nitrate-Reductase, Glutamine-Synthetaseand Glutamate-Deshydrogenase Activities as aMeasure of Potential Inorganic Nitrogen Utilization byPhytoplankton in Eutrophic Lake Aydat (France)M. F. CHARPIN, C. M ALLET, M . RICHARDOTandj. DEVAUXLaboratoire de Biologie comparee des Protistes, URACNRS 1944, Universite Blaise Pascal , 631 77 AubiereCedex

Dur ing the combi ned nitrogen-depleted summer stra tificationof eutrophic lakes phytoplank ton have been shown to para­doxally exhibit substantia l levels in biomass and primaryproduction suggesting that phytoplankton populations devel­oped enhanced nutrient-uptake capacities or enzymatic adap­tat ion to environmental constra ints. In order to assessvariations in inorganic nitrogen-utilization abilities of phyto­plankton community, activiti es of several enzymes involved innitrogen-assimilation were determined over the phytoplank­ton seasonal succession in eutrophic Lake Aydat. Nitrate-re­duct ase (NR) activity was used to estimate the potentialnitrate-assimilation by cells and the sum of GS (glutamine­synthetase) and GDH (glutamate-dehydrogenase) activitiesas an estimate of potent ial combined inorganic-nit rogen as­similation. The enzymatic activities and several biomass para­meters exhibited similar spatial and temporal distribut ionpatterns. The enzymatic activities were homogeneously dis­tributed from the surface to 10m as water temperatu rewas vertically homogenous. However the therm al stra tifica­tion induced higher levels in the epilimnion than in deeplayers. GDH activities showed punctual variations, whileGS-activity was detected all over the year. Therefore, theGS/GOGAT pathway seemed to be the main pathway of in­organ ic nitrogen assimilation in phytoplankton corroboratingnumerous laboratory experiments. However, when a GDHactivity was detected , it exceeded about sixfold the GS activ­ity. The NR activities were periodicall y distributed. In addi­tion, the results showed that nitrate accounted for about 30%of the inorganic nitrogen assimilated by the phytopl anktoncommunity of Lake Aydat.Clearly, the indirect estimat ions of the inorganic nitro genwhich is uptaken by phytoplankt on yielded higher inorganicnitrogen utilization rat es than assimilation rates (i.e., calcu­lated from enzymatic activities of GDH and GS).

38Chromatin Comparative Study of Colpoda inflataStarved Cells and Increasingly Aging Resting Cysts,Using Microphotometric Image Analysis MethodsM. G. CHESSA, P. PELLl, G. NICOLO * , M. U. DELMONTECORRADO and E. M ARGALLO ' f

Ist ituto di Zoologia, Universita di Genova, and"Servizio di Anatomia e Citoistologia Patologica,Istituto Nazionale per la Ricerca suI Cancro, Genova,Italia

Previous cytofluorometric results showed macronuclear DNAsynthesis activity taking place in Co/poda inflata during tran­sition from starved cell to resting cyst, as well as throughout along-term encystment. Th is accounts for a chrom atin com­parative study both of the macronucle us and the macronu­clear extrusion bodies, in Feulgen-thionin stained starvedcells and increasingly aging resting cysts. The Becton Dickin-

son CAS 200 Image Analysis System supplied with Cell Mea­surement Program (CMP) and Quantitative DNA Analysis(QDA) software modules were used. Moreover, the studywas also referred to micronuclear chromatin aspects, basingupon the evidence that resting encystment can possibly reju­venate senescent cell lines in Colpodidae.This research was supported by M.U.R.S.T. 40% 1994 grant.

39Signal Transduction in Tetrahymena thermophilaRequired for Cell Survival and Proliferation. Role ofInsulin, Nitric Oxide and Cyclic GMPS. T. CHRISTENSEN and K. KEMPInstitute of Medical Biology, Department of Anatomyand Cell Biology, Odense University, Campusvej 55,5230 Odense M, Denmark

Tetrahymena thermophila do not grow simply because it hasadequate nutrition, but seem also to require autocrine signalsfor survival and/or proliferation. This requirement manifestsitself when cells are inoculated into low density cultures inculture flasks in a nutritionally complete, synthetic mediumwhere the amounts of the cell-produced signals or growthfactors become too dilute in the medium for the cells to re­spond and they rapidly die. The identity of the growth factorsare so far unknown, but we suggest that they are insulin-likeand may act through the activation of an NO-dependent (so­luble) guanylate cyclase. Thus, we have shown that additionof insulin in nanomolar concentrations as well as cell-freesamples of conditioned medium rescues T. thermophila fromdying and promote cell proliferation, and it has been reportedthat Tetrahymena produce and release insulin-like materialwith bioactivity in adipocytes. Insulin and nitroprusside (acompound releasing nitric oxide, NO) have been shown tostimulate production of endogenous cGMP in Tetrahyme­na, and we have demonstrated that also nitroprusside, proto­porphyrin IX, free fatty acids and 8-bromo cGMP addedseparately to low density cultures rescues the cells from dy­ing. In agreement with this hypothesis, cells in cultures bothin the absence and in the presence of insulin supplementedwith either N-methyl-L-arginine (NMA) (which inhibits theproduction of NO via the NO-synthase), or methylen blue(MB) (which blocks the activity of soluble guanylate cy­clase) cannot go through cell division. Addition of either ni­troprusside, protoporphyrin IX or 8-bromo cGMP bypassesthe inhibitory effects of NMA, but only 8-bromo cGMP iscapable of eliminating the effects of MB. We have also foundthat insulin produces a biphasic response in T. thermophila bystimulating cell survival and proliferation in two separateconcentration intervals: at micromolar to high nanomolar le­vels and at low picomolar to femtomolar levels. The reasonsfor this pattern are unknown, but one might construe that thebiphasic response is due to receptors with different affinitystates for insulin, or to events of an inhibitory/desentitizationbetween separate receptor systems.

Abstracts . 413

40Comparison of Catalase and Glutathione PeroxidaseActivities in Babesia hylomysci Obtained in vivo andin Babesia divergens Cultivated in vitroG. CLAREBouTl, B. GAMAINr, c. SLOMIANNY\E. PRECIGOUT2, A. GORENFLOT2 and D. DIVEl

lINSERM U42, 369 rue Jules Guesde, B.P. 39, 59651Villeneuve d' Ascq cedex, France. 2Laboratoire deBiologie Cellulaire, UFR des Sciences Pharmaceutiqueset Biologiques, 15 Avenue Charles Flahault, 34060Montpellier, France

The existence of an endogenous SOD in B. hylomysci andB. divergens implies the existence of enzyme(s) able to de­stroy hydrogen peroxide produced by the SOD activity. Wesearched for catalase and glutathione peroxidase activitiesin these two species.B. hylomysci was produced by experimental infection ofSwiss mice. B. divergens was cultivated in vitro on humanred blood cells (RBC).After isolation and purification of parasites from their hostRBC, a catalase activity was found in the two species. Theactivity was very low in B. hylomysci, and very importantin B. divergens.A glutathione peroxidase selenodependent was also found inparasites. Contrarily to catalase, its activity was much moreimportant in B. hylomysci than in B. divergens.Similar results were observed previously with rodent Plasmo­dium species obtained in vivo and P. [alciparum cultivated invitro.The balance between glutathione peroxydase and catalase ac­tivities observed in Plasmodium and Babesia may be largelydependent on the expression of the glutathione peroxidase,which is itself dependent on selenium available for the para­site.

41Ultrastructural Features of the Life Cycle ofMicrosporidia belonging to the Genus Tuzetia fromEphemeropteraD. CODREANU-BALCESCUBiological Institute, Bucarest, Splaiul Independentzei296, P.O. BOX 56-53, Romania

In the different rivers in the mountains of the South Carpatesin Romania, the adipose tissue of ephemeran nymphs are in­vaded by two micros poridian species, reported to belong tothe genus Tuzetia by their uninucleated sporoblasts andspores, which are individually enveloped in sporophorous ve­sicles produced late in the sporal morphogenesis and which isdifferent from a sporophorous vesicle of the sporont.Tuzetia ecdyonuri n. sp. (in Ecdyonurus venosus) the uninu­cleated rounded schizonts (4.5 11m x 3.5 11m) are limited by aunit membrane and they are laying directly in the host cyto­plasm; no diplokarya. The schizonts are dumbbell-shaped orflat plasmodia with 2-4 nuclei. They divide in a rosette-likemanner to produce the future sporonts. These (3.5 Il x 3 IIIare secreting on their surface a small number of spots ofdense material, which become confluent to a continous sacin the sporoblasts become elongated. At the beginning of thisstage the sporoblasts are released by rosette-like budding.Spores are pyriform-ovoid (5 Il x 2.5 11), 6-7 coils of the po­lar filament.

414 . Abstracts

Comparing the developmental characteristics of T. lipotropbaCodr., Codr. Bale., 1975 (in Rhithrogenasemicolorata) andthis species, we can conclude that the Tuzetia from the ephe­merids provides new features from those of the ]uzetia speciesforms Cyclopides and Simuliides. Future ultrastructural stu­dies will recognise another species, probably, reported to thegenus Nosema.

42Characteristics of AnaerobesG. COOMBSParasitology Laboratory, Institute of Biomedical andLife Sciences, University of Glasgow, Joseph BlackBuilding, Glasgow G12 8QQ, Scotland, UK

Protists that lack mitochondria have generally been regardedas anaerobes. Such organisms characteristically reside in en­vironments either lacking oxygen or where its concentrationis very low. Protists with these features are a phylogeneticallydiverse group and their metabolic adaptations differ consid­erably. Recent data confirm the great variability of anaerobesand that this lifestyle may not be restricted to amitochondrialorganisms. There have also been advances in ideas on the evo­lutionary origin of some of the organisms and cell structures.This presentation will provide a brief overview on the mainmetabolic characteristics of anaerobes, their occurrence andfunctional significance, and some of the recent data that havequestioned the validity of traditional views.

43"Glycogenosomes" in Giardia intestinalisTrophozoites and Cysts?D. S. CROSS and J. G. ZALITISSchool of Biochemistry and Molecular Genetics,University of N.S.W., Sydney, N.S.W. 2052, Australia

Giardia intestinalis trophozoites have been shown to containglycogen granules. During encystation, glycogen was the en­ergy reserve and precursor for cyst synthesis, survival and ex­cystation. Therefore activity and regulation of the enzymes ofglycogen metabolism are crucial for the survival of Giardia.The trophozoites and cysts have glycogen (~ 1.5 - 2 umoles/mg protein). 14C glucose showed nett glycogen synthesis introphozoites. In cysts there was no glucose uptake. The activ­ities of glycogen phosphorylase, phosphoglucomutase, UDP­glucose pyrophosphorylase and UDP-G glycogen synthasecould be assayed in 16,000 g supernatant fraction of the so­nicated trophozoites. Centrifugation at 110,000 g resulted in100% of the synthase, 70% of phosphorylase and 40% ofphosphoglucomutase being sedimented. This distributionwas maintained after purification of the "glassy" glycogen.No UDP-G pyrophosphorylase sedimented. The cysts didnot have glycogen synthase activity, phosphorylase activitywas very low, phosphoglucomutase and UDP-G pyro-phos­phorylase activities were to trophozoites with a similar distri­bution.All trophozoite glycogen synthase activity was tightly boundto a very high molecular weight "glycogen" fraction (glyco­gen/protein ratio 4:1). Its fractionation on density gradientsresulted in its separation into four visible bands. The mostdense band had glycogen and glycogen synthase activity.The purified glycogen band, when denatured and electrophor-

esed pon SDS PAGE, contained three major proteins of 55,65and 160 kD. Because no allosteric regulation was observedand trophozoites have some degree of "futile cycling" withrespect to glycogen turnover, control was "loose". Cysts haveno synthase or "futile cycling" and both enzymes of glycogendegradation are bound to glycogen, the degradation of whichappears to be tightly regulated. In many organisms not only arethe enzymes bound to glycogen but also the regulatory proteinkinases and phosphatases. Therefore it is possible that a pro­cess as crucial as glycogen metabolism in Giardia is also regu­lated in situ on a "glycogenosome".

44Localization of y-tubulin in the Mitotic and MeioticNuclei of Euplotes octocarinatusS. CURTENAZt, M. WRIGHT2 and K. HECKMANNllInstitut fur Allgemeine Zoologie und Genetik,Universitat Munster, D-48149 Munster, Germany;2Laboratoire de Pharmacologie et Toxicologiefondamentales, CNRS, F-31400 Toulouse, France

A polyclonal antibody raised against a y-tubulin sequence ofAspergillus showed the presence of y-tubulin in the corticalMTOCs (basal bodies of dorsal cilia, cirri and membra­nelles) and also in the interphase micronucleus, which exhi­bits a spotted distribution of y-tubulin (Liang et al.,submitted).In using the same antibody during stages of conjugation, wereport here the localization of y-tubulin in the intranuclearmitotic and meiotic spindles assembled without visible polarstructures. As mitosis and meiosis show the same patterns oflabeling, the following results are valid for both types of nu­clear division. In the prophase micronucleus, the labeling isweaker than in interphase, but remains punctuated. Whenthe chromatin condenses during metaphase into 4-5 thickaggregates that span as longitudinal bands from one poleof the spindle to the other, the y-tubulin exhibits a longitudi­nal distribution along the chromatin. At the beginning of ana­phase, this labeling condenses into an equatorial plane,between the two migrating clusters of chromosomes. Thisequatorial labeling becomes thinner until it disappears in lateanaphase. At this time, both extremities of the spindle arestrongly labeled. Finally, in telophase the two daughter nucleialready show the spotted interphase labeling, while a reloca­lization of y-tubulin results in a strong decoration of the in­termediate portion of the long spindle that separates the twonuclei.

45Isolation and Characterization of the ChaperoninCCTll Subunit Gene in TetrahymenaL. CYRNEl,2, H. SOARESt, A. C. CARDOSO!, P. GUERREIRO l

and C. RODRIGUES-POUSADA1

lLaborat6rio de Genetica Molecular, InstitutoGulbenkian de Ciencia, Apartado 14, 2781 OeirasCodex Portugal, 2Dep. de Quimica e Bioquimica daFaculdade de Ciencias da Universidade de Lisboa,Campo Grande, 1700 Lisboa, Portugal

The chaperonin containing TCPl (CCT) is an heteromericcomplex that in mammalian cytosol has about 850­900 kDa. This complex is composed of seven to nine distinct

polypeptides in the size range 52- 65 kDa (named CCTa,CCT I3, ...CCTTJ) encoded by a family of related genes. Thiscomplex seems to be involved in the maturation of the activetubul in (Kubota et aI., 1994; Ellis et aI., 1991 ). In our labora­tory, we have been involved in the study of the CCT genefamily in Tetrahymena pyriformis and Tetrahymena thermo­phila. Therefore , we have characterized the CITy gene, andstudied its expression duri ng reciliation (Soares et aI., 1994).In order to complete the analysis of th is gene family in Tetra­hymena, we have construc ted several oligonucleotides, basedon the conserved sequences among the CCT subunit genes.Using PCR techniques, we have been able to produce homo­logous probes, in order to search for other Tetrahym ena CCT­subunit sequences. Th e DNA fragment s so far obt ained con­tain part of the gene coding for the CCTTJ subunit in Tetra­hym ena pyriformis and Tetrahym ena thermophila. Thesefragments were sequenced and characterized; the predictedaminoacid sequences show an identity of about 60% to themouse CCTTJ protein, one of the seven divergent subunitsof the chaperonin containing TCP1. Experiments are in pro­gress aimed to isolate the full gene coding for these subunits,and to study the expression patt ern of the CCTTJ gene, dur ingreciliat ion and conjugation of Tetrahymena cells.References: Kubota , H. , Hynes, G., Carne, A., Ashworth, A.and Willison, K. (1994), Curro BioI. 4, 89- 99; Ellis, R. J.andVan der Vies, S. M. (1991), Annu. Rev. Biochem. 60, 32 1­347; Soares, H. , Penque, D., Mouta, C. and Rodrigues­Pousada , C. (1994), J. BioI. Chern. 269, 29299- 29307.

46Is Hi Histone Present in Blepharisma japonicumMacronucleus?T. D. LuccHE1, M. DURANTE2 and M. SALVINI3

Dipartimento Scienze Ambiente e Territorio,Universita di Pisa I , Dipartirnento Biologia delle PoanteAgrarie, Universita di Pisa' , Scuola Normale Superioredi Pisa 3, Pisa, Ital y

Since a peptide having typical H I physico-chemical chara cter­istics had not been definitely pointed out in the macronucleusof Blepharisma japonicum by electrophoretic techniques,furth er analyses, using high performance liquid chromatogra­phy, have been carried out to separate total basic macronuc­lear prote ins from this ciliate. This procedure has allowed usto identify two peptide s whose retent ion times are comp ar­able to those of Hl and its phosphorylated form, both in calfthymus and Tetrahym ena pyriformis. Both peptides have highmolecular weight (between 35 KDa and 45 KDa) and appearimmunologically related to the HI histone of T. pyriformis.Nevertheless, hybrid ization analyses on Southern blots haveshown that in macronuclear DNA of B. japonicum there is nonucleotidic sequence comparable to that of Tetrahym enatherm ophila HI histon e gene. At the same time we have per­formed a structural analysis of B. [aponicum macronuclearchromatin by studying the thermal denaturation of chroma­tin extrac ted from 1 day and 5 days starved cells. Dat a ob­tained have pro ved that chromat in extra cted from 1 daystarved cells is less stabilized than chro matin extra cted from5 days starved cells, which is supposed to be less transcripti on­ally act ive and also to have a higher cont ent of HI than chro­mat in extracted from 1 day starved cells. These results haveallowed us to suppos e that B. [aponicum macronuclear chro ­matin is not entirely lacking in a peptide with chromatin sta­bilizing function even though the chromatin has resulted less

Abstracts ' 415

stabilized than that of T. pyriformis analysed with the sametechnique. This hypoth esis is also suggested by the thermaldenaturation profil es of B. japonicum macronuclear chro ma­tin reconstituited with one molecule of Hl histone ofT. pyriformis for repetit ion unit .

47New Insights into the Mode of Action of theIonophores on Eimeria SporozoitesP. DAszAKI and C. A. KING .'School of Life Sciences, Kingston University, SurreyKTl 2EE, UK & 2Department of Biology, UniversityCollege London, Gower Street, London, UK

Ionophores are antibiotics which are able to trap cations andmove freely across lipid bilayers. Three ionophores, lasaloc id,monensin and salinomycin have been used for over 20 years asanticoccidials. They are fed proph ylactically to broiler chicksand are now respon sible for over 80% of the US anticoccidialdrug sales. Ionophores have a range of effects in different cellsystems and their ant icoccidial mode of action is uncertain.The ionophores are poorly absorbed into the gut cells andthus it is thought that they act primarily on the invasivestages (sporozoites, merozoites) in the gut lumen prior tohost cell invasion.Using in vitro video-microscopy of living Eimeria sporozoi resand statistical TEM studies in vivo, the effect of the ionophorelasalocid has been analysed. Initially the drug blocks zoitemotility: 100 % at a concentra tion one order of magnitud elower than the recommended proph ylactic dose, with 50%blocked at more than 2 orders of magnitude lower. A secondeffect is swelling of the zoite mitochondria which occurs invivo even at the intrac ellular drug concent rations. Finallythe zoite outer membranes bleb and the cells rap idly burstdue to influx of water.

48Invasion of Polarised Cultured Cells by Eimeria sp.SporozoitesP. DAszAKt, S. J. BALLI, C. A. KING2 and R. M. PITTIL01

'School of Life Sciences, Kingston University,Kingston-u-Thames, Sur rey KTl 2EE, UK &2Department of Biology, University College, GowerStreet , London, UK

Coccidia of the genus Eime ria can invade and develop withinenterocy tes of the intestinal and caecal mucosa of poult ry.Various in vitro systems have been used to examine targetcell invasion and the different developm ental stages of Eimer­ia, and to test anticoccidial drugs. The chorio-allanto ic mem­brane of chick embryos, chick kidney primary cell culture andtransformed cell lines have been used extensively in these stu­dies, however these host cells do not have well-defined micro­villi and lack the thick layer of cortical filaments found inintestinal enterocytes. The pathology dur ing host cell inva­sion and effects of anticoccidial drugs therefore differ sub­stantially from the in vivo situation.Using a correlated video-microsco py and electron microscopytechnique, we have studied the effects of host cell invasion byEimeria sporozoites in a cell line derived from human embryointestinal enterocytes, which possesses a layer of large micro­villi. These cells are grown on coverslips and are polarised

416 . Abstracts

such that the mitochondria are proximal to the microvilli,lying above the nuclei. The system is thu s a more useful mod­el for examin ation of the target cell invasion by Eimeria.

49Three Dimensional Molecular Modelling of Iron-SODof Plasmodium falciparumM. DAUCHEZ!, A. J. P. ALIXI and D. DIVE2

'Laboraroire de Spectroscopi es et StructuresBiomoleculaires, URCA, IN SERM U314, 45 rueCognacq Jay, 51092 Reims, France. 2INSERM U42 ,369 ru e Jul es Gu esde, B.P. 39, 59651 Villeneuve d'Ascqcedex, Fran ce

Macromolecular modelling permitted us to construct a thre edimensional model of one monomer of the iron-superoxidedismutase (SOD) of P. [alciparum. This model is determinedfrom 1D data (predictio n methods using the sequence, alig­ments and comparison of 65 known Fe/Mn SODs), 2D struc­ture (Hydrophobic Cluster Analysis) to 3D model (3Dhomology-based computer assisted modelling with knownX-ray structures "Prote in Data Bank"). For 1D and 2D ana­lyses, calculations were done on a PC computer with home­made programs (ID) and with HCA, Dori ane Ce (2D). For the3D structur e determination, all computations were performedon a Silicon Graphics workstation (R440-Elan) using BIO­SYM products: HOMOLOGY and DISCOVER softwa res.The initial model was determined by sta rting from known3D-structures of Pseudom onas ovalis and Escherichia coliFe-SODs and from Thermus thermopbilus Mn -SOD, leadingto well known Structurall y Conserved Regions (SCRs) andthen building Variable Regions. This model was then opti­mized using molecular mechanic procedures in vacuum, innon-expl icit solvent (e=80 and 4r ). During the minimizationprocess, a specific attention was dra wn on the behaviour ofthe SCRs, on the residues involved in the chelation of the me­tal and on the interface with the second monomer.

50Cystogenesis of th e Virulent RH Strain of Toxoplasmagondii in RatsC. DE CHAMPS, J. RICARD, A. BELMEGENAI, H. PELLOUXand P. AMBROISE-T HOMASLaboratoire de Parasitologie, Faculte de Medecine,63001 Clermont-Ferrand. Departeme nt deParasitologie-Mycologie Medicale et Moleculaire,CNRS EP 78, Faculte de M edecine Grenoble,38706 La Tronche

The RH strain of Toxoplasma gondii was isolated in 1939from a child with a lethal encephalitis. This strain exhibitedhigh virulence after succcessive inoculat ions in mice. At­tempts to obtain brain cysts from non-immuni sed rats havebeen rarel y successful, but if obtained, would offer an experi­mental model for studying cystogenesis and the reap pearanceof virulence as observed in AIDS. Ten adult male Fischer rats(IFFA-CREDO- L'Arbresle, France) were inoculated with 107

tachyzoites of the RH strain from mouse peritoneal fluid. Fivecontrol rats were inoculated with serum. Serological controlswere done before inoculat ion and thr ee weeks afterwa rds bydirect agglutination (Toxo-Screen DA biolvlerieux, Ma rcy -

I'Etoile, France). Infection by RH strain was shown by sero­conversion in the ten infected rats and not in the five controls.Four rats and two controls were killed by barbituric overdoseand brain samples taken. Cysts were ext racted from homoge­nates on disconti nuous Percoll gradients . One typical cyst andseveral broken cysts forms were observed in two of the fourRH rats, which confirms the RH strains cystogenesis in non­immun ised Fischer rats.Further studies with this model (which will requ ire a largernumbe r of cysts) will allow characterization of parametersinvolved in virulence and cystogenesis of RH T.gondii strain.

51Sequence Variation in the Ribosomal InternalTranscribed Spacer, Including 5.8S Ribosomal DNA,of Naegleria spp.J. F. DEJONCKHEEREDepartment of M icrobiology, Inst itu te of H ygiene andEpidemiology, BI050 Brussels, Belgium

The ribosomal internal transcribed spacer (ITS), includ ing the5.85 ribosomal DN A, has been amplified by the polymerasechain reaction (PCR) for the 10 described species of the amoe­boflagellate genus Na egleria. Because it is a species complexN. gruberi was not included . Whilst the majority of the PCRproducts obtained had a size between 400 to 500 bp, a pro­duct of approx . 850 bp was obtained from N. jadini andN. min or. Except for N. fow leri, no size difference was ob­served from strains of the same species - nor restr iction frag­ment length polymorph ism (RFLP). In strains of thepathogenic N. fowleri isolated from different continents, 4different sizes of amplified ITS were observed. RFLP analysisindicated that deletions and/or insertio ns had only occurred inthe ITS of N. fowleri. The ITS and 5.8S ribosomal DNA ofdifferent N. fowleri strains and one representa tive, mostlythe type strain, of all other Naegleria spp. were sequencedand aligned. Based on these alignments it is possible to designprimers specific for the pathogenic species N. fowleri . Thesequences of the ITS confirmed the species separatio ns basedon SSUrDNA sequences.

52Feeding Mechanisms in Free-living and ParasiticSpecies of Ciliate Protozoa from Marine Bival veMolluscs of Commercial InterestF. DELA FUENTE, M. RIUS, H. SALVADO and J. M. AMIGOLaborat ori de Protozoologi, Departament de BiologiaAnimal, Universitat de Barcelona, 08028 Barcelona

For a period of 12 months, the protozoan (mainly ciliates)populations living in bivalve molluscs Ostrea edulis, My tilusedulis and venerupis decussatus have been studied, analysing1800 individuals from Alfacs Bay and a mollusc depuratingfacility, both in Ebre Delta (NW Mediterranean).We determined 7 ciliate species in O. edulis (2 free living cili­ates, and 5 parasitic), 8 ciliates in M. edul is (2 free living and 6parasites) and 5 species in V. decussatus (1 free living and 4parasites).The efficiency of mollusc depur at ion process (a process thateliminates bacteria and algae from mollusc for human food)in the elimination of ciliate protozoa is of about 91% inO. edulis and V. decussatus and 86% in M. edulis. However

the efficiency of the process may change along the year de­pending on the type of feeding strategy of the ciliates andits relation with its host. Furthermore the depuration processsignificantly modified the structure of ciliate populations,thus while in non depurated molluscs bacteriophagous-histo­phagous and obligate bacteriophagous ciliates are predomi­nant (more than 50% of the total number of ciliates in allcases), in depurated samples they became minoritary.

53Diffuse Cutaneous Infection Caused by a LowerTrypanosomatid in an HIV Positive PatientJ. P. DEDETt, B. ROCHE2, F. PRATLONG 1, D. CALES-QUIST2,

J. JOUANNELLE2, J.-c. BENICHOU3 and M. HUERRE3

lLaboratoire Parasitologie, Faculte de Medecine,Montpellier, France; 2Centre Hospitalo-Universitairede Fort-de-France, Martinique, France and 3InstitutPasteur, Paris, France

The trypanosomatids are protozoa including well known hu­man parasites, such as Trypanosoma and Leishmania. Withinthe same family, the lower trypanosomatids are monoxenousparasites of insects, which, up to now, have never been re­ported infecting humans.The authors present a parasite isolated from disseminatedskin nodules on a HIV positive patient from MartiniqueIsland (French West Indies). The parasites was studied by iso­enzymatic characterization and transmission electronmicro­scopy processed on both culture forms and patient skinbiopsy.It was isoenzymatically different from all New and Old WorldLeishmania species, and from Trypanosoma and Sauroleish­mania genus. Ultrastructural observations led to its assign­ment as a lower trypanosomatid.

54Immunolocalization and Partial Characterization ofProteins from the Microsporidium Encepbalizozooncuniculi, an AIDS Opportunistic ParasiteF. DELBAC, F. DUFFIEUX, D. DAVID, G. METENIER,G. PRENSIER and C. VIVARESProtistologie moleculaire et cellulaire des parasitesopportunistes, LBCP, URA CNRS 1944, Univ. B.Pascal, F-63177 Aubiere Cedex, France

We have investigated the ultrastructural localization of pro­teins in the spore of Encephalitozoon cuniculi, using micepolyclonal antibodies directed against different proteinbands separated by SDS-PAGE. First results have been ob­tained for three types of cellular components: the endospore(anti-30 kD), the plasma membrane (anti-33 kD) and the po­lar tube (anti-ISO kD).Characteristical patterns of glycosylated proteins have beenvisualized on blots after treatment with four different lectins(Con A, PNA, RCA, WGA). All these lectins bind to a major52 kD-band while none bind to the 33-kD band ascribed tothe plasma membrane.Six Cav-binding proteins (28, 35,42,47,50 and 52 kD) havebeen identified by autoradiography following incubation with45Ca, the predominant one corresponding to a 52 kD-band.

Abstracts . 417

Western blotting with commercial polyclonal antibodiesagainst a C-terminal peptide of actin did reveal the presenceof a 42-kD protein. An unusual spectrin analog might corre­spond to a 50-kD protein which is localized close to the plas­ma membrane and within the polar tube.Taken together, these data may be of a peculiar interest fordeveloping specific probes to facilitate the detection of micro­sporidia in AIDS patients.

55Dynamic and Composition of the MicrotubularCytoskeleton in a Primitive Group of Protists: TheTrichomonadsP. DELGADO, G. BRUGEROLLE and E. VISCOGLIOSILaboratoire de Biologie Comparee des Protistes, URACNRS 1944, Avenue des Landais, 63177 Aubiere,France

Trichomonads constitute a primitive group of amitochondri­ate parasitic protists belonging to the phylum Parabasala. Inthe recent years, Trichomonas vaginalis has emerged as themost common sexually transmitted disease of parasitic ori­gin. Division in T. vaginalis, as in all trichomonads, is ofthe cryptopleuromitotic type with a conspicuous extranu­clear spindle (or paradesmosis). The nuclear envelope doesnot disappear during mitosis. These protists possess a mi­crotubular cytoskeleton composed of the axostyle-pelta com­plex, constituting an axial skeleton, and the recurrent andanterior flagella. At the onset of division, the parental axo­style-pelta complex depolymerizes and new axostyles gradu­ally increase in size in each daughter kinetid beforecytokinesis. We also observed the segregation of the parentalflagella followed by an equilibration of the number of flagellain each daughter flagellar apparatus. Two atractophores,which are structures appended to the basal bodies and whichplay the role of centrosomes, polarize the paradesmosis andcontain y-tubulin. However this paradesmosis does not seemthe main motor of the flagellar apparatus bipartition since thegriseofulvin, a drug inhibiting the polymerization of the par­adesmosis in dividing cells, does not block the bipartition. Wehave also approached the analysis of tubulin isoforms inT. vaginalis using specific antibodies. So far we do not detecttyrosylated and polyglycylated isoforms in trichomonads. Incontrast, during the cell cycle, all the microtubular structuresare acetylated including the new axostyles and the parade­smosis. The glutamylated tubulin isoforms would be mainlyconcentrated in a large pericinetosomal region and in the par­adesmosis. The action of microtubule inhibitors such as no­codazole and other drugs has been undertaken as well as theelucidation of the aminoacid sequence of the y-tubulin.

418 . Abstracts

56Cell Surface Topological Pattern of Lectin Receptors inParamecium primaurelia Mating Types, Evaluated byFluorescence Resonance Energy Transfer (FRET)ImagingM. U. DELMO!'lTE CORRADO, G. BOTTIROLI*,D. LOCATELLI" and G. TAGLIAFIERROOIstituto di Zoologia, Universita di Genova, "Centro diStudio per l'Istochimica, C.N.R., Pavia, °Istituto diAnatomia Comparata, Universita di Genova, Italia

The involvement of Limulus polyphemus agglutinin (LPA)and wheat germ agglutinin (WGA) surface membrane recep­tors in the first phases of Paramecium primaurelia conjuga­tion, has been inferred by the finding of pairing inhibitionand splitting, after incubating mating type I or mating typeII cells with LPA or WGA before their mixing. Distributionaland structural characteristics both of LPA and WGA bindingsites on the somatic membrane region engaged in conjugation,were studied in situ via FRET-based spectroscopy combinedwith fluorescence imaging systems. FITC and Texas-Red (TR)fluorochromes were employed as the donor/acceptor coupleof excitation energy, respectively. FRET efficiency was evalu­ated by imaging analysis systems showing the topological pat­tern of the process. Differences in energy transfer efficiency,found in fixed cells double-labelled with FITC-LPA andTR-WGA, revealed that different patterns of these lectin re­ceptors characterize the mating type I and mating type II cellmembrane region engaged in conjugation. This research wassupported by M.U.R.S.T. 40 % 1993-94 grants.

57Protozoan Diseases of Fish in Mediterranean MarineAquacultureA. DIAMANTl and 1. PAPERNA 2

lNational Center for Mariculture, IsraelOceanographic and Limnological Research Ltd., Eilatand 2Paculty of Agriculture, Hebrew University ofJerusalem, Rehovot, Israel

Protozoan infections pose great potential danger to marinefish aquaculture. Endoparasitic protozoans, such as Myxo­sporeans, are becoming increasingly abundant and gainingimportance as fish pathogens in the Mediterranean withthe development and spread of mariculture facilities in theregion. Sphaerospora dicentrarchi and S. testicularis, bothparasites of Dicentrarchus labrax, now abound in this spe­cies in the western basin while Myxidium leei afflicts Sparusaurata and at least one other sparid (Puntazzo puntazzo) inthe eastern Mediterranean. Myxosporean prevalence infarmed fish coincides with a corresponding spread of infec­tions amongst feral fish populations. It is suggested that theseparasites share a common intermediate host vector, while anincrease in the occurrence of potential intermediate hosts(oligochaetes) may be linked with the eutrophication nor­mally associated with aquaculture and the constantly dete­riorating environmental conditions in both Mediterraneanbasins. Transportation of fish culture seed and fingerlings be­tween countries within the region, coupled with unsatisfac­tory sanitary control measures , tends to exacerbate theproblem. Although the pathogenicity of S. dicentrarchi insea bass is (still?) moderate, there is evidence of severe kidneydamage related with Sphaerospora in grey mullet both in Italy

and Israel. M. leei, originally discovered in Cyprus, is nowalso found regularl y in Israel and Greece and is emergingas a serious pathogen of S. aurata both in pond and netpenculture. Integumental fish parasites also display interestingnew manifestations. Past studies have shown that the ciliateCrypto caryon irritans and dinoflagellate Amyloodinium ocel­latum are both typically warmwater species. A. ocellatum,and a highly pathogenic form of Cryptocaryon, have bothbeen reported to cause severe disease outbreaks in S. auratain subtropical eastern Mediterranean fish farms. It now ap­pears that the small cyprinodont fish Aphanius dispar, whichflourishes in hypersaline and hyperthermic coastal marineswamps in the Mediterranean and Red Sea, is a natural reser­voir for A. ocellatum. Evidently we are being confronted withincreasingly cold tolerant forms of these protists. It is unclearwhether these merely represent different populations, orwhether they are distinct strains, subspecies or species. Is itpossible that we are witnessing a process of selective adapta­tion that results from the massive expansion of mariculturedevelopment in the region?

58Soluble and Membrane-bound Polypeptide­pheromone Isoforms of the Ciliate Euplotes raikovi areControlled by the Same Maronuclear GeneG. DI GIUSEPPE, A. LA TERZA, C. MICELI and P. LUPORINIDepartment of Molecular, Cellular, and AnimalBiology, University of Camerino, 62032, Camerino(MC), Italy

Sequence comparison of the type-Il cell macronuclear geneencoding pheromone Er-2 with several eDNA clones, ob­tained by RT-PCR, produced evidence that mRNAs of thisgene may differ from one another for the alternative elimina­tion of introns located close to the 5' ends. One mRNA speciesis produced after the elimination of a 23-base intron (witheukaryotic splice junction consensus sequences ) and specifiesthe precursor of pheromone Er-Z; another species, that main­tains the 23-base intron, encodes a larger polypeptide of 144amino acids, of which the 75 ones at the carboxyl terminusare identical to the Er-2 precursor.The expression of two distinct mRNAs by the Er-2 macronu­clear gene was further proved by transfecting (via electropora­tion) this entire macronuclear gene into E. raikovi cell typeslacking the Er-Zgene. Transfected cell lines were identified bytheir new ability of secreting Er-2 pheromone and shown cap­able of expressing mRNA for the 144-amino acid Er-2 iso­form. As this isoform contains a potential transmembranedomain (represented by an internal hydrophobic sequence)and is recognized by anti -Er-2 antibodies on membrane frac­tions of type ll cells, it is likely retained in membrane.Overall, these findings confirm and extend to the whole pher­omone gene family previous evidence, obtained from studiesof type-I cells, that a single macro nuclear gene controls theproduction of soluble and membrane-bound pheromone iso­forms by a mechanism of alternative splicing.

59Occurrence of Cryptosporidia in Immunocompetentand Immunodeficient PatientsO. DITRICHInstitute of Parasitology ASCR, 370 05 CeskeBudejovice, Czech Republic

Although Cryptosporidium parvum has been known for al­most 20 years as causative agent of human infection and isintensively studied as one of the most important opportunis­tic parasites of immunodeficient patients, the prevalence ofinfections within immunocompetent human population is dif­ficult to determine. Serological studies indicate that theoccurrence of cryptosporidia in "healthy populations" is un­derestimated, since many cases are not detected by routinecoprological examination. Children under 5 years of ageare the most susceptible age group because of their immatureimmune system. However, the infections of newborn childrenunder 1 year of age are rare, at least in the countries with highhygiene standard. In most cases of children's infection, theinfected animals (especially calves) could be recognized asthe source of infection. Massive outbreaks caused by contami­nated drinking water, when inhabitants regardless of their ageor immune status suffer from symptomatic cryptosporidiosissuggest the existence of strains of cryptosporidia with in­creased virulence. In immunodeficient humans, especiallyin HIV positive patients who developed AIDS, cryptospori­dia can cause serious watery diarrhea with consequent disse­mination of infection to various organs and metabolicbreakdown. However, spontaneous improvement of persist­ing diarrhea and resolution of the infection has been ob­served in some patients. Although some differences wereobserved among human isolates of cryptosporidia (oocystsize, antigenic profile, infectivity to animal models), spe­cies-specific primers for PCR of SSUrRNA showed that allstrains tested belonged to Cryptosporidium parvum sensulato.

60SODs in ProtozoaD. DIVEINSERM U42, 369 rue Jules Guesde, B.P. 39, 59651Villeneuve d' Ascq cedex, France

All living cells are equipped with antioxidant enzymes in or­der to detoxify active oxygen species (02°- , H2, 020HO).Superoxide dismutase (SOD) is the first enzyme of the sys­tem, able to convert superoxide anion to H 202. Three cate­gories of SODs are known according to their cofactor metals:Cu/Zn SODs, Mn SODs and Fe SODs, the two last categoriesare structurally related. The survey of SODs characterized inprotozoa show that most species have a Fe-SOD. Cu/ZnSODs have been found only in Tetrahymena and in Eimer­ia. A Mn-SOD was shown in Eimeria. On the basis of theprotein sequences, it is to distinguish more precisely the dif­ferent SODs, particularly Mn-and Fe-SODs which are closetogether, but unfortunately, not many sequences of protozo­an SODs are available. Alignment of 65 known Fe and Mn­SODs showed that all protozoan Fe-SODs (excepted one)were localized in the same Fe-SOD family and close to­gether. Tetrahymena Fe-SOD appears unique, by its highhomology with mycobacteria and yeasts Mn-SODs. Notmany results are available about the regulation, of SODsin protozoa. The most precise informations concern

Abstracts . 419

E. histolytica. As SODs of the main parasitic protozoa aredifferent (iron-dependent) from the host enzymes, the possi­bility of a drug design was proposed some years ago. If thesimilarities in the structure of the active sites of Fe- andMn- SODs have been considered as an obstacle to target se­lectively the Fe-SODs, the difference of sensitivity of the twoenzymes to hydrogen peroxide can be the basis for the strategyof the drug design. Comparisons of the 3D structures of thetwo categories of SODs may enable to find the structureswhich can be selectively targetted.

61Calonympha grassii: An Ideal Organism in the Searchfor Kinetosome DNAM. DOLAN and L. MARGULISDepartment of Biology, University of MassachusettsAmherst, MA 01003 USA

The symbiogenetic hypothesis of undulipodial origin predictsthe presence of centriole/kinetosome DNA in cells capable offorming mature kinetosomes. Most early lineages of eukar­yotes (eg. retortamonads, diplomonads, trichomonads) con­tain karyomastigonts, nuclei with undulipodia (eukaryoticflagella) and associated fibers and tubules. The karyomasti­gonts of members of the order Trichomonadida, for exam­ple, contain a nucleus, four undulipodia, paraxonemalfibers, parabasal fibers, the pelta-axostyle complex and otherstructures, Within this order the multinucleate genus Calo­nympha is distinct because it contains both karyomastigontsand akaryomastigonts which are the same as karyomastigontsexcept that they lack nuclei. Thus they are ideal organisms fortesting this hypothesis. Because these protists are also amito­chondriate, any DNA found in these cells must necessarily beassociated with the nuclei, symbiotic organisms or otherstructures. We have used fluorescent nucleic acid stains(DAPI, acridine orange and Hoechst vital stain) to seek kine­tosome-associated DNA of the akaryomastigonts of Calo­nympha grassii from Cryptotermes brevis, a dry woodeating termite from Florida. We have tested against false po­sitives with DNase and RNase pretreatment.

62Isoenzymes of Malate Dehydrogenase(Decarboxylating) in Hydrogenosomes ofTrichomonas vaginalis: Purification and PartialCharacterizationT. DRMOTA, J. KULOA and J. TACHEZYDepartment of Parasitology, Faculty of Science,Charles University, Vinicna 7, 128 44 Prague, CzechRepublic

Isoenzymes of malate dehydrogenase (decarboxylating) ofdifferent charges were separated from a hydrogenosome-richfraction of the T. vagina/is homogenate by ion-exchange chro­matography. Positively charged form at pH 7 was purifiedusing cation-exchange chromatography (CM-Sepharose).The enzymatic activity with maximum at pH 9 utilizedNAD+ (Km = 6 ~M) preferentially to NADP+(Km =125 ~M) in the presence of malate. The Km for malate was103 ~M and 439 ~M in the presence of NAD+ andNADP+, respectively. A negatively charged isoenzyme of simi­lar kinetic properties was isolated at pH 7.3 using anion-ex-

420 . Abstracts

change (Q-Sepharose) and Red-Sepharose chromatography.The Km for NAD+ and for NADP+ in the presence of malatewas 6/lM and 145/lM, respectively. Km for malate was 95 and790 in the presence of NAD+ and NADP+, respectively. Thisisoenzyme displayed broad pH optimum for its activity ran­ging from 6.6 to 10.2. Apparent subunit size of both isoen­zymes was 60 kDa on SDS-PAGE and native molecularmass determined by HPLC gel filtration was 234 kDa.Localization of malate dehydrogenase (decarboxylating) inhydrogenosomes showed the presence of the enzyme activityin both non-sedimentable and membrane fractions. Isoelectricfocusing revealed that positively charged isoenzyme (pl >7)was present preferentially in the hydrogenosomal matrixwhile the form of pI < 7 was associated with the membranefraction. Further studies are required to elucidate physiologi­cal role of the isoenzymes in T. vaginalis hydrogenosomes.

63Immunological Cross-reactivity Between Proteinsfrom Two Microsporidia: Glugea atherinae andEncephalitozoon cuniculiF. DUFFIEUX, F. DELBAC, D. DAVID, G. METENIER andC. VIVARESProtistologie moleculaire et cellulaire des parasitesopportunistes, LBCP, URA CNRS 1944, Univ. B.Pascal, F-63177 Aubiere Cedex, France

Mice antisera were raised against total SDS-solubilized pro­teins from broken spores of two microsporidian species: Glu­gea atberinae, a fish parasite, and Encephalitozoon cuniculi,an AIDS opportunistic parasite.For each species, corresponding antiserum reacts with a largenumber of bands of electrophoretic patterns as shown byWestern blotting.Antisera against Encephalitozoon proteins cross-react with afew Glugea protein bands which are located between 160 and30 kD. Cross-reactivity has been ascertained by indirect im­munofluorescence and electron microscope immunocyto­chemistry.Moreover, an antiserum against a 35-kD protein ofE. cuniculi specifically recognizes a 34-kD protein ofG. atherinae. For both species, such a protein is localizedin the polar tube. For the first time, we show that two differ­ent micro sporidia share common antigenic characteristics fora protein component of the extrusion apparatus.No cross-reactivity has been observed using an antiserumagainst a 25-kD Glugea protein which is shown to be loca­lized in the lamellar polaroplast.

64Epigenetic Regulation of Programmed GenomicRearrangements in Paramecium tetraureliaS. DUHARCOURT and E. MEYERLaboratoire de Cenetique Moleculaire, ENS, 46 rued' Ulm, 75005 Paris

Ciliates are characterized by the presence in each cell of twotypes of nuclei. Micronuclei are transcriptionally silent germ­line nuclei, which undergo meiosis during sexual events toproduce the gametic nuclei. Macronuclei are highly poly­ploid somatic nuclei, in which all vegetative transcriptiontakes place; they are lost during sexual events. After fertiliza-

tion, new macronuclei differentiate from mitotic products ofthe zygotic nucleus. The process of macronuclear differentia­tion involves spectacular rearrangements of the genome,which are of two types: fragmentation of germline chromo­somes, followed by the addition of new telomeres, and pre­cise excision of numerous internal sequence elements,either single-copy or repeated, from most coding sequencesand intergenic regions.In P. tetraurelia, the position of the region where telomeresare added after chromosomal fragmentation is not entirelydetermined by cis-acting sequence elements of the germlinegenome. Indeed, it is sensitive to the structure of the parental(pre-zygotic) macronuclear genome. Transformation of thepre-zygotic macronucleus with cloned macronuclear se­quences can result in novel sequence-specific telomere posi­tions in the new macronucleus developing in the samecytoplasm, suggesting that these rearrangements are modu­lated by the pairing of sequences from the pre-zygotic macro­nucleus with the germline sequence being processed.A similar epigenetic regulation can also apply to the othertype of rearrangement identified in Paramecium, the somaticexcision of numerous Internal Eliminated Sequences (shortnon-coding single-copy elements) from the germline gen­ome. The presence of a particular IES sequence in the paren­tal macronucleus results in the inhibition of its own excisionin the macronucleus of the following sexual generation. Thenon-excision of this IESis thus maternally inherited. Differentmechanistic models can be proposed on the basis of a quan­titative analysis of the effect of transformation of the pre­zygotic macronucleus with wild-type or in vitro-modifiedsequences.

65Chromosomal Polymorphism in Leishmania (Viannia)braziliensis and Leishmania (Viannia) peruviana:Functional Significance?J.-c. DUJARDINa, A.-L. BANULS b, K. VICTORab,

J. AREvALoc, A. LLANos-CuENTAsc, M. TIBAYRENCb andD. LE RAya

a Laboratory of Protozoology, Institute of TropicalMedicine "Prince Leopold", 155 Nationalestraat, B­2000 Antwerp, Belgium; b Genetique des Parasites etVecteurs, UMR CNRS-ORSTOM 9926, ORSTOM,BP5045, F-34032 Montpellier, France; C Instituto deMedicina Tropical "Alexander von Humboldt",Universidad Peruana Cayetano Heredia, AP4314,Lima 100, Peru

Allopatric populations of 1. (Y.) braziliensis (Amazonian for­est) and 1. (V:) peruviana (Andes) were sampled across Peruwithin biogeographical units! (BGUs). Karyotype and isoen­zyme analyses were carried out in order to (i) compare bothgenomic and genetic polymorphisms and (ii) test the hypo­thesis of adaptative significance of chromosomal variabil­ity. Out of nine chromosomes studied, 5 were conservedand 4 were polymorphic. The two species were discriminatedby size of pLb-134Sp chromosome while the other chromo­somes displayed intra-specific polyrnorphism-. Due to itshigher polymorphism (measured on the polymorphic chro­mosomes with a special algorithm.'] and to the eco-geographi­cal heterogeneity of the Andes, 1. peruviana populations wereselected to test our hypothesis. Firstly, following PrincipalComponent Analysis of karyotype data, 1. peruviana popu-

lations clustered as karyodemes according to their BGU oforigin-. Secondly, comparison of karyotypical divergenceand isoenzyme variation showed that eco-geographical differ­ences explained 8% only of the total enzymatic variation(measured on 16 loci), and 62.3% of the total karyotypicaldivergence (measured on 4 chromosomes). Thirdly, chromo­somal polymorphism was correlated with variation of somephenotypic characters (i) mucosal metastasis potentiality ofL. braziliensis and, within L. peruviana (ii) lesion size (Lla­nos-Cuentas and Davies, personal communication) and (iii)multiplication rate in vitro. Fourthly, genes coding for keyfunctions (gp63) were rearranged in one of the polymorphicchromosomes (pLb-134Sp)4,5. These four observations sup­port the hypothesis of adaptative significance for the variabil­ity of the chromosomes under study. Further work is inprogress in order to identify other sequences involved in chro­mosomal size polymorphism, to check for functional altera­tions due to genomic rearrangements, and to compare in asimilar way L. braziliensis isolates from cutaneous and muco­sal lesions. lLamas, G. 1982. In Biol. Divers in the Tropics,pp. 336-357. 2Dujardin, J. c. et al. 1993. Ann. Trop. Med.Paras., 87: 335-347. 3Dujardin, J. c. et al. 1995. Parasitol­ogy, 110: 21-30. "Dujardin, J. c. et al. 1994. Ann. Trop.Med .. Paras., 88: 445-448. 5Victoir, K. et al. 1995. Parasitol­ogy, III press.

66The Variant Surface Glycoprotein of Trypanosomabrucei: Biosynthesis, Processing, IntracellularTransport and Fate During Antigenic VariationM. DUSZENICOBiochemical Institute, University of Tubingen,D-72076 Tiibingen, Germany

African trypanosomes, which cause sleeping sickness and se­vere livestock diseases, are covered by a densely packed sur­fac~ coat consisting of about 10 7 copies of a single protein, thevan,ant surface glJ:coprotein (VSG). This coat serves as a pro­tective barrier against all means of the unspecific immune sys­~em of the host. On the other hand, VSG is highlyimmunogenic and leads to the formation of specific antibo­dies, which induce killing of all parasites expressing the re­spective antigen. A persisting parasitemia is thus onlyensured by the sequential expression of up to several hundredimI?unologically different VSG types (antigenic variation),which are encoded within the parasite's genome. Formationand maintenance of the coat is a highly dynamic processwhich includes intracellular transport of newly formedVSG ?1olecules via the conserved exocytotic pathway, endo­cytosis and membrane recycling as well as shedding and in­tracellular degradation. Membrane traffic in trypanosomesas well as VSG biosynthesis and possible functions of the gly­cosyl-phosphatidyl-inositol (GPI) membrane anchor will besummarized and discussed.

Abstracts . 421

67Response of Horse Intestinal Ciliates on Starvation ofthe HostG. DVOINOS and O. TIMOSHENKOInstitute of Zoology, Kiev, 262601 Ukraine

Dynamics of ciliate excretion in faces of two wild horses (maleand female) was examined under the ten day starvation ex­periment dealing with the research on horse internal para­sites. In order to sample ciliates, squeezed fecal liquid wasfixed by 10% formol solution every day.Two days before the trial number of ciliate species and theirdensity per ml were 24 and 2.1 x 106,28 and 2.4 x 106 for themale and female, respectively. Host starvation resulted indrastic reduction of both indices and increasing in numberof semi-damaged specimens. Buetshliidae and Paraisotrichi­dae species disappeared first, then Blepharocorythidae fol­lowed by Cycloposthiidae. The last remaining species werethose with thick cuticle, strengthened by additional "case,"folds or skeleton, such as Bundleia triangularis, Tetratoxumexcavatum, T. parvum [. sulcatum, Cycloposthium spp. De­creasing in number of entodiniomorphes correlated with in­creasing of suctorian number.Resumption of host feeding resulted in recovery of ciliatecomposition in opposite sequence. One week after that bothindices were 80% and 81% of initial figures as for ciliate den­sity, and 72% and 57% as for the number of species for themale and female, respectively.The influence of host starvation on horse ciliates closely re­sembles that on the ruminant ciliates (Ferber, 1928). There­fore, short time starvation does not cause dramatic changesin protozoal status of the hosts.

68Pathogenicity of Some Protozoan Parasites of CulturedFishesI. DYKOvAInstitute of Parasitology, ~cademy of Sciences of theCzech ~epublic, 370 05 Ceske Budejovice, CzechRepublic

The unders~anding .of pathog.enicity of some groups of proto­zoa~ parasites of fishes has Improved over recent years pri­manly due to advanced histological studies of infections inwild and farmed freshwater and marine fishes. The wide vari­ety ~f hitherto known tissue injuries caused by myxosporeanshas increased by new data on alterations of capillary com­plexes .(~et'Yorks) in eye and swim bladder and on changesIII specific sites of vascular, nervous and urinary systems. In­tracell~lar development of plasmodial stages was revealed inepithelial cells of renal tubules in early stage of infection.Newly recognized ,al~erations by extrasporogonic life cyclestages changed existing concept of pathogenicity of some"coe~ozoic" myxosporeans. Pathogenic potential of amoe­bae III the development of proliferative gill disease wasproved by the pathogenetic studies of gill lesions. Free-livingamoebae were recognized as agents of systemic infections inf:eshwater fishes .. New data emerged on the nature and pos­Sible~xte~t of leslOn~ caused by previously known protozoanparasites III farmed fishes. Among the recent most instructiveexamples of protozoan infections that can influence profit-

422 . Abstracts

ability of intensive fish cultures, turbot (Scophthalmus max­imus) infections with micro sporidia, amoebae and histopha­gous ciliates can be quoted.

69Intraspecific Variation within a Parapbysomonasimperforata Population] . ECCLESTON-PARRYDivision of Biological Sciences, Lancaster University,Lancaster, UK

Strain variability amongst the heterotrophic nanoflagellateshas received very little attention despite evidence for its exis­tence in other aquatic organisms such as cladocerans, fish,ciliates and bacteria. Although the latter have means for pro­moting genetic exchange within and between populations itappears that sexual reproduction may not be an essential pre­requisite for inducing strain variabil ity. Sexual reproductionin heterotrophic flagellates has not been demonstrated and yetthere is evidence of variability within a single species. Choi &Peters (1992) demonstrated that two distinct ecotypes of theflagellate Paraphysomonas imperforata had optimum specificgrowth rates at different temp eratures. But is this physiologi­cal adaptation of a single strain to a given environment or is itthe existence of different strains of the same species?Thi s poste r presents the results obta ined from a preliminarystudy which examined physiological differences between fourParaphysom onas imperforata strains, all of which were iso­lated from the same location at the same time.

70The Ultrastructure of Occupied and UnoccupiedTrichocyst Docking Sites in Pseudomicrothoraxdubius: What Morphological Modifications Occur atthe Docking Site During DockingK. EISLER* and R . K. PECK " *"Universitat Tubingen, Spezielle Zoologie, Auf derMorgenstelle 28, D-72076 Tubingen, FRG;**University of Geneva, Proristologie, Pavilion desIsotopes, 20 Bd. d'Yvoy, CH-1211 Geneva 4,Switzerland

A combined freeze-fracture and thin-section analysis employ­ing a fixation-contrasting procedure that improves resolutionof memb ranous structures has been used on P. dubius cellsthat either possess or do not possess trichocysts, or on cellssynchronized to produ ce high numbers of tr ichocyst dockingevents. Cells cultivated without Se contain no ejectable ma­ture trichocysts docked in the cell cortex, therefore these cellscan be used to examine unoccup ied docking sites and com­pare them with occupied ones in cells cultivated with Se. Inmost other ciliates, there are alveolar sacs underneath theplasma membrane, and alveolar septa are observed wheresacs meet one another. In P. dubius, however, the cortical al­veolar system is not comp osed of distinct alveolar sacs. In­stead, it consists of a continuous alveolar space that istranspierced by channel s that are circular in cross-sectionand that join inner and outer alveolar membranes together.No septa are observed. In occupied tr ichocyst docking sitessuch alveolar channels are located above the tr ichocysttips. Here the epiplasm is also lacking. As a consequence,the membrane surrounding the trichocyst reaches nearly to

the plasma membrane, in sufficient proximity to allow fusionof the 2 membrane systems during exocytosis. In unoccup iedtrichocyst dockin g sites, however, we have verified by seria lsectioning that the epiplasmic layer is not interrupted andno alveolar channels are present . Thus, a docking trichocystmust penetrate the epiplasm and a channel must be createdthrough the alveolus before it can appro ach the plasma mem­brane in its final, docked position . This cannot be achieved bysimply pushing aside the alveolar bord ers as is assumed tooccur during docking in Paramecium and other ciliates withdistinct alveolar sacs and a discontinuous epipla sm at thedocking site. These results illust rate the first and last stepsof what appears to be a highly dynam ic process of tr ichocystdocking. We are curr entl y exa mining cells that were placed inSc-supplernented medium to induce synchronous tr ichocystmaturation and docking, and then fixed for electron micro­scopy during the period when a high number of dockingevents should be observable. We hope to obtain a step by stepsequence of the details of the docking process itself.

71The Paroral Membrane, its Implication inMorphogenesis and its Importance in Ciliate EvolutionK. EISLERUniversitat Tiibingen, Spezielle Zoologie, Auf derMorgenstelle 2 8, D 72076 Tubingen, FRG

In recent years results on ciliate stomatogenesis clearly de­mon strate that gyrnnostome ciliates probably have evolvedfrom ancestors equipped with an elaborate oral cilature. Si­multaneously it could be shown by molecular data that cili­ates with a true paroral and adoral ciliature branch off veryearly in the ciliate tree. Thu s one may go one step furth erproposing the paroral membrane of extant ciliates, rath erthan somatic kineties, to be the remnant of the very first ci­liature of the ciliate ancestor. Although highly modified inadult cells, a basic pattern of the paroral membrane can berecognized, at least during those stages of stomatogenesiswhen the new cytopharyngeal apparatus is formed. The par­oral membrane basicall y is composed of kinetosomal pa irsorientated perpendicular to the longitudinal axis of the entireorganelle. The anterior-posterior axis of the posterior kineto­some of each pair is orientated perpendicular to the longitu­dinal axis of the paroral membrane. The anterior kinetosomesshow variable orientations and even may change orientationduring stomatogenesis. A paroral membrane composed likethis is not only found in man y ciliate taxa like the kar yorelic­rids, heterotrichs, hypotrichs, nassulids, hymenostomes, pros­tom es, colpodids and peritrichs, it also plays a cent ral partduring stomatogenesis. Prob ably in all ciliates that havenot completely lost their paroral ciliature the paroral mem­brane is involved in the form ation of the cytopharyngeal ap­paratus. In man y ciliates the paroral membrane provides theoral anlagen for the opisthe. In some ciliates it wa s observedthat the paroral membr ane provides new somatic structures.Due to its broad distr ibuti on and its important ontogeneticfunctions, it is assumed that the paroral membrane alsoplayed a central part in ciliate evolution: The first step inthe evolution of the ciliate kinetome was the formation ofthe paroral membr ane as a single row of paired kinero­somes, evolved from the flagella of a dinoflagell ate-like ances­tor and responsible for locomot ion, ingestion and form at ionof the cytopharyngeal tube. In a second step somatic kinetieswere formed from the right row of kinetosomes of the paroral

membrane as a result of a longitudinal splitting of the paroralmembrane and a subsequent migrat ion of the forming kinetyinto the somatic cortex. Finally, in a th ird step the adoral or­ganelles evolved from somatic kinetids left of the oral area.Consequently the ancestral type of sto matogenesis was a buc­cokinetal one derived from the mode the flagellate ancestorused to distribute its kinetosomes to the offspring cells. Theseconclusions, based on compara tive studies on ciliate morph o­genesis, are corroborated by molecular data from other la­boratories.

72Heterotrophic Flagellates in SoilF. EKELUND and R. R0NNDepartment of Population Biology, ZoologicalInstitute, University of Copenhagen,Universitetsparken 15, DK2100 0. e-mail:fekelund@zi.ku.dk

The diversity of heterotrophic flagellates in soil have beenstudied by two different methods: the traditional, indirect"culture dilution method " and the direct "coverslip meth­od"; which - to our knowledge - never has been applied tosoil flagellates before. Flagellates commonly observed bythe culture dilution method include: Heteromita globosa, spe­cies of Cercomo nas and Spumella, Dimastigella trypanifor­mis, and Hyperamoeba sp. Th ese forms fit nicely into thetraditi onal picture of a soil flagellate: that is a small moreor less amoeboid organism, without any elaborate surfacestructures.When the soil flagellate commu nity was studied by the direct"coverslip method ", a numbe r of forms common to aquaticsystems were observed; forms which have never or only veryrarely been reported from soil. These included among othersseveral choanoflagellates and severa l species of Bicosoeca.The reported observations shows that the soil flagellate diver­sity - as revealed by the traditional culture techniques - is onlya subset of the tot al soil flagellate community; and that thesampling method has a huge impact on the observed speciescomposition in samples of heterotrophic soil flagellates.

73Heterotrophic Flagellates in Soil, and their Potential asBioindicatorsF. EKELUND and S. CHRISTENSENDepartment of Population Biology, ZoologicalInstitute, University of Copenhagen,Universitetsparken 15, DK2100 0 . e-mail:fekelund@zi.ku.dk

Despite some obvious advantages, heterotrophic soil flagel­lates have been used very little as bioindicators. Besides thequalitie s soil protozoa have in genera l (see Foissner 1994,in Soil Protozoa, ed. J. F. Darb yshire), heterotrophic flagel­lates have some further advantages, including a high geneticand ultrastructural diversity, which almo st encompass thevariation displayed by all eucaryotic organisms; in many si­tuation s they are the numerically dominant group of soil pro­tozoa. Furthermore counts of small heterotrophic flagellatesand amoebae will give an integrated measurement of bacterialproduction over time; a quantit y which gives a good indica­tion of soil fertility and is otherwise very difficult to measure .

Abstra cts . 423

Preliminary studies suggest a high sensitivity of heterotrophicflagellates against some xeno biotics: four species of small het­erotrophic flagellates, Cercomonas minimus, C. granulifera,Dimastigella trypaniformis, and Hyp eramoeba sp. showeda much higher sensitivity against the fungicide fenpropi­morph than the ciliate Colpoda sp., and the naked amoebaAcanthamoeba sp.Repeated samplings, which will reveal successional pat terns,should be preferred to single observations, because ecosystemdisturbance may result in both increased and decreased flagel­late numbers. Effects on flagellate diversity will yield moreinformation than counts but is hampered by lack of knowl­edge of the species compositio n of the soil flagellate commu­nity.

74Experimental Human Microsporidiosis inImmunocompetent and Immunocompromised MiceBALB/cY. EL FAKHRY", T. VAN GOOL'~ ", C. OMBROUCK",1. DESPORTES-LIVAGE* and M . GENTlLlNI*"Unite INSERM 313, Centre Hospitalie r Universitairede la Pitie-Salpetriere, 91 Bd de l'Hopital, 7563 4 Pari scedex 13 , France,*"Department of M edical M icro biology, Acad emicM edical Center, University of Amsterdam,Meibergdreef 9, 1105 AZ , Amsterdam, TheN etherlands

Objective: Obtention of a murine model for studying protec­tive immune responses to human microsporidioses and forscreening immunological and therapeutic agents.Methodology: Spores of Septata intestinalis obtained by invitro culture on RK13 cells were used to infect 2 groups ofBALB/c mice. One group was immunocompetent. Immun o­suppression of the other group was induced by intraperito­neal inoculation of 20 ul of hydrocortisone acetat e twice aweek. Each mouse was administered perorally 2 x 107

spores. Immunocompromised and immunocompetent miceBALBlc which were given sterile PBS were used for con­trols. The spores shedding was evaluated in feces by usingUvitex 2B staining.Results: 1. Both groups develop a microsporidian infection.2. The number of spores collected in the feces was signifi­cantly higher in immun ocompromised animals than in thoseimmunocompetent. 3. In most animals the shedding began onday 7 and peaked on day 14 post infection. Two other peakswere also obtained on days 21 and 28.Conclusion: Hum an microspor idia may develop infection inmice. The para sitemia appears to be more important in im­munocompromised animals. The period icity of the sporesshedding suggests that the life-cycle of Septata intestinalisis completed within about 7 days.

424 . Abstracts

75Recent Developments of Myxosporeans in FishM. EL-MATBOULI and R. W. H OFFMANNInstitute of Zoology, Fish Biology and Fish Diseases,University of Munich , Germany

About] 400 species of myxosporeans have been recorded in46 genera. Most of them are para sites of fish. The relativelylow pathogenicity of most myxosporeans is interpreted by along history of adaptat ion.All information on the life cycle of these parasites were basedon the sporogonic phase in spontaneously infected fish host.Therefore attempts to transmit the myxosporean spores aswell as searches for vectors, secondary hosts and the actualinfectious form have taken place. We have been studyingthe mode of transmission of five myxosp oreans (Myxoboluscerebralis, M. cotti, M. pavlovskii, M. carassii, Hoferelluscarassii). In all cases actinosporean could be proved to bethe stage infectiou s for fish. Penetration of Triactin omyxonsporopl asm (Actinosporea) of Myx obolus cerebralis thr oughskin, fins, buccal cavity and gills have been demonstrated ex­perimentally in rain bow trout. Furthermore the mult iplica­tion-stages of penetrated triactinomyxo n-sporoplasms reachthe car tilage via periph eral nerves and the central nervoussystem (CNS). This is in contrast to the assumption thatthe agents reach the cartilage via blood, lymph and/or coelo­mic fluid. During the first hour following penetrat ion, thesporoplasm migrates between the epidermal cells. Then, itenters the epitheli a and multipli es intracellularly. Thesestages migrates deeper into the subcut is, then through the per­ipheral nerves and CNS. After about 2 ] days the parasitesreach the head cartilages. During their migration they alsomult iply to increase parasite numb ers. The ultrastructur e ofthe proliferative phase (presporogonic developm ent ) andthe sporogo nic phase of the life cycle is shown in detail.

76Preliminary Study on Glycolytic Metabolism ofCryptosporidium parvumE. ENTRALA, M. SANCHEZ-MORENO and C. MASCARODept. Parasitologia, Fac. Ciencias, InstitutoBiotecnologia, Severo Ochoa sin , 18071-Granad a,Spain

Cesium chloride pur ified oocysts were obtained from sponta­neously infected newborn calves. After excystat ion, sporo­zoites were homogenised and the activity of enzymesrelated to the glycolytic pathway measured. In the sameway, the following assays were performed: Amyloglucosi­dase, amylases, glucosidases and phosphoglucomutase;pyruvate decarboxylase, alcohol dehydrogenase , phosphoe­nolp yruvate carboxykinase and car boxylyase, malatedehydrogenase, "malic" enzyme, pyruvate carboxylase, pyr­uvate dehydrogenase, fumarase, succinate dehydrog enase andisocitrate dehydrogenase. We also study the electroph oret icmotility on isoelectrofocusing gels of the main enzymes, aswell as the intracellular distribution using different ial centri ­fugation. The results suggest that the main energy source atthe infective stage of C. paruum depends on previously storedamylopectin, being unable to use exogeno us glucose or othersugars. Tricarboxylic acid cycle was not detectable at thisstage, and the main end-product of the Embden-Meyerhofpath way of glycolysis appears to be lactate.

77Interactions Between Different Groups of Ciliates in anActivated-Sludge Plant Loaded with CommunalSewageM. ETfLDepart ment of Zoology, AG Heinig , Philipps­University of M arburg, Marburg, Germany

Regulat ing the species composition ciliated pro tozoa play animporta nt role in the community of organisms in activatedsludge.Changes in size and species comp osition of the ciliate popula­tion have been determined in sequentia l samples of mixedliquor twice a week, taken over one year, from activatedsludge of the communal treatment plant of Marburg-Cappel.Usually the protozoan community was dominated by peri­trich ciliates of the genera Epistylis and Vorticella. It turnedout that the population density of the peritrichs was affectedby predation and by infestation with parasites. The majorconsumers were ciliates of the genera Amphileptus and Lito­notus; the parasites belonged to the genera Manuelophryaand Endosphaera.Furth ermore, the regulat ions of the popu lation dynamics havemainl y been attributed to fluctuat ions in the amount of theaffluent.

78Looking for Actin through the Protists' WorldJ. F. FAHRNIDepartment of Animal Biology and Zoology,University of Gen eva, CH-1224 Chene-Bougeries,Switzerland

Actin was identified in several species of Proti sts. Total cellextracts were separated by 1D SDS-PAGE then immuno­blotted with the anti-actin monoclonal antibody ]LA 20(Lin, 198 1). Some examples: among the Mastigophora,Chlorogo nium and Pandorina show 1 band at 41 kD, Vol­vox 1 band at 42 kD. Among the Sarcodina, Grom ia shows1 band at 42 kD, Amoeba 1 band at 43 kD and Actinosphaer­ium 1 band at 44 kD. Among the Dinoflagellates, Glenodi­nium and Amphidinium show 1 band at 42 kD,Prorocentrum 1 band at 43 kD and Gonioaulax 1 band at44 kD. Among the Ciliophora, Pseudom icroth orax shows 1band at 38 kD, Euplotes shows no band at about 40 kD,but several band s between 60 and 120 kD, Sty lonichia has1 band at 40 kD, Urostyla has 1 band at 43 kD, Stentorhas 1 band at 40 kD, Spirostomum and Blepbarisma have1 band at 42 kD, Climacos tomum has 1 band at 43 kDand Loxodes has 1 band at 46 kD. Finally, among the Fora­minifera, Triloculina shows 1 band at 46 kD, Allogromia has2 major bands at 43 and 45 kD while Rosalina has 1 majorband at 43 kD and 1 minor band at 45 kD. In conclusion, thisscreening reveals that, contrary to the Metazoa which showgenerally 1 actin band at about 42 kD, the Prot ists exhibitactins of different MW, ranging from 38 to 46 kD, and thatsome species possess 2 or 3 actins of different MW.

79A new High Speed Video Camera for Protozoology andCell BiologyJ. FEBVREl, F. BOUFFAULTI,2, M. PAINDAVOINE2 andC. FEBVRE-CHEVALIERIIURA 671, CNRS: Biologie Cellulaire Marine 06230,Villefranche-sur-Mer, France.2Laboratoire LIESIB. Faculte des Sciences. 6 bdGabriel, Dijon, France

The high speed video camera constructed in our laboratoriescan be used as a pre-industrial model. The recording speed canbe adjusted from 50 fr.s" to 600 fr.s- 1• This camera can beeither record tens of images with a higher resolution or a longsequence of frames with a low spatial resolution. In the lastcase, the images stored in a CCIR standard can be read with astandard magnetoscope. The available formats are 380 x 244or 190 x 244 pixels at 600 images S-l.

Using this camera, it has been possible 1) to confirm that thevery rapid microtubular destabilization in the heliozoanActinocoryne contractilis consists of a fragmentation process2) to show that the lag time between the stimulus and thecontractile response is less than 2 ms which indicates thatthe biochemical message induced by the Ca 2+ influx is remark­ably short.

80Preliminary Study of the Intertidal EpibenthicProtozoan Communitites in Three Areas of the Bay ofBiscay (Spain)D. FERNANDEZ, G. FERNANDEZ-LEBORANS andA. NOVILLOLaboratorio de Biologia General, Departamento deBiologia Animal I (Zoologia), Facultad de Biologia,Universidad Complutense, 28040 Madrid, Spain

Samples from three areas of the Cantabrian coast: Castro Ur­diales, Santofia e Isla, were collected from November 1994 toJanuary 1995, in order to analyze the presence of differentprotozoan groups, the abiotic factors and the possible impactof heavy metals on the structure of the communities. Sedimentwas sampled to a depth of 5 em. The density and biomass ofeach protozoan species was obtained, and also, by means offluorescence, the density of bacteria. In the interstitial waterthe following factors were measured: pH, oxyde-reductionpotential (mV), dissolved solids (rng.l :"), temperature (QC),phosphates (ug.l"), nitrates (ug.l :"}, nitrites (ug.l "), salin­ity (%.), dissolved oxygen (rng.l r '] and levels oflead and cad­mium. In addition a granulometric analysis of the sediment,and the amount of organic matter, was carried out. 211 pro­tozoan species were found, with a predominance of bacteri­vore (37%), photoautotroph (30%) and non-selective (25%)trophic groups. The density of protozoans was 0.9-2.0 x 10 3

ind.crn ? (Castro Urdiales), 1.4-4.3 x 10 3 ind.cm- (Santona)and 0.9-3.9 x 103 ind.cmv- (Isla). Protozoan biomasses were0.04-3 x 10 3 mg.rnr? d.w. (Castro Urdiales), 0.2-52 x 10 3

mg.mr- d.w. (Santotia), and 0.01-47 mg.rn r- d.w. (Isla). Thecanonical analysis showed significant differences betweenSantofia and the other two areas. The principal componentsanalyses using abiotic and biotic variables indicated that themost important differences were due to the salinity change

Abstracts . 425

observed in Santofia (January). No pollution caused by cad­mium was detected and the levels of lead in water have fluc­tuated between 1 and 13.25 ug.l :".

81Changes in a Human Personality Profile Induced byChronic ToxoplasmosisJ. FLEGR, P. KODYM and S. ZITKOVADep. Parasitol., Charles Univ, Vinicna 7, Prague 12844,Czech. Rep.

The effect of parasites on host behavior was proved in manyhost-parasite systems. Because the induced behavioral pat­terns often promote parasite transmission, the behaviour­modification activity is usually considered as a sophisticatedproduct of the parasite evolution aimed at the host manipula­tion rather than an accidental by product of other physiolo­gical activities of the parasite. A strong influence of a parasiteon host behavior has been observed in different Toxoplasma­mouse and Toxoplasma-rat models. Recently the existence ofstatistically significant differences in two personality factors(the Superego strength and the Pretension, [1)) has been ob­served on a random sample of health students with and with­out anti-Toxoplasma cellular immunity (assayed with IDHT)[2). To reveal whether toxoplasmosis induces the personalityfactors shift or whether certain combination of personalityfactors influences the probability to acquire the Toxoplasmagondii infection, we examined the personality profiles of 164patients with the acute toxoplasmosis diagnosed during past13 years. After the elimination of the effect of age, the regres­sion analysis shoved the existence of positive correlation be­tween length of latent toxoplasmosis and the intensity of theSuperego strength shift (p =.017). There was no significantcorrelation between the duration of infection and any of fif­teen other studied personality factors, including the Proten­sion. The present study confirmed the existence of thetoxoplasmosis-associated Superego strength shift (decreaseof a willingness to accept group moral standards). More­over, it proved that the decrease was induced by latentT. gondii infection, rather than the acquisition rate of infec­tion being positively influenced by the low value of the Super­ego strength. 1 Cattell, R. B. 1970, Handbook for the sixteenpersonality factors questionnaire (16PF). Institute for Per­sonality and Ability Testing, Champain, 115 pp. 2 Flegr,J. & Hrdy, 1. 1994, Folia Parasitologica 41, 122-126.

82Fine Structural Specializations in a JumpingPeritrichous Ciliate, Hastatella radians Erlanger, 1890(Ciliophora, Peritrichia)I. FOISSNER and W. FOISSNERUniversitat Salzburg, Institute fur Pflanzenphysiologieund Zoologie, Hellbrunnerstrasse 34, A-S020Salzburg, Austria

Hastatella radians is a rare planktonic ciliate living in tempor­ary pools and in the pelagial of lakes and slowly running, largerivers. It lacks a stalk but possesses an anterior and equatorialgirdle of mobile spines continuous with the somatic cortex.The length and number of the spines decreases drastically,i.e. by 50% and more in laboratory cultures, obviously dueto the lack of environmental stress. Previous light micro-

426 . Abstracts

scopic stud ies have suggested that the spines are passivelymoved by contractions of the cell and/or of individual rnyo­nemes, thereb y producing the conspicuous jumps driving thecell through the medium. Howe ver, our electron microscopicinvestigations suggest that the spines can move independentlyof the myonemes, because they cont ain specialized structureslacking in other peritrichs, viz. subcort ical fibres and micro­tubul es. The closely packed fibres extend underneath the epi­plasm and have a complicated periodic structure reminiscentof that known from flagellar rootl ets. Underneath the striatedfibres is a layer of loosely arranged microtubules extending tothe top of the spines. The general ult rastructure of H. radiansis very similar to that of other peritrichs. There is, for instance,a scopula organelle composed of short cilia lacking the centralmicrotu bule pair and the axosome. The oral infraciliatureconsists of ciliated adorals and a paroral having only the dis­tal basal bodies of the dikinetids ciliated.

83Ciliate Phylogeny Inferred from OntogenyW. F OISSNER

Universitat Salzburg, In st itut fur Zoologie,H ellbrunnerstrasse 34, A-S020 Sa lzburg, Austria

The phenomenology of ontogenesis in ciliated protozoa hasbeen reviewed, with emphasis on stomatogenic data pub­lished between 1870 and 1993. Thr ee basic types of fission(homotheto-genic, enantiotropic, parallel), two basic modesof division (active, cystic), and five main modes of sto mato­genesis (apokinetal, parakinetal, buccokinetal, telok inetal,mixok inetal) were distinguished. Within the main stoma to­genic patterns several subtypes occur, some of which, how­ever, possibly evolved convergently in different ciliategroup s. There is an urgent need for refined studies, especiallyin most heterotrichs, thigmotrichs, apostomes and prostoma­tids and in all karyorelictids, chonotrichs and rhynchodids.Hennig's cladistic method was applied to the ontoge neticdata and several morphological features as well as molecularmarkers. Although it was not possible to determine all char­acter states unequ ivocally and to harmonize all data, the c1a­dogram suggests main pathways in ciliate evolution and threemajor conclusions: (i) A subphyletic division of the Cilio­phora based on a cyrto s or rhabdos type of oral app aratusis not supported; (ii) Some stomatogenic modes evolvedeither convergently or are only superficially similar, viz. bylight microscopy; (iii) The "eociliate" possibly possessedthe following character constellation: a dividing, homomer­ous macronucleu s without a reorganizat ion band ; a cyrtos­type ora l apparatus with adoral membranelles and apar oral membrane; somatic dikinetids with postciliodesma­ta; homothetogenic fission, and buccok inetal stomatogenesis.

84Tropical Soil Protozoan Di versity: The Ciliates(Protozoa, Ciliophora) of a Giant Pancake, Etosha inNamibia (Southwest Africa)W. FOISSNER

Universitat Salzburg, Institut fur Z oologie,Hellbrunnerstrasse 34, A-S020 Sa lzburg, Austria

12 soil samples were investigated for ciliates from the centreand periphery of the Etosha Pan. The pan soil is a very specialmixture of salt, clay, and lime having a pH range of about8.0 - 9.7; the air-dried mixture is like a stone, but quickly dou­bles its volume and becomes a fluffy pancake when it is re­wetted. Most of the soil is covered with a more or lessdistinct layer of filamentous cyanobacteria . 153 ciliate spe­cies were found, 53(!) of them were new to science. Most be­long to one of the following group s: hypotrichs (43 species),colpodids (35), gymnostomatids (33), nassulids (15). Thehigh number and frequency of nassulid ciliates, usually spar­sely occurring in soil, is obviously related to the comm onnessof cyanobacteria, which are their preferred food. A transectfrom the pan to the surrounding savanna showed that the saltshrub (Suaeda) region has the highest species richness and thenum ber of species sharply decreases abo ve pH 8.6: pan centre(saline desert, pH 9.7-8 .7, 9-21 species), Suaeda zone(pH 8.6 -8.4, 43-57 species), thorn bush savanna (about1 km distant from pan margin, pH 7.7, 28 species), Mopane(Colophospermum) savanna (about 15 km distant from panmargin, pH 7.7, 37 species). Refined ecological researchabout these special ciliate communities is pressingly neededbut difficult to realize because of the high numb er of new,not yet described species. (The help of Dr. Lindeque, directo rof the Ecological Research Stat ion of the Etosha NationalPark; is greatly acknowledged.)

85Dr. Merkle's Life Crystals and Chondrianas, a Chancefor Cancer and HIV Patients or Dividing and ExcystingColpodid Ciliates?W. F OISSNER

Universitat Salzburg, Institut fur Zoologie,Hellbrunnerstrasse 34, A-SOlO Salzburg, Austria

George Merkle, Ph.D., a nuclear physicist from the USA,sta tes in a 55 min video presentat ion: "This program docu­ments a major breakth rough in medical science and molecu­lar biology. For the first time, living, intelligent, pre-cellularorganisms, from which all life form s evolved have becomevisible. The discovery point s directly to a new genera tionof biologists and physicians, who will have the ability to pre­vent the diseases that now afflict human kind". The discov­eries mentioned are "Life Crystal s", developing from "freeenergy", and organ isms which he calls "Chondriana" . A care­ful ana lysis of the film, which is of poor techn ical quality,proved the following: The Life Crystals are bacteria andthe "superintelligent and polymorphic Chondriana", whichare the "precursor to our killer T-8 cells and lymph ocytes",are trophonts, dividing, and excysting ciliates of the generaColpoda and Pseudoplatyophrya. Thu s, Dr. Merkle's "dis­covery" is nonsense and would be not worth to be men­tioned. Unfortunately, he sells a serum containing theseLife Crystals and Chondria nas and mentio ns that the serumhas been successfully applied in many countries. The serum is

applied intravenously (as I know from a physician who tried itin a self-experiment - and got a sepsis) and should heal alldiseases, including cancer and AIDS. A microscopic investiga­tion of the serum showed that it contains a lot of bacteria,yeast, and ciliates. Thus, if it is injected, very likely a heavytoxaemia and eventually death will result.

86Infraciliature and Phylogeny of Trachelocercids(Ciliophora, Karyorelictea)W. FOISSNER' and J. DRAGESC02lUniversitat Salzburg, Institut fur Zoologie,Hellbrunnerstrasse 34, A-5020 Salzburg, Austria,2Bd du Grand Devois, F-34980 Saint-Clement-de­Riviere, France

Taxonomy of trachelocercid ciliates is bewildering and bur­dened with many poor species descriptions, even from recenttimes. The type species, Trachelocerca sagitta Muller, 1786,fixed by Ehrenberg (1840), has been largely ignored and isthus poorly known. Silver impregnation has been rarely ap­plied, possibly because of methodological problems. Usinga new fixative and Wilbert's protargol technique, we got ex­cellent preparations and could distinguish four organizationtypes. Type A: trachelocercids with a "compound" paroralcomposed of 2-4 rows of dikinetids; it is interrupted onthe left side, where a complicated "brush organelle" resideshighly reminiscent of that found in prostomatids. This typeis definitely different from the following types which havea "simple" paroral composed of a single row of dikinetids.Type B: trachelocercids with simple paroral and brush orga­nelle. Type C: like type B, but without brush organelle. TypeD: like type C, but with a conspicuous tuft of cilia (possibly ahighly modified brush) near the centre of the apical end, i.e.within the presumptive oral opening. Each of these types re­presents at least a distinct genus, type A will be separated atfamily level. Its multi-rowed paroral is reminiscent of the lox­odid buccal kineties, like the circular kinety extending on theleft side. Thus, trachelocercids and loxodids are very likelysister groups, i.e. have a common ancestor.

87Metabakuella sp., a New Hypotrichous Ciliate(Ciliophora, Hypotrichida) Occurring in FreshwaterBenthos?C. FRANCO", G. ESTEBAN'''' and C. TELLEZ""*"Estacion Biogeol6gica "EI Ventorrillo", MuseoNacional de Ciencias Naturales, CSIC, Madrid~"'Institute of Freshwater Ecology, WindermereLaboratory, Ambleside, Cumbria LA22 aLP, UK.**"Departamento de Microbiologia III, Facultad deCiencias Biol6gicas, U.C.M

The morphology of a new hypotrichous ciliate, Metabakuellasp. is described. It was found in the benthos of a freshwaterstream in Guadarrama (Madrid, Spain). The description isbased on live observations, protargol (Wilbert, 1975) and sil­ver carbonated (Fernandez-Galiano, 1994) organisms. Meta­bakuella sp. is elliptical, rounded at both ends, dorsoventrallyflattened, flexible and 135 -310 x 35 -62.5 urn in size. It has5- 8 buccal cirri, 4- 6 frontal cirri, 2 frontoterminal cirri, 26-

Abstracts . 427

36 midventral cirri arranged in 13-18 pairs that end belowthe cytostome, and 7 obliquely arranged cirral rows at theposterior end of the midventral row. The anterior part ofthe midventral row forms a "bicorona" with the frontalcirri. It also has 2 right marginal rows, 2 left marginalrows, 8-11 transversal cirri and 3 dorsal kineties. Caudal cir­ri are lacking. It has more than 100 macronuclear fragmentsand 4 -12 elliptical micronuclei. Contractile vacuole at levelof cytostome. The adoral zone of membranelles, about 40%of body length, consists of 46 membranelles on average. Itfeeds on bacteria, flagellates and small ciliates.Metabakuella sp. is probably a new species: the arrangementof midventral cirri and ventral rows of cirri and the number ofmarginal rows of cirri, frontal cirri and frontoterminal cirriseem different than in other similar hypotrichs. A revisionof the Subfamily Bakuellinae Jankowski, 1979, is also in­cluded.

88Evidence for a Selenodependent GlutathionePeroxidase in PlasmodiumB. GAMAINl, J. ARNAUD2, A. FAVIER2, D. CAMUSl,D. DIVE! and C. SLOMIANNy 1

lINSERM U42, 369 rue Jules Guesde, 59651Villeneuve d' Ascq cedex, France; 2Laboratoire deBiochimie C, Centre Hospitalier Regional etUniversitaire de Grenoble, 8P217, 38043 Grenoblecedex 9, France

Glutathione peroxidase (GPx), a key enzyme involved in thedetoxification of many peroxides was investigated in P. yoeliiin vivo and P. falciparum in vitro. We showed the presence ofan endogenous GPx activity in these two Plasmodia species.Enzymatic assays and the use of specific substrates and inhi­bitors allowed us to precise that the detected activity was se­leno-dependent, As this activity was shown to be smaller inP. falciparum than in P. yoelii, we hypothesized and then ver­ified by dosage that there was a severe selenium deficiency inculture medium and culture red blood cells which could beresponsible for this difference. After selenium supplementa­tion with sodium selenite on one hand and selenocystineon the other hand, we observed an increased growth ofP. falciparum only in the first supplemented medium whereashigher GPx activities were noted in parasites grown in the twosupplemented media. As the host cell is unable to synthesizenew proteins, these results provide further evidence of an en­dogenous parasitic selenodependent glutathione peroxidasein Plasmodium falciparum.

89Protein Processing and Secretory Granule Biogenesis inParameciumN. GARREAU DE LOUBRESSE, M. C. GAUTIER, L. MADEDDUand L. SPERLINGCentre de Genetique Moleculaire, CNRS,91198 Gif-sur-Yvette, FRANCE

Paramecium, like other ciliates, has a regulated secretory sys­tem featuring architecturally complex secretory storage gran­ules known as trichocysts. The protein contents of thegranules has a crystalline organization yet is built up froma heterogeneous family of immunologically related small

428 . Abstracts

acidic polypeptides. These polypeptides are the products ofproteolytic maturarion of precursor proteins encoded by alarge multigene family. We have cloned and sequenced genescoding for three different precursor proteins and comparisonof the sequences provides information on the different proces­sing reactions required to produce the mature polypeptides ofthe crystalline trichocyst matrix. This information, along withrecent studies of protein processing and membrane traffic insecretory mutants unable to produce functional trichocysts,allows us to propose a model in which distinct protein pro­cessing reactions control key steps in trichocyst biogenesis.

90Does the Large Subunit Ribosomal RNA Sequence ofTrichomonas vaginalis Improve the Resolution of theBase of the Eukaryotic Tree?A. GERMOT1, H. PHILIPPE2, N. PUCHOT' andH. LE GUYADER2

'Laboratoire de Biologie Comparee des Protistes, URA1944, Universite Blaise Pascal - Clermont-Fd II, lesCezeaux, 63177 Aubiere cedex, France. 2Lahoratoirede Biologie Cellulaire 4, Bat. 444, URA 1134,Universite Paris-Sud, 91405 Orsay cede x, France

Microsporidia, diplomonads and trichomonads are threeamitochondrial groups which seem to have diverged veryearly during the evolution of eukaryotes, as evidenced by se­quences comparison, especially of small subunit rRNAs(SSU,18S). But their relative order of emergence is not clearlyresolved, principally the position of diplomonads versus tri­chomonads. Presently, the SSU rRNA database is the mostcomplete one (about 400 eukaryotic species). The large sub­unit rRNA (LSU,28S) sequences are known for 40 eukaryoticspecies which are almost all present in the SSU database, ex­cept for microsporidia and trichomonads. In order to improvethe resolution of the base of the eukaryotic tree, we havedecided to sequence the LSU rONA of Trichomonas vagina­lis. Using 5' terminal region of Trichomonas vaginalis LSU(obtained by PCR) as probe, we have screened a ",ZAP geno­mic library (kindly provided by Patricia Johnson, Universityof California). One clone contained the whole ribosomal clus­ter (about 8 kbp). The gene of the LSU rRNA has been se­quenced on both strands. Its length (2753 bp) is close tothe ones of prokaryotes and of the diplomonad Giardia, asalready observed with SSU rRNA; for one of the divergentdomain (D2), it exhibits the smallest one known so far in pro­karyotes and eukaryotes. Its GC content (about 46 %) isweakly biased. Phylogenies were constructed using SSU,LSU and SSU+LSU databases, by neighbor-joining and parsi­mony methods. LSU sequences (about 2000 aligned nucleo­tides) confirm the early emergence of Trichomonasvaginalis within eukaryotes, but unfortunately do not im­prove the resolution of the relative emergence of diplomo­nads and trichomonads. Even if we use SSU and LSU(about 3000 aligned nucleotides and 40 species) the phyloge­netic inference is the same that those obtained from SSU(about 1000 nucleotides and 100 species). This result couldbe due to the small number of LSU rRNA sequences for spe­cies at the base of the tree. Increasing the number of nucleo­tides is efficient only if the number of species remains thesame. Then, the further step would be to sequence LSUrDNA of species emerging at the base of the eukaryotic tree.

91Cellular Compartimentalization in Epixenosomcs(Epibionts of Euplotidium itoi)M. A. GIAMBELLUCA, E. TAITI and G. ROSATIDip. di Scienze dell' Ambiente e Territorio, via A. Volta4, 56126 Pisa, Italia

Epixenosomes, even in their mature form, lack membranouscell organelles but have a complex cellular organization. Anultrastructural and cytochemical study has been carried out toanalyze in more detail the various epixenosomal structures.Different compartments have been evidenced: 1) The apicalzone, not delimited by a true membrane nor by a thinner en­velope, as definitely demonstrated by freeze etching prepara­tion. It is however, well defined by its electrondensity andcontains DNA, basic proteins and a peripheral glycosilatedlayer. 2) An oval, electrondense body containing glycoproteicmaterials surrounded by a thin "limit" . 3) The extrusive ap­paratus (EA) immersed in a glycoproteic matrix differentfrom the remaining cytoplasm. The EA is delimited by a thinglycosidated layer and, following freeze etching procedureappears as a well distinct body. The localization of some en­zymatic complexes was then analyzed. Our attention wasfirstly focused on acid phosphatase and peroxidases, en­zymes that in eukaryotic cells are localized in well deter­mined cellular organelles . A precise localization has beenfound for each of them in relation with the above mentionedcompartments. Besides we applied a technique for adenylatecyclase localization: this enzyme too has a constant localiza­tion at the cell membrane level and along the external andinternal limits of EA.

92Contribution to the Understanding of theDinoflagellate Chromosome Structure: A MolecularApproachB. GILLET, G. LENAERs, Y. BHAUD and M.-O. SOYER­GOBILLARDDeparternent de Biologie Cellulaire et Moleculaire,Observatoire Oceanologique de Banyuls-sur-mcr, URACNRS N° 117, Universite Paris 6, BP44, F-66651Banyuls sur Mer, France

For most species of dinoflagellate protists, the genomic ma­terial is characterized by a high amount of DNA (7.0 x10 to base pairs/nucleus for Prorocentrum micans Ehr., 1.4x 10 to base pairs/nucleus for Crypthecodinium cohnii B.)(1) compacted in a hundred chromosomes permanently con­densed and devoid of any longitudinal differentiation (2). Thearchitecture of nucleofilaments, organized in a super twisteddouble helix (1) is maintained by divalent cations (3) andstructural RNAs (4). The DNA composition reveals the pre­sence of a rare base (5-Hydroxymethyluracil) which substi­tutes 68% of thymines in P. micans (5). Previously, wehave shown the presence of long circular molecules (6) bymeans of molecular DNA spreadings.Taking into account these original characteristics, we haveanalysed the genomic DNA of P. micans on agarose gels. ThisDNA appears to be shared in two sub-types, one of high mo­lecular weight (HMW DNA) which stays on the well edge,and one migrating as a DNA of around 25 kb (25 kbDNA). Subsequent purification of the HMW DNA on glassbeads gave rise to the 25 kb DNA, suggesting that it consists

of a dense compaction of rhe 25 kb DNA subtype. This com­pact ion is independent of the presence of structural RNA ordivalent cations, as neither treatment with RNAse and/norwith EDTA+EGTA modifies the level of compaction. Futurework will focus on the genetic and structural functions of this25 kb DNA.Ref. (1) Haapala O. K. & Soyer M. 0., 1973, Nature, 244 ,195-1 97; (2) id, Hereditas, 1974, 78, 14 1- 145; (3) Herzog,M. & Soyer, M. O. 1983, Eur. ]. Cell BioI., 30, 33 - 41; (4)Soyer, M. O. & Herzog, M. , 1985, id, 36(2) 334-342; (5)Herzog et al., 1984, Or igins of Life, 13(3-4), 205-215;(6), Haapala, O. K. & Soyer, M. 0., 1974, Hereditas, 76,83- 94.

93Role of Interferon-y Against Invasion by Toxoplasmagondii in a Human Monocytic Cell Line (THP1):Implication of the Parasite's Secreto Phospholipase AZand Evidence for a Direct Interaction Parasite- HumanCytokineJ. E. GOMEZ MARIN*, N. BENBERNOU, A. BONHOMME,M. GUENOUNOU and J. M. PINONLaboratory of Parasitology and Equipe 4 INSERMU-314, CH U Reims and Laboratory of Cellul arMadiators, Faculty of Pharmacy, Universite de Reims(France). "Depositary of a doctoral fellowship fromColciencias (Colombia)

Interferon-v (IFN-y) is a major cytokine involved in protec­tion against Toxoplasma gondii. We have examined the roleof IFNy in protection agains t T.gondii in the mono cytoid cellline THPl. The addit ion of IFNy dur ing cultures of infectedTHPI cells, reduced, in a dose dependent manner, the numberof para sitized cells without affecting the intracellular mut ipli­cat ion during the first 24 hours. Th is reduction was more im­portant in the presence of bacterial lipopolysaccharide (LPS).After 48 hours infected cell cultures treated with IFNy and/orLPSshowed a parasitostatic effect (reduction in the numb er ofintracellular parasites in comparison to controls). We exam­ined the role of secretory phospholipase-A2 (sPLA2) in theprocess of invasion in our model, and its relat ion with theIFNy and LPS induced reduction of parasitized cells. Thetreatment of cells or parasites, 10 minutes prior to infec­tion , with 4-p-bromophenacylbromide, a specific inhibitorof sPLA2, significantly reduced the number of infectedcells. Th is reduction was more importa nt when parasiteswere treated. The addition of exogen sPLA2 from Naja najavenom did not interfere with the protective effect of IFNy,and , surprisingly, confered prote ction when used alone.The activity of PLA2 was measured through a colorimetrictest in supernatants from parasites maintained in culture med­ium without cells and in the presence of IFNy+LPS. We founda significant reduct ion in the sPLA2 activity of the paras ite.Using an indicator of metabolic reducti on we found a signif­icant enhancement of the redox activity of the parasite in pre­sence of IFNy. Our data suggest that IFNy can pro videprotection not only through reducti on of intracellul ar multi­plication, but also by reducing the numb er of infected cells,indicat ing an effect on process of invasion. These results giveadditional evidence of direct inter action between protozoanpar asites and human cytokines.

Abstracts . 429

94Studies on Encystment and Excystment of thePlasmodial Rhizopode Reticulomyxa filosaG. GOTHE, K. J. B6HM, U. GOTHE and E. UNGERInstitute of Molecular Biotechnology, Research Groupof Electron Microscopy and Molecular Cytology,Beutenbergstr. 11, D-07708 Jena, Germany

It has been shown that the plasmodial rhizopode Reticule­myxa filosa produces cysts which showed a dimorphic seaso­nal trend over thr ee years: Wall-less cysts (uncovered cysts)were formed throughout the year in response to deterior ationof general environmenta l conditions. These cysts could notwithstand pressure, freezing, prolonged contact with air, ordrying. Impro vement of culture conditions is a trigger me­chanism for excystation of uncovered cysts.More resistant cysts with a thick bilayered wall (coveredcysts) developed only in summer and were able to endu re pres­sure, complete dehydr at ion, and freezing. Trigger mechan­isms for encysta tion and excystati on of the covered cystsare enigmatical up to now.With help of time lapse video light microscopy and transmis­sion electron microscopy the role of these two types of cysts inthe life cycle of Reticulomyxa filosa is discussed in relat ion toseasonal dispersion events.

95Comments on Phylogenetic Trees reconstructed fromN on-molecular Data, and on the Origin of CiliatesJ. GRAINURA CNRS 1944, BioI. comparee des Protistes, Univ.Blaise Pascal , Les Cezeaux, 631 77 Aubiere-Cedex

In a recent attempt at reconstructing a phylogeny of ciliates,de Puytorac et a1. (1994) used data covering 56 species and 23morph ological, nuclear, morphogenetical and ultr astru cturalcharacters. Two valuable trees were obtained which identify 5main groups; in tree A, two main branches early separate, oneleading to 2 sister-groups (1: Karyorclictids, Het erotrichs,Spirotri chs; 2: Colpodids), the second leading to the 3 othergroups (including Nassophorea, Oligohymenophorea, andKinetofragminophorea); in tree D, group 1 separates early,the 4 other groups differenciate later. These 2 trees are com­pared with tho se from other authors and their validity is dis­cussed with regard to the evolution of some chara cters: 1) thenuclei, whose probable history (first, 2 diploid mitot ic nuclei;then for 1 nucleus, paradiploidy but no division, and finallypolyploidy with amitotic division ) fits well with tree D, notwith tree A; 2) the position of the oral opening: the two treesagree with primitiveness of the ventral mouth; 3) the somaticcort ical cytoskeleton (CSK): the supposed primitive state(with at once 3 basic components: microtubules, non-actinmicro filaments, epiplasrn) gives rise not only to lines whichexclusively privileged one of the 3 components (hypothesisof Fleury et aI., 1992 ), but also recently appeared lines inwhich 2 (Litostomatea) or the 3 components (Vestibuli­ferea, Entodiniomorphida) are conserved. So from the "pro­tociliate " or "eociliare", different characters evolved withdifferent rates and it is impossible to find an actual paralle­lism between respective evolution of the cortical CSK, nu­clei, position and structure of the oral apparatus. As theemergence of ciliates seems to occur at a uniqu e recenttime, we have to hypothesize on the origin of ciliates and

430 . Abstracts

realisation of the protociliate. Phylogenetic relationships be­tween ciliates and dinoflagellates were postulated, whichwere confirmed by some molecular phypogenies but in someother similar studie s ciliat es and dinoflagellates do not appearas sister-gro ups. Eisler's hypothesis (1992) focused on the firstappearance of the paroral membrane, which secondarily givesthe somatic ciliature, from which the adoral ciliature differ­ent iates. However, if we consider ciliates of the first emergingline (He terotricha for ex.) whi ch are the best candidates toreflect, in the ir own ontoge ny, the ciliate ancestor onto­geny, we can observe that the buccal ciliatu re originates fromthe somatic one and that the sequence of events is tot ally thereverse of Eisler's sequence.

96Leishmania in AIDSM. GRAMICCIALaboratorio di Parassitologia, Istituto Superiore diSanita , Viale Regina Elena 299 , 00161 Rome, Italy

Leishrnanial infections in HIV-infected individuals, only 70%being AIDS patients, are a well-recognized phen omenon incountries of the Mediterranean basin: over 90% of some600 cases reported worldwide being observed in Spain, Italyand France . Thi s is interpreted as a con sequence of sharpoverlapping of the viral epidemic and the endemic status ofvisceral (Vl.) and cutaneous (Cl.) leishmaniasis, both cau sedby Leishmania infantum in this area . Almost all M editerra­nean co-infection cases described displayed visceral dissemi­nation of parasites. Some 130 Leishmania stocks obtainedfrom these patients have been examined by isoenzyme electro ­phoretic analysis and found to belong to 16 L. infantum zy­mod emes, of wh ich some are known agents of Cl, and someare new zymodemes. In Italy only, 11 zymodemes have beenident ified among 38 HIV-infected individuals with VL, repre­senting about one th ird of all co-infect ion cases recorded inth is country. Only one zymodeme, detected in 22 patients,is a common agent of Med iterranean VL in HIV-negative in­dividuals; five zymodemes, isolated from 11 patients, usuall ycause simple, self resolving Cl; five zymodemes, from 5 pa­tients, belong to unique genotypes which have not been pr~­

viously reported from either VL or CL cases . In

immunocompetent individuals. Thi s last group of parasitesshowed reassortment patterns within electromorphs fre­quently observed in dermot rop ic L. infantum zymodernes,which could be interpreted as a consequence of genenc recom­bination. Geographical anal ysis would suggest uneven distr i­but ion of L. infantum genotypes in Italy, the highestzymodeme heterogeneity being observed in Sicily.Onl y scanty information is available from other New and O~d

World areas endemic for leishmaniasis. A few VL cases In

HIV-infected ind ividuals were found to be caused by classicaldermotropic species, L. braziliensis and L. tropica. Work sup­ported by the Italian Mini stry of Health, AIDS Projects 1994and 1995.

97Plasmodium [alciparum SOD as a Potential DrugTarget: Expression of the Recombinant EnzymeS. GRATEPANCHE\ D . T OUATI2, J. M. CAMADR0 2,J.JACQUEMONT3 and D. DIVE1

lINSERM U42, 369 rue Jules Guesde, B.P. 39, 59651Villeneuve d' Ascq ced ex , France. 2Institut JacqueMonod, 2 Place Jussieu, Tour 43, 75251 Pariscedex OS, France. 3IN RA, 369 rue Jules Guesde,B.P. 39, 59651 Villeneuve d'Ascq , France

Th e endo genous SOD of P. [alciparum is iron dependent, sodifferent from its two host enzymes (respectively Cu/Zn andMn-dependent). In order to define specific inhibitors of th isenzyme, its structure has to be studied precisely. Two strate­gies are possible: 1) the stud y of the structure of the protein byphysical approaches; 2) the construction of a homologousmodel based on the structures of known iron-SODs.Th e physical approach of the structure of P. falciparum SODneeds a large quantity of the pure protein. As this enzymeconstitutes only a very small part of the total proteins ofthe parasite, the production of a recombinant protein wasa good strategy. The coding region of the cDNA and the 3/end were amplified by PCR with prim ers including two re­striction sites not present in the coding part of the cDNAand inserted in an adeq uate plasmid for expression . AnE. coli strain from which the sodA and sodB genes were in­activated was then transformed. Th e construction allowed usto obtain an active protein, which was different from the tw oenzymes of E. coli, and was recognized by an antibody spe­cific of the N-terminal part of P. [alciparum SOD subunit .A mass production of the recomb inant enzyme was plann edusing a fermentor. Th is allowed us to obtain a large amount ofthe protein both for bioph ysical approac hes and further tocrista llize it in order to perform an X-ra y cristallographystudy of the enzyme structure.

98The Motile Behaviour of Cell Fragments Obtainedfrom Amoeba proteus depends on F-actin SpatialArrangementL. GREBECKA and P. POMORSKIDepartment of Cell Biology, Nencki Institute ofExperimental Biology, Warsaw, PL 02093

The mechanisms of cell motility can be investigat ed by study­ing the different fragments of mot ile cells made in a variety ofways . The surgical bisection of a polarized Amoe ba proteuswith a microneedle produces tw o halves which differ in theamount of F-actin and its distribution . The difference be­tween F-act in arrangement in the anterior and posteri or nu­cleated , motile fragm ent s of amo eba determines somesignificant variation of their locomotory beha viour.Separately investigated anucleated fragments of Amoeba pro­teus are motionless because the depr essive effects of the ab­sence of the nucleus on the organization of thecytos keleton and cell adhesion. Application of polylysinecoated glass substrat e shows, that the recovery of adhesionand locomotory act ivity by these fragments is accompaniedby rearrangement of F-acr in, previously randomly diffusedin the ir cytoplasm deprived of nucleus.

99Influence of Surface Wettability and Charge on theAdhesion and Locomotion of Amoeba proteusA. GREBECKI, L. GREBECKA, W. KtOPOCKA andJ. KOLODZIEjCZYKNencki Institute of Experimental Biology, Warsaw,PL 02093

Recovery of locomotive morphology and resumption ofmovement by floating heterotactic cells of Amoeba proteuswere investigated on untreated glass surface, and on modi­fied substrata which had variable wettability and/or electro­static charge. All amoebae readhere to the glass within 12 minand recover the locomotive (polytactic) morphology. The rateof resuming locomotion does not significantly change on styr­ene/methyl methacrylate copolymers with different density ofhydrophilic sulfonic groups bound to their surface. It meansthat amoebae are indifferent to a variable wettability of sub­stratum, in contrast to the tissue cells which adhere better tomore hydrophilic surfaces.On the other hand, amoebae readhere as soon as after 3 minto the positively charged surface of glass coated with polyly­sine, are strongly flattened and move 2112 times slower than onthe glass. The floating amoebae never read here to the nega­tively charged gelatin gel and never resume locomotion.Maximally 25% become polytactic, but their pseudopodiaslip over the substratum and cannot move the cell.The prompt reaction of amoebae to the positively or nega­tively charged surfaces, probably depends on very low ionicstrength in their medium. Their indifference to the wettabilityof substratum is puzzling. Polylysine and gelatin substrataseem to be suitable for the study of adhesion dependent mo­tor functions of amoebae.

100The Enigma of the Refractile Body in EimeriaG. GREIFl, A. WECK-HEIMANN2 and R. ENTZEROTH2

IBayer AG, Institut fur Parasitologie, 0-51368Leverkusen, Bayerwerk;2TU-Dresden, Institut fur Zoologie, 0-01062 Dresden

Chicken coccidiosis caused by protozoan parasites of thegenus Eimeria is a widespread disease of major economic im­portance to the poultry industry. The refractile bodies are themost prominent organelles seen in sporozoites of Eimeria butstill their antigen composition and function remains poorlyunderstood. Recently two components of refractile bodywere identified by molecular cloning and sequencing: aspar­tyl-protease (Laurent et al. 1994) and trans hydrogenase(Vermeulen et al. 1993).We raised monoclonal antibodies (mab) against fractionizedsporozoite antigens of Eimeria tenella. On western blots onemab G12C4 (IgG2b) identified two polypeptide bands ofabout 28-31 kDa under reduced and nonreduced condi­tions. These proteins were distributed inside the two refrac­tile bodies as shown by immunofluorescence studies (IFAT) onacetone fixed Eimeria tenella sporozoites using confocal laserscanning microscopy. The specific label of refractile bodyorganelles was confirmed by immunogold labeling of para­formaldehyde fixed Eimeria tenella sporozoites in electronmi­croscopy. Interestingly a similar staining was also achieved byR-648 (Molecular Probes) a fluorescencemarker with highaffinity for the endoplasmatic reticulum.

Abstracts . 431

We followed immunofluorescence morphology of refractilebody antigens also during invasion and further developmentin primary chicken kidney cells (PCKC). 24 hours after hostcell invasion (h p.i.) the intracellular sporozoite has lost onerefractile body. Diffusion of refractile body antigen could bevisualized throughout the trophozoite stage but also in thecytoplasma of infected cells. In developing schizont stage72 h p.i. random scattering of refractile body antigens wasseen. After the first schizogony a small intensely stained re­fractile body organelle was observerd in mature first genera­tion merozoites and in young trophozoites of the secondgeneration. During further development fluorescence gradu­ally disappeared and was not seen in second generation schi­zonts. A similar expression of refractile body proteins couldbe followed by mab G12C4 on western blots prepared bySDS-PAGE of different stages, confirming a crucial role ofrefractile body antigens in the early stages of intracellular de­velopment (24-96 h p.i.). The importance of these antigenswill require the identification and expression of their eDNAclones.

101Cytoskeleton Activities During Capping of Receptorsin Entamoeba histolyticaN. GUILLEN, P. ARHETS and P. SANSONETTIUnite de Pathogenic Microbienne Moleculaire,INSERM U 389, Institut Pasteur, 28, Rue du Dr. Roux,75724 Paris Cedex 15, France

In response to external stimuli, Entamoeba histolytica, theagent responsible for amoebiasis, glides as a polarized cellthat presents a frontal pseudopod and the posterior uroid,a membrane appendix that accumulates capped ligand-recep­tor complexes. It is generally accepted that the cytoskeleton isinvolved in movement and capping activity of the cells. Bio­chemical analysis of purified caps from amoebae have shownthe presence of two receptors undergoing capping by incuba­tion with concanavalin A. One of them corresponds to theGal/GaiNAc lectin and the other corresponds to the ADHenzyme. A study of cytoskeleton dynamics has shown thatmyosin II and actin are present within protein complexesformed during capping of membrane receptors. Drugs thataffect actin polymerisation or myosin II phosphorylationhave no effect on receptor's patches formation. In contrast,they inhibit translocation of these patches to the rear partof the cell leading to the absence of uroid formation. Theseresults are consistent with the hypothesis that cytoskeletalfunctions are required only for the translocation of patchesduring capping.

102Tubulin Isotypes, Microtubule Assembly andMicrotubule Mediated Events in the Cell Cycle andLife Cycle of Trypanosoma bruceiK. GULLSchool of Biological Sciences, University ofManchester, 2.205 Stopford Building, Oxford Road,Manchester M13 9PT, U.K.

Cells of the African trypanosome, T. brucei, have a very de­fined cell shape and arrangement of organelles. However, du­plication events in the cell cycle and changes of cell type

432 . Abstracts

during the life cycle of the parasite involve major reorganisa­tions of cell shape and the position of these organelles. Muchof the control of cellular shape and form in this parasite is adirect result of the action of the microtubule cytoskeleton.The major alpha and beta tubulin components of the micro­tubules are encoded by a multi-gene family, whilst we haverecently identified a third, more minor component, gammatubulin in this organism. The alpha tubulin protein can under­go a number of reversible post translational modificationssuch as acetylation and detyrosination. Production of suchspecific isotypes is intimately coordinated with the construc­tion and turnover of particular microtubule structures such asthe flagellum, mitotic spindle and sub-pellicular corset. Ex­periments involving the use of specific and highly effectivemicrotubule inhibitors such as Rhizoxin have revealed thatprocesses such as kinetoplast segregation in the cell cycle, cellcleavage and kinetoplast reorientation during the blood­stream to procyclic cell form transformation are microtubulemediated events.

103Chemotherapy of Human and Animal Coccidia; Needsand PerspectivesA. HABERKORNInstitute of Parasitology, Animal Health Division,Bayer AG, D-51368 Leverkusen, Germany

A number of highly-effective drugs are available for the con­trol of coccidiosis in poultry. Increasing problems with resis­tance have led to treatment schemes intended to make itsdevelopment more difficult. To this end the anticoccidialdrugs used are changed in shuttle or rotation programmes.The important discovery and development of new anticocci­dials belonging to completely new chemical classes is slowand unpredictable.In addition to a new polyether, semduramycin, in recent yearsan azauracil derivative (diclazuril) has been developed, andafter many years a new drug for therapy (toltrazuril), a sym­metrical triazinetrione derivative. The increasing possibilitiesfor vaccination may result in new aspects for the use of che­motherapeutics. The sector of mammalian coccidioses is ofincreasing interest. The methods of treating Cryptospori­dium infections in animals and in AIDS patients are still com­pletely unsatisfactory.In toxoplasmosis the therapeutic care and treatment of trans­placental infections and the treatment of infections whichhave got out of control because of immunodeficiency (e.g.in HIV) is in the forefront of attempts at improvement. Inthe veterinary medicine sector the possibilities of vaccina­tion, perhaps in combination with the use of drugs, showsome interesting prospects. Attempts to treat Neospora infec­tions are still in the initial stages. The lack of informationabout the complete life cycle is making animal studies in par­ticular more difficult. In addition, chemotherapy and prophy­laxis of sarcocystoses will be discussed, and some aspects ofthe treatment of wild- and zoo-animal coccidioses will be in­dicated.

104Plastid-like Organelles in Anaerobic FlagellatesJ. H. P. HACKSTEIN1, H. SCHUBERT2, J. ROSENBERG3,M. V. D. BERG\ S. BRUL1, J. DERKSENS and H. e. P.MATTHI]S21Dept. Microbiology and Evolutionary Biology, and"Dept. Experimental Botany, Fac. Sci., CatholicUniversity of Nijmegen, NL-6525 ED Nijmegen,2Laboratorium voor Microbiologie, University ofAmsterdam, Nieuwe Achtergracht 127, NL-I018 WSAmsterdam, Dept. Animal Physiology, Ruhr­Universitat, D-44780 Bochum, Germany, "Dept.Biochemistry (Fac. Medicine), E.e. Slater Institute,University of Amsterdam, Meibergdreef 15, NL-ll05AZ Amsterdam, The Netherlands

Psalteriomonas lanterna is a heterotrophic, free-living, micro­aerophilic amoebomastigote. It lacks dictyosomes, microbo­dies, mitochondria, and classical plastids. However, in bothmastigote and amoeba stages of this organism, we discovereda novel organelle that we call "Thylakosome". This organellecontains DNA, the photosystems PS I and PS II, and traces ofchlorophyll a. Gold-labelled antisera against cytochrome oxi­dase (COX) and ribulose bisphosphate carboxylase (Rubisco)reveal the presence of both enzymes. These attributes charac­terize the thylakosome either as a highly specialized cyanelleor a totally new type of plastid. However, its photosyntheticproperties are seemingly rudimentary because the flagellatescan grow and divide for a number of generations in completedarkness. Consequently, the organelle might have functions incellular metabolism that are not light-dependent. The rela­tionship to the plastid-type organelles of several parasitic Api­complexa is discussed.

105Biogenesis of Hydrogenosomes in PsalteriomonaslanternaJ. H. P. HACKSTEIN1, J. ROSENBERG2, e. A. M. BRoERs1,e. K. STUMM1 and G. D. VOGELS 1

1Dept. Microbiology and Evolutionary Biology, Fac.Sci., Catholic University of Nijmegen, NL-6525 EDNijmegen, 2Dept. Animal Physiology, Ruhr­Universitat, D-44780 Bochum, Germany

The free-living, microaerophilic amoebomastigote Psalterio­monas lanterna harbours two populations of hydrogeno­somes. The mature, active organelles form a prominentcomplex in the center of the mastigote cell, whereas develop­ing hydrogenosomes are scattered over the periphery of thecell. These hydrogenosomes do not exhibit hydrogenase activ­ity, whereas those forming the complex react with BSTP, in­dicating hydrogenase activity. Both types of hydrogenosomesare stained by a FITC-labelled hydrogenase antiserum. Thefailure of DAPI or ethidium bromide staining indicates theabsence of nucleic acids. Electron microscopy also fails to re­veal the presence of ribosomes or tubular or membraneousstructures in the matrix of both types of hydrogenosomes.The population of peripheral hydrogenosomes containsmany sausage-like organelles that are surrounded by one ortwo cisterns of rough endoplasmatic reticulum. Measure­ments suggest that these propagation stages bud off a centralvesicle that is in intimate contact with the endoplasmatic re-

ticulum . Thus, our studies cannot support a mitoch ondrialdescent or recent endosy mbiotic or igin of the hydrogeno­somes of Psalteriomonas lant erna.

106Internalization of Legionella pneumophila by Free­living Amoeba: Flow Cytometry and MicroscopicalCharacterizationC. H ARFt, S. GOFFINET t, D. A. COl.IN2, M. FABREJ ,

O. M EUNIER2 and H. M ONTEIL2

Laboratoire d 'Hydrobiologie de I'Environnernent ', ­In st itut de Bacteriologie/ , - Service de M icroscopieElectronique', Universite Louis Pasteur - Strasbourg67000, France

Flow cytometry is a potentially powerful tool for analysingthe interactions of intracellular bacteria and free-living amoe­ba at a cellular level. We established parameter s for labellingvirulent and avirulent L. pneumop hila serogroup 1 with a li­poph ilic fluorophore and used flow cyromerry to investiga teinternalization by axenic Aca nthamoeba patinensis and Nae ­gleria clarki. Removal of the externa l bacteria was performedby washing, and contro lled by sodium azide incubation, dif­ferentiating between bound and internalized particles. Fineadjustment of thresholds on light scatter channels was usedto fractionate cells according to cell volume. The presentflow cytometric method was used in conjunction withphase-contrast combi ned to fluorescent microscopy and elec­tro n microscopy providing confirmation of L. pn eumophilainternalizat ion. Flow-cytometry histograms demonstrated in­ternalizat ion beginning within 5 minutes of coculture, as wellas intracellular surviva l of L. pn eum opbila serogroup 1 inA. palestinensis and N. clarki . Results also showed the het­erogeneity of amoeba popul ations in bacterial uptake andtheir intracellular surviva l. Intracellular growth seems to bedependent on the adapta tion of a small fraction of bacteriain amoeba popul at ions bear ing special surface or cellularproperties .

107Defensive Function of the Red Pigment in BlepharismajaponicumT. H ARUMOTO!, H. 110 2, K. Z ENFUKU2 a nd A. MI YAKEJ

IDept. Bio!. , Nara Wom en's Univ., N ara , Japan,2Faculty Sci., O saka Ci ty Univ., Osaka, Japan, 3Dept .Cell BioI. , Univ. Camerino, Camerino, Italy

The heterot rich ciliate Blepharisma [aponicum has pigmentedextrusomes , pigment granules (PGs), which function as defen­sive organelles against the predatory ciliate Dileptus margar­itifer (Miyake et al. 1990, Europ. J. Proti stol. 25, 310- 315 ).We purified the red pigment in PGs by silica gel TLC (15%MeOH/CH 2CI2 ) to obtain two sampl es A and B (Rf: 0.25 and0.2 0, respectively). Both samples were toxic toD. margaritifer and many other ciliates. Samples A and Bkilled a half of the 10 dilept i suspended in 250 III of 5MB(a balanced solution) in 1 day at 3.4 and 2.3 ug/ml , respec­tively (in darkness except dur ing handling and observa­tion ). LDso of the sample A, similarly measured for otherciliates, is as follows (ug/rnl): Lembadion bullinum (1.4), Di­din ium nasutum (2.1), Param ecium caudatum (3.0), Parame­cium tetra urelia (3.2), Colpidium sp. (4.7), Eup lotes

Abstracts . 433

oetocarinatus (5.0), Stentor polymorphus (16), Stentor coer­uleus (50), B. [apo nicu m, both red and albino (> 80). Thesample A at 10 ug/rnl induced an immedia te strong backwardswimming in dilept i. We conclude that the red pigment par­ticipates in the defensive function of PGs in B. jap on icum.

108Gravitaxis of Loxodes and ParameciumR. H EMMERSBACH and R. D ONATHDLR-Institute fo r Ae rosp ace M edicine, 51147 Koln,Germany

Cravitac tic protozoa offer an advant age in stud ying how thephysical signal of gravity is transformed into a physiologicalresponse. In the case of Loxodes, a specialized vacuole (M ul­Ier Organelle) is under discussion as to its function in gravityperception . In contrast to a specialized gravireceptor, com­mon cellular part s are thought to serve this function in Para­m ecium. The gravitactic behavior of both ciliates was studiedunder var ied stimulus conditions - ranging from simulatedand actua l near-weightlessness, to 1 x g, to hypergravity. Un­der controlled cond itions at 1 x g Param ecium shows a nega­tive gravi tax is, while Lox odes shows a positive or negativegravitaxis or a bimodal distribution . Switches in the sign ofgravitaxis were only observed in case of Loxodes. Both cili­ates show a random distr ibution und er gravity-free conditionsindicating that gravity actually is the stimulus for the observedbehavior at 1 x g. Under hypergravity conditions - tested on aslow rotating centrifuge microscope (NIZEMI) - gravitaxispersists and can be even induced in less or iented sampleswith­in seconds. Experiments using inhibito rs and media with ad­justed density are under investigat ion . Results will bediscussed in respect to possible perception mechanisms.

109Chromosomal Rearrangements in Plasmodiumfalciparum Leading to Amplification or Deletion of theRESA Gene in Different HostsR. HERNANDEZ-RIVAS, K. HI NTERBERG and A. SCHERFExper imental Parasitology Unit , URA 1960, InstitutPasteur, Paris, France

Karyotype and mapp ing studies of P. [alciparum chro mo­somes have shown that subtelomeric regions are highly poly­morphic. Ant igen genes genera lly map to these fragilechro moso me regions. Deletion , duplicati on and transloca­tion of large subtelomeric regions largely contribute to karyo ­typic polymorphism and allelic diversity.In the present study we investigated whether rearrangments ofsubtelomeric genes occur in natural parasite popul ations. Inseveral clinical isolates from West African pati ents, we de­tected a RESA related gene family dispersed on four differentchro moso mes. The different members of RESA map to thesubtelo meric regions of chromosomes 1,2, 11 and 14, sug­gesting that they or iginate from dupli cative translocat ionof the subtelomeric chro mosome 1 region carrying the RESAgene. Parado xically, RESA gene deletion on chromosome 1seems to facilita te the adapta tion of P. [alciparum strai ns tothe experimental monkey host Saim iri sciureus. These resultsillustrate the plasticity of the parasite genome.

434 . Abstracts

110Incidence and Differential Distribution of Folliculinaviridis, a Ciliate Epibiont, on some Crustacean SpeciesM.]. HERRERO and G. FERNANDEZ-LEBORANSLaboratorio de Biologia General, Departamento deBiologia Animal I (Zoologia), Facultad de Biologia,Universidad Complutense, 28040 Madrid, Spain

A study has been made of the incidence and differential dis­tribution of Folliculina viridis (Wright, 1858) Faure-Fremiet1936, a heterotrich ciliate epibiont, on some decapod crusta­ceans whose habitats are muddy sea beds of 6-28 m depth.Decapods were collected in two sites on the Catalan coast,Vilanova i la Geltru and San Carlos de la Rapita (Spain),in February, March, April and May 1993, using a beam trawlsystem known locally as "rastell", Samples were fixed in asolution of 3% forma line in sea water. The anatomical unitsof each decapod were separated and analyzed, isolating pro­tozoans for further study. A statistical study (principal com­ponents and correlation analyses) was carried out in order toexplain the distribution on each anatomical unit.Of the 16 crustacean species analyzed,S showed individualsfrom this group of ciliates, only on decapods collected in Feb­ruary, March and May. There were three incidence categories,based on the number of F. viridis found on each decapod: low« 50), moderate (50-100), and high (> 100). Some specime­nens of braquiure decapods, such as Atelecyclus rotundatusOlivy, 1792, Parthenope augulifrons Latreille, 1825, andEthusa mascarone Herbst, 1785, as well as the hermit crabPaguristes oculatus, showed a moderate or low density ofthis ciliate. The decapods with the highest incidence be­longed to Pagurus prideaux, indicating a certain degree ofepibiosis specifity. In this last there was a noticeable coinci­dence with respect to the areas colonized by F. viridis. Mostof heterotrichs were located in the more protected areas of thebasibiont, such as the hollows of the carapace surface andabdomen, and on the tergites near the thorax of the walkinglegs, on their internal edges or protected by rows of setae.

111In vitro Culture on Pleistophora sp. from an AIDSPatientW. S. HOLLISTER, E. U. CANNING1, E. WEIDNER1,

D.]. MARRIOTT', A. S. FIELD2, S. T. MILLIKEN2,

J. HARKNESS2 and J.KENCHlDepartment of Biology, Imperial College, London,England2St. Vincents' Hospital, Sydney, Australia

A case of microsporidian myositis due to Pleistophora sp. hasbeen diagnosed in an AIDS patient in Australia. The patienthad been HIV-positive since 1991 and his CD4 count was6 x 106/ml at the time of presentation in January, 1995 witha 3-month history of generalised myalgia with shooting pains.The muscles were tender, there was generalised wasting, fixedflexion deformities of both forearms and shortening ofAchilles tendon. The Pleistophora sp. infection was diag­nosed from a muscle biopsy taken 24. 01. 95 and cultureswere initiated from a repeat biopsy on 11. 02. 95. The musclebiopsy was teased apart and trypsinised to release the spores,some of which were purified on 50% Percol!. Purified sporesand the muscle residue were added to cell cultures of RK13(rabbit kidney), COSI (African green monkey kidney),

MDCK (canine kidney) and myoblasts. Purified spores werealso inoculated intramuscularly and intraperitoneally intoathymic mice. Growth of the parasite was prolific in COS1and RK13 cells and in the nude mice at the site of intramus­cular inoculation. This represents the first in vitro culture ofPleistophora sp. and paves the way for extensive investiga­tions on the human infection. All stages were surroundedby a thick electron dense surface coat which extended as sin­uous projections into the host cell cytoplasm. Meronts had upto about 6 nuclei and divided by plasmotomy into smallersegments. In sporogony the surface coat separated from themultinucleate sporogonial plasmodium to form a sporophor­ous vesicle within which division into sporoblasts and ma­turation of spores took place. The number of sporesranged from four to 16 or more per vesicle. The sporeshad 10-12 coils of the polar tube, an extensive polaroplastof close-packed membranes and a thick exposure.

112Nuclear Ribosomal DNA Variability in a Free-livingPopulation of Ammonia spp. (Foraminifera) fromCamargue, FranceM. HOLZMANN!, W. E. PILLERland]. PAWLOWSKI2

llnstitut fur Palaontologie der Universitat Wien, A­t 090 Wien, Austria,2Department of Animal Biology and Zoology,University of Geneva, CH-t224 Chene-Bougeries,Switzerland

The genetic variability in a population of free-living forami­nifera of the genus Ammonia collected in Camargue, was ex­amined by amplifying and sequencing a 450 bp fragment ofthe large subunit ribosomal DNA (LSD rDNA) in 20 speci­mens. Two different groups of sequences were obtained, cor­responding to two different morphotypes that were identifiedas Ammonia tepida (Cushman, 1926) and Ammonia beccarii(Linne, 1758). The sequence similarity between both groupsranges from 61-68%. The similarity of the sequences insidethe group "beccarii" reaches 95 -99%, while inside the group"tepida" two types of sequences can be distinguished, whichare similar in about 84-89%. The two types of "tepida" can­not be separated by morphological characters. The sequencessimilarity of the one type ranges from 82-89%, while in theother type it ranges from 94-99%. The distinction ofA. tepida and A. beccarii was also possible through analysisof the PCR products that were cut with restriction enzymeSea I.

113The Fixation and Staining of Karyorelictid CiliatesS. HOPE and D. M. L. ROBERTSDepartment of Zoology, The Natural HistoryMuseum, Cromwell Road, London SW7 SBD, UK

All but one genus of karyorelictid ciliates are marine, living ininterstitial spaces in sand. They can be extracted live by meansof Uhlig's ice extraction but tend to be flexible, highly con­tractile and terribly fragile. Details of their ciliation havebeen described from stained specimens for only one spe­cies, because they are notoriously difficult to fix, tendingto burst at the slightest provocation. The effect of numerousfixatives in a variety of buffers was studied with successful

fixation achieved with Raikov's fixative (based on mercuricchloride and gluteraldehyde) and ZFA (based on osmium,acetic acid and formalin). Silver impregnation was wholly un­successful with silver nitrate; silver carbonate consistentlyover-stained the cells; protargol staining was successful. Im­munostaining with commercial anti alpha-tubulin was alsosuccessful, although some difficulty was experienced withthe fragility of permeabilised cells.

114The Major Epiplasmic Proteins of Ciliates areArticulins; a new Family of Cytoskeletal Proteins inProtistsI. HUITENLAUCH, N. GEISLER, U. PLESSMANN,R. K. PECK1, K. WEBER and R. STICK2

Max Planck Institute for Biophysical Chemistry,Department of Biochemistry, P.O. Box 2841, D-37018Gottingen, FRG. IDept. of Zoology and Animal Biol.,Univ. of Geneva; 2Inst. f. Biochemie u. Molek.Zellbiol., Univ. Gottingen

The cytoskeleton of certain protists comprises an extensivemembrane skeleton, the epiplasm, which contributes to thecell shape and patterning of the species specific cortical archi­tecture. The isolated epiplasm of the ciliated protist Pseudo­micro thorax dubius consists of two major groups of proteinswith molecular masses of 78-80 kD and 11-13 kD respec­tively. To characterize the structure of these proteins peptidesequences of two major polypeptides (78 - 80 kD) as well as aeDNA representing the entire coding sequence of a minor andhitherto unidentified component (60 kD; p60) of the epiplasmhave been determined. All three polypeptides share sequencesimilarities. They contain repeated valine and proline rich mo­tifs of 12 residues with the consensus VPVP-V-V-V-. In p60the central core domain consists of 24 tandemly repeatedVPV-motifs. Within the repeat motifs positively and nega­tively charged residues when present show an alternating pat­tern in register with the V- and P-positions. Infraredspectroscopic measurements of recombinant p60 indicate30% ~-sheet. Electron microscopy reveals short filamentouspolymers with a rather homogenous diameter (about 20 nm)but variable lengths. The small polymers form thicker fila­ments, ribbons, and larger sheets or tubes. A core domainsimilar to that of P. dubius p60 is also found in the recentlydescribed epiplasmic proteins of the flagellate Euglena, the socalled articulins. Our results show that the members of thisprotein family are not restricted to flagellates but are also pre­sent in the distantly related ciliates where they are major con­stituents of the epiplasm. Comparison of flagellate and ciliatearticulins highlights common features of this novel family ofcytoskeletal proteins.

115An EM Analysis of the Surface Pattern of InterphasicParamecium: Interactions of the Associated Basal­bodies Cytoskeletal Ribbons and their EnvironmentF.IFTODEBiol, Cell. 4, Bat. 444, Univ. Paris-Sud, 91405 OrsayCedex, France

Previously available ultrustructural data on Paramecium cor­tex have been extended by using the tannic acid method(Eichenlaub-Ritter and Tucker, 1984, Nature, 307, 60) on

Abstracts . 435

permeabilized cells, allowing a good visualisation of the finestructure of the basal bodies (BBs) and associated fibers. Themain new insights are: 1- In a pair, the anterior BBis stronglybound to the kinetodesmal fiber (Kd) coming from the poster­ior BB, by "bones-nodes connectors"; the Kd is also con­nected to the post-ciliary (PC) microtubular ribbon by aspecific "fingered connector" (also found in other ciliates).The origin of paratene microtubular bundles associated toBBs of the anterior invariant and oral fields is shown. 2­The three rootlets, the microtubular ones (PC and transverseTR), and the Kd fibers, are attached to the epiplasmic scalesaccording a specific different linkage pattern. 3- An "epi­plasmic ciliary domain", enclosed in the epiplasm itself, en­circles the BB: this domain is often interrupted thus leavingspace along the BB, possibly for forwarding material to theaxoneme. 4- The PC (and TR) microtubules numbers, thetotal length of the BBs and the number of cartwheel (Cw)elements are variable according to the regions of the cell cor­tex, the greatest numbers (6 PC microtubules, 5 Cwelements)and the largest BBs belonging to the invariant oral or corticalfields.These data clearly show the specific modes of anchoring therootlets to the epiplasmic scales, and provide new interpreta­tions of the disassembly-reassembly events of cytoskeletal ele­ments during division, in relation to specific staining ofsubclasses of cortical microtubules close to membranes withan antibody directed to a new type of tubulin post-transla­tional modification.

116Effects of Copper and Cadmium on Astasia longaP. IRATO, R. DE GABRIEL!, E. PICCINNI and V. ALBERGONIDepartment of Biology, University of Padova, ViaTrieste 75, 35121, Padova, Italy

Metallothioneins (MTs), cysteine-rich metal-binding pro­teins, whose functions are the detoxification and intracellu­lar regulation of certain metals, are present in manyinvertebrate and vertebrate organisms. With the aim of study­ing the effects of Cu and Cd in Astasia longa, we treated thisorganism with various concentrations of these metals. Astasiawas grown in Euglena gracilis medium (EGM) at 28°C. Inthis experimental condition, the duplication time of controlcells was about 16 h. In the literature, several duplicationtimes ranging from 8.8 h to 25.5 h are reported, accordingto the medium used. In Astasia, as in Euglena, Cu is bettertolerated than Cd; in fact, the duplication time of cells treatedwith 10 ug/ml of Cu is the same as in controls. A higher con­centration of Cu (20 ug/ml) affects the growth rate. Cd re­duces the growth rate according to the concentration used.A dose of 0.5 ug/ml increases the duplication time to 27 h.Cell accumulation is very low: Astasia treated with 10 and20 ug Culml accumulates about 85 and 110 ~g!g dry wt re­spectively and the metal is found mainly in the particulatefraction. Cells treated with 0.5 ug Cd/ml accumulate about150 ug Cd/g dry wt and the metal is found in both particulateand soluble fraction. No soluble chelating proteins werefound to be induced by metals in Astasia, whereas Cu andCd binding proteins were purified in Euglena. In conclu­sion, it appears that chelating proteins with functions similarto those of MTs but with different amino acid composition ­chelatins - may be induced in Euglena but not in Astasia(Piccinni et al., Compo Biochem. Physiol., 82C, 29, 1985;Gingrich et al., Environ. Health Perspect., 65, 77, 1986). In-

436 . Abstracts

stead in the ciliate Tetrahymena proteins with a certain degreeof homology with MTs of pluricellular organisms are induced(Piccinni et aI., Europ. J. Biochem., 226, 853, 1994). GrantsMURST 40%.

117Proteolysis and Amino Acid Metabolism of Anaerobes.Good Drug Targets?J. W. IRVINEParasitology Laboratory, Institute of Biomedical andLife Sciences, University of Glasgow, Joseph BlackBuilding, Glasgow G12 8QQ, Scotland, UK

Many parasitic anaerobic protists have been found to possesshigh proteolytic activity and to release multiple proteinasesinto their environment. Studies on the more abundant en­zymes, which are of the cysteine type, have shown that theparasites' enzymes differ significantly from those of mam­mals and they have been suggested as suitable targets for che­motherapeutic exploitation. This presentation will summarisethe evidence for and against this contention, recent advancesin our understanding of the roles and characteristics of pro­teolysis and amino acid metabolism in these anaerobes, andthe approaches being adopted to exploit these peculiarities ofthe parasitic anaerobes.

118The Body-scales of Prymnesiophyceae: PhylogeneticalImplications, Especially for Imantonia rotundaReynolds (1974)M.-C. JANINUniv. P.-et-M. Curie et CNRS,Labo. de Micropaleontol., 4 PI. Jussieu (T15 E4),75252 Paris cedex as, France

The Prymnesiophyceae are usually considered as a homoge­neous group including extant and fossil protists, amongwhich the coccolithophorid algae, whose calcitic secretions(coccoliths) playa prominent part in geology (as rock consti­tuent as well as biostratigraphical or palaeoenvironmentalmarkers).The coccoliths (sole remain of extinct species of Prymnesio­phyceae and related taxa) obviously result, in living species,from the calcification of organic scales similar to the unminer­alized body-scales covering the cell of many Prymnesiophy­ceae and other algae. Consequently, comparison betweenthe morphology of organic scales and that of coccoliths orother calcareous nannofossils seems a suitable method for in­vestigation of phylogenetic links among unicellular algae.According to the few observations published, the organicscales secreted by Prymnesiophyceae are circular to ellipticplates with a characteristic ornamentation of radiating ridgesasymmetrically arranged in four quadrants. Comparable out­line, asymmetry and ornamentation in quadrants is also foundin the central part of many coccoliths, in agreement with thetaxonomic attribution classically proposed for the corre­sponding organisms. On the contrary, the scale type of sometaxa, especially the living not-calcifying Imantonia rotunda(Isochrysidales), suggests affinities with algal groups otherthan the Prymnesiophyceae.

119Spectrin and Other Cytoskeletal Proteins in CiliateCellsR. JANISCHDepartment of Biology, Med. Faculty, MasarykUniversity, 66243 Brno, Czech Republic

The infusorians Paramecium caudatum, Tetrahymena pyri­formis and Blepharisma unduland americanum were investi­gated for the presence of spectrin, myosin, tropomyosin,filamin, and vimentin by indirect immunofluorescence withthe use of polyclonal and monoclonal antibodies. Before ap­plication of antibodies, the protozoan cells were treated with0.05 -0.5% Triton X 100 in microtubule-stabilizing bufferfollowed by fixation with 2-4% paraformaldehyde.The detection of spectrin did not show any marked differencesin the intensity of fluorescence related to the use of differentprimary antibodies. In Paramecium, positive findings wereseen on the whole surface of the plasma membrane, with par­ticularly strong fluorescence of the ridges which surroundedthe kinetids situated above the outer lattice. Strong positiveresults were also obtained in the areas of the oral apparatusand the cytoprocts and, especially, in the epiplasm of subpel­licular alveoli, in the area of the pores of contractile vacuolesand in the membranes of their central cisternae. In Blephar­isma, spectrin was demonstrated all over its complex surfaceand in Tetrahymena pyriformis positive fluorescence was de­tected in the plasma membrane and the inner alveolar mem­brane, most probably originating in the area of epiplasm. Inall infusorians tested, spectrin was detected in ciliary mem­brane. In Paramecium, spectrin distribution was also fol­lowed in fragments at 2, 10, 30, 60, 120, and 360 minafter merotomy. Within 30 min of merotomy, accumulationof spectrin was observed at the site of defect in a thin layerof cytoplasm under the newly-formed plasma membrane.Myosin and tropomyosin were colocalized with actindetected previously in the injured pole of each fragment.Filamin was demonstrated in the cortical skeleton, accompa­nying cytoplasmic filaments even in the in areas where, inprevious experiments, anti-actin antibody failed to demon­strate actin. Accumulation of filamin in the injured areawas seen at 5 -30 min after merotomy. In the intact infusor­ian cortex, vimentin was found associated with the outer la­tice, pores of contractile vacuoles, cytoproct, kinetosomesand was also attached to the membranes of trichocysts includ­ing those which were artificially dilated in the cytoplasm. Nochanges in its distribution were observed in relation to cellinjury by fragmentation.

120Lithium Chloride and Pattern Formation inTetrahymena thermophilaM. JERKA-DzIADOSZ"o and J. FRANKEL"*Department of Biological Sciences, The University ofIowa, Iowa City, Iowa, USA and °Department of CellBiology, M. Nencki Institute of Exp. Biology, PolishAcademy of Sciences, Warsaw, Poland

In multicellular systems, lithium ions modify the specificationof cell fates. We have analyzed the effects of Li- on intracel­lular patterning in Tetrahymena thermophila. LiCI does notaffect the locations of the oral primordium, contractile va­cuole pores, cytoproct and fission zone. Exposure to Li ions

does not modify the phenotypes of pattern mutants: janA,disA, hpo2. LiCI affects differentially early stages of oral de­velopment: initially it retards regression of oral primordia,which is followed by redevelopment and an excessive prolif­eration of new basal bodies leading to a hypertrophy of theoral ciliature. The oral pattern mimics the membranellar-pat­tern D (mpD) mutation, whose phenotype is grossly enhancedby Li ions. These effects were partially reversed by myo-ino­sitol but not by neomycin. Thus, although lithium ions havemajor cellular effects on Tetrahymena, they do not influencethe specification of the body plan in a manner analogous tothat observed in multicellular organisms, and may work inpart through mechanisms other than the classical inositol­phosphate cycle.

121Using GFP-expressing E. coli to Measure IngestionRates in Mixotrophic Algae and CiliatesH. L. J. ]ONESt, R. MITRA2 and C. M. SILVA2

INERC Centre for Population Biology, ImperialCollege at Silwood Park, Ascot, Berkshire, SL5 7PY;2Palo Alto Institute for Molecular Medicine, 2462Wyandotte Street, Mountain View, CA 94043, USA

The limitations of using fluorescent microspheres and alsofluorescently labelled or radiolabelled bacteria to determinethe ingestion rates of protists has often been discussed. Thepresent technique involves the use of a live, fluorescing bac­terial prey source. The bacteria, a BL21 (de3) E. coli strain, istransformed by electroporation with a plasmid encoded withGFP. The instant the bacteria are ingested by the protist theylose their fluorescence. Ingestion rates can therefore be deter­mined by measuring the reduction in fluorescence as the preypopulation is ingested, or alternatively by direct counts of thebacteria population using bacterial counting chambers andfluorescent microscopy. Mixotrophic algae and ciliates havebeen used to test this method.

122Bacteria-Protozoan-Interactions: Strategies on BothSidesK. JURGENSMax-Planck-Institut fur Limnologie, P.O. Box 165,D-24302 PIon, Germany

Predator-prey relationships between bacteria and bacterivor­ous protozoans are assumed to have a long evolutionary his­tory. Thus, the main selection forces, i.e. optimizing predatoravoidance by bacteria and the foraging efficiency of bacteri­vores, must have played an important role in the evolutionarydevelopment of bacteria and their consumers. Although theseaspects of protozoan-bacteria-interactions have only poorlybeen investigated, the general importance of this point ofview is confirmed by recent evidence for manifold mechan­isms of bacteria to reduce mortality due to protozoan preda­tion. These comprise morphological, chemical andbehavioural defenses as well as growth in spatial refuges.Field and laboratory experiments have shown that naturalbacterial communities can respond to increased protozoangrazing by phenotypic and genotypic changes towards graz­ing-resistant forms. The planktonic protozoans with thestrongest potential to exert grazing pressure and trigger these

Abstracts· 437

changes in bacterial community structure are heterotrophic-nanoflagellates (HNF). Despite having very distinct taxono­mical origins, the majority of HNF-species which feed on sus­pended bacteria can be seen as a functional group. Theirfunctional morphology and foraging behaviour are designedto maximize the net energy intake and the survival in a fluc­tuating environment. The most important adaptations are anincrease of capture efficiency for larger sized bacteria and, byraptorial feeding flagellates, chemically mediated prey selec­tion. Some species seem to have a behavioural flexibility infood selection which is consistent with optimal foragingand optimal diet theories, developed from studies with highermetazoan predators.

123Ultrastructural Data on Triactinomyxon ignotumStole, 1899 (Actinomyxidia, Myxozoa)G. KABREt, N. SAKITI2 and A. MARQUES3

lLaboratoire de Zoologie, EA.S.T., Universite deOuagadougou, Burkina Faso. 2Universite Nationale duBenin, Laboratoire de Zoologie, Cotonou, Republiquedu Benin. 3Laboratoire de Parasitologie etImmunologie, Universite Montpellier II, 34095Montpellier Cedex, France

The ultrastructural observations of Triactinomyxon ignotumStoic, 1899, parasite of Tubifex tubifex show a pansporocystenclosing two lines of cells, Ci and ~. The presence of synap­tonemal complexes and of polar bodies reveals a meiosis andthe formation of gametic cells (8 «-cells and 8 ~-cells). Theirwill unite in pairs inside the pansporocyst to form 8 zygoteswhich will form eight separate spores. When mature, eachspore is constituted by three episporal cells, three capsularcells containing each a polar filament, a sporoplasm com­posed of a syncytium with eight cellular elements set in line.This confirms the reality of a sexual phase in the developmentof Actinomyxidia.

124Whole Oral Apparatus Migrates in cdaK Mutant ofTetrahymena thermophila; Call for Pushing PullingForce for this DislocationJ. KACZANOWSKA, T. BURAKOWSKI and A. KACZANOWSKIDepartment of Cytophysiology, Warsaw University,Warsaw 00-927, Poland

The oral apparatuses (old GAl and newly formed GA2) stayin situ during divisional morphogenesis of Tetrahymena. Incontrast in dividing cdaK (cell division arrest) mutant ofT. thermophila (of dr Frankel) the GA2 is formed at usualplace and then is displaced beneath GAL This dislocationis not due to the oral replacement at new site or due to resorp­tion of area between GAl and GA2.Formation of the fission line in cdaK cells is delayed and ante­riorly shifted on dorsal side (as detected with antibodiesanti-B and antifenestrin, Nelsen, 1994). Migration of newGA2 is associated with the furrowing of this obliquely lo­cated fission line. During furrowing an oblique fission linetakes perpendicular position separating two daughter cellsof different sizes. It is assumed that ultimately transversal fis­sion furrow mechanically drags upwards the GA2 againstneighbouring cortex. Hence dislocation of the GA2 in cdaK

438 . Abstracts

mutant of T. thermophila is passive and is forced by furrow ingof the oblique fission line. 1. Nelsen et al. (1994 ) J. Euk. Mi­crobiol. 41: 483.

125Etoposide, an Antitopoisomerase II Inhibitor InducedPronuclear Failure (Apoptosis of all Post-meioticNuclei) During Conjugation of TetrahymenathermophilaA. KACZANOWSKI, A. DOMARADZKA and J. KACZNOWSKADepartment of Cytophysiology, Warsaw University,Warsaw 00-927/1, Poland

Topoisomerase II (topo II) is an enzyme that regulate s tran­cient breakage and rejoining of double stranded DNA, andcondensation of chromatin. Etoposide inhibits removing oftopo II from its DNA binding sites on DNA and may inducechromosomes fragmentation.In conjugating Tetrahymena thermophila three products ofmeiosis are resorbed. Onl y one of them is rescued and dividesyielding two pronuclei , except the micronucleary defective" *,,strains in which all four postmeiot ic nuclei are lost. Eto­poside applied to conjugat ing T. thermophila interfered withmeiosis I and induced fragmenting and extrusion into the cy­toplasm of chromosomal fragments and then resorption (nu­clear apoptosis) of all four postmeiot ic nuclei. Nuclearconfigurations of etoposide-treated conjugants mimickedconjugation of " * "strai ns (phenocopy).Conjugant s of Tetrahymena may be useful for bioassay fortopo-II-reactive inhibitors and etoposide may be used for in­ducing of chromosomal var iant s with large delations in Tet­rahym ena.

126Comparison of Normal and Giant Cells of twoExconjugant Subclones of Climacostomum virensB. KARAJANInstitute of Cytology, 194064 St-Petersburg, Russia

Two subclones of one exconjugant clone of the ciliate Clima­costomum uirens, isolated more than two years ago, werecompared by electron microscopy of both thin sections andspread preparations of macronuclei , pulsed field gel electro­phoresis and cytoph otometry of the macronuclear DNA.These subclones differ morphologically (normal vs. giant spe­cimens) by fission rate (the subclone of giant cells has a longercell cycle), and by feeding behavior (giant cells are able tointraclonal cannibalism).No aber rations of the form and mode of division of themacronucleus occur in either subclone. In either subclone,almost all chromatin of the interphasic macronuclei is orga­nized into compact bodies, 80 - 160 nm in diamet er, whichbecome dispersed in solutions of low ionic strength: periph­eral rad ial loops formed by nucleosome fibrils appear aroundthem.The size of macronuclear DNA is also similar: two types ofmolecules, in the ranges of 150 -200 kbp and 600­800 kbp, predominate in both subclones.It has been shown photometrically that the overall quantity ofDNA in individual macronuclei is more variable and at aver­age 1.7 times greater in giant cells than in normal ones. It isalso more than double in a cell cycle.

Although the role of the macronucleus in the emergence ofgigantism and cannibalism is not yet clear, it is supposed thatincreased macro nuclear DNA content of giant cells may playa part in maintenance of this type of cell organization for along rime,

127Suspended Particulate Matter and AssociatedPlanktonic Protists in Coastal WatersG. M. KENNAwAyand G. N OVARINODepartment of Zoology, The Natural HistoryMuseum, London SW7 SBD, England

Direct observations using bright field and scanning electronmicroscopy indicate that at certain times of the year sus­pended particulate matter (SPM) in coastal waters has a largebiological component.Water samples from 1.0 metre above the sediment/wate r in­terface were taken with modified Quisset tubes, which collecthorizont al water samples with minimal disturbance to SPM.The abundance, size, diversity and biological composition ofSPM with different settling velocities were examined in theIrish Sea. Our preliminary findings indicate that 1) we wereexamining the subunits which make up larger floes; 2) speciesassociated with floes reflected the most common species in thewa ter column; 3) biological floes have a characteristic andconservative matrix of bacter ia, cysts and nanoflagellates;and 4) the species diversity of associated planktonic proti stsappeared to be relat ively low.

128Pattern of the Phosphorylated Structures inMorphostatic Tetrahymena thermophileM. KIERSNOWSKA and K. GOLINSKADepartment of Cytophysiology, Warsaw University,Warsaw 00-927 and N enck i Institute of ExperimentalBiology, Warsaw 02-093, Poland

Mitotic proteins monocl onal antibody (MPM-2) against a fa­mily of phosphorylated polypeptides and antitubulin anti­body were used to compare patterns of microtubules andphosphoproteins in the cytoskeleton of morphostat ic Tetra-hymena. .By the use of the immunogold labelling technique it has beenshown that the MPM-2 antibody binds to: 1) proximal part sof somatic and oral basal bodies and cilia, 2) the contractilevacuole pore radiating filaments and cytoproctal slit, 3) kine­todesma and 4) structures associated with the system connect­ing microtubules with membranes (fuzzy linkers), 5) maximalMPM-2 binding sites are localized at the bottom of the oralpouch in the specialized cytop lasm where the cytostomallip,the striated fine filamentous reticulum, the microtubules ofthe ribbed wall and the microtubules of deep fiber merge.It is assumed that in Tetrahym ena, both the filaments involvedin the maintenance of three-dimensional sculpturing of oralpouch and cortical ridges; and systems associated with con­traction/motility are preferentially phosphorylated.

129Cell Motility in the ApicomplexaC. A. KING

Department of Biology, University College, GowerStreet, London WCIE.6BT, England

Gliding motility has been recorded in a large number of api­complexan protozoans but it is not a ubiquitous feature of thephylum. High speeds of gliding can be ach~e.ve~ - s?me of thehighest for substratum-dependent cell motility m biology, Wehave recorded speeds: - Lum.s"! for Plasmodium sporo­zoites, 4-10 um.s"! for Gregarina trophozoites, 2­20 urn.s"! for Eimeria sporozoites, and 20-50 um.s :' forPorospora gigantea .trophoz?ite.s. The .lack .o~ observ~ble

change in cytoplasmic orgamzation durmg gliding providesno obvious clues to mechanisms - hence many hypotheseshave been proposed.Generally gliding is unidirectional with the ."anterior" ~nd

leading. Addition of microbeads leads to their translocationover the surface of the zoite frequently producing a cap ofbeads at the posterior end. This led to the p~o'p'osal tha~ cellsurface linear motor(s) were present. The inhibition of glidingmotility by cytochalasin drugs coupled with the detection ofactin in cells by immunofluorescence and m cell extracts byWestern blotting suggested an actin-based motor presumablycoupled with myosin. This mechanochemical enzyme hasbeen detected in Toxoplasma and Gregarina.Translocation of various sizes of beads over the surface ofGregarina trophozoites provided valuable data to buildmodel(s) for the proposed cell surface linear motor.

130The Antimicrosporidial Activity of Albendazole isPotentiated by CimetidineB. KOUDELA and J. VAVRf.Inst. Parasitol., 37005 Ceske Budejovice, CzechRepublic and Dept. Parasitol., Charles University,Prague, Czech Republic

Albendazole an anthelmintic drug acting as microtubular in­hibitor is ad effective chemotherapeutic agent against somemicrosporidia. We have investigated if its effectivity can .bepotentiated by cimetidine, an anti-p~ptlc ulcer dru?, whichis known to increase the concentration of the actrve formof albendazole (albendazole sulphoxide) in tissues and bodyfluids. The antimicrosporidial activity of albendazole in com­bination with cimetidine was tested against Encephalitozooncuniculi in scm mice. A total of 25 eight-week-old scmmice were administered perorally 10 7 spores of the murineisolate (EC2) of E. cuniculi. At ten days post infection(DPI), twenty animals were given orally albendazole (ZEN­TEL suspension, Laboratories Smith Klifole & French,France). Four dose regimens of albendazole in combinationwith cimetidine (Primamet, LEK, Ljubljana, Slovenia) wereused: 5 mg/kg of albendazole daily (5 mice), 5 mg/kg of albe~­dazole twice daily (5 mice), 5 mg/kg of albendazole dally mcombination with 20mg/kg/day of cimetidine (5 mice) and 5mg/kg of albendazole twice daily in co~bination with 20 mg/kg/day of cimetidine (5 mice). Five moculated scm miceserved as a untreated control. The efficacy of albendazolewas evaluated by daily observation for clinical signs or deathdue to microsporidiosis. Control untreated SCID mice inocu­lated perorally with E. cuniculi were the first to develop clin-

Abstracts . 439

ical signs of wasting and lethargy at 22 days and died 25 - 28days p.i. All dose regimens of albendazole glve~ for 2 weekseliminated E. cuniculi infections from SCID mice. After ces­sation of albendazole treatment on DPI 24, the microspori­diosis however recurred in all albendazole-treated mice.Mice ~reated with albendazole alone died in the third weekafter discontinuation of treatment. Mice treated with alben­dazole in combinations with cimetidine survived for fiveweeks after discontinuation of treatment. The present studyindicates that cimetidine potentiates the antimicr.ospori~ial

activity of albendazole. This is of importance for improvmgthe treatment of human microsporidiosis in immunodeficientpatients.

131Evidence that Phototactic Orientation of CertainCiliates is Dependent on the Presence of ConspicuousOrganellesH.-W. KUHLMANN

Institut fur Allgemeine Zoologie und Genetik,Schlossplatz 5, D-48149 Munster, Germany

Phototaxis which means movement of motile microorgan­isms with respect to the light direction, has been describedfor about a dozen ciliate species. Most of them are character­ized by conspicuous organelles as e.g. a coloured spot ?r 'stig­ma' (found in Chlamydodon rnnemosyne, Nassula citreai, a'cup-like organelle', formed of alternating layers of crystalsand cytoplasm (found in Porposton;a notatum), o.r a watch:glass-like, light-refractive body, the organelle of Lieberkuhn(found in all species of the ge~era. Oph.ryogle~a ~ndIchthyophthirius). While phototactic onentation of typicalcells' that carry these kinds of organelles, has already beenanal;sed (r values of 0.7-0.9 were genera!ly determined),'exceptional individuals' of the same species, that lackedthese organelles, remained unexplored up to the presentstudy. While in C. mnemosyne stigma-deficient cells were oc­casionally found after a period of heavy starvation and thecup-like organelle of P. notatum was spontaneously decom­posed in several cells during the divisions of a tomont, theorganelle of Lieberkiihn had to be removed from therontsof O. catenula and other species by micromanipulation. Ori­entation tests with exceptional cells were performed underidentical experimental conditions as applied for typical cellsbefore, with the two exceptions that single cells were testedinstead of a few hundred cells in parallel and that the cells,which had a tendency to settle down in the experimentalchamber were slightly irritated before the light was switchedon. It was found that phototactic responses were either dra­matically reduced in the exceptional cells (in stigma-free cellsof C. mnemosyne and in the dividing cells of P. notatum,r values 0.2-0.4), or even completely lost (in watchglass-de­ficient cells of Ophryoglena, r values <0.2/micromanipulatedcontrol cells with watchglass organelle, r values 0.4-0.7).The results indicate that specialized organelles have somefunction in phototactic orientation of at least a few ciliates.

440 . Abstracts

132The Hydrogenosome and Metronidazole Resistance inTrichomonadsJ. KULOA, E. TOMKovA and J. TACHEZYDepartment of Parasitology, Charles University,Vinicna 7, 12844 Prague 2, Czech Republic

The hydrogenosome, a double membrane bound organelleinvolved in pyruvate metabolism, is the critical compartmentof a trichomonad cell, where antimicrobial properties of me­tronidazole are activated. Metabolic reduction of the drug ,accompanied with the release of cytotoxic radicals, ismediated by ferred oxin, an iron sulphur protein of low redoxpotential, which tran sports electrons generated by hydrog e­nosomal oxidoreductases to the drug. Trichomonads can de­velop resistance to metronidazole either by decrea sed oxygenscavenging leading to interference of int racellula r O 2 withreduction of the dru g (aerobic resistance) , or by eliminationof electron generating pathways of the hydrogenosomal me­ta bolism (anaerobic resistance). Our studies on the in vitrodevelopment of drug-resistance in Trichomonas vaginalishave shown that the two types of resistance, believed to beunrelated, belong to a single multi step process. Trichomo­nads expo sed to a sublethal drug pressure developed firstthe aerobic resistance. Gradual development of the anaerobicresistance followed thereafter, involving changes in activitiesand expression of several hydr ogenosomal enzymes and fer­redoxin. The activ ity of pyruvate:ferredoxin oxidore ductase(PFOR) disappe ared early and the loss did not result in acqui­sition of the full anaerobic resistance, as it is in T. foetus. Byusing EPR spectroscopy, we demonstrated release of metroni­dazole free radicals by T. uaginalis stra ins that lacked PFORactivity. Our furth er studies revealed that T. uaginalis pos­sesses an additional system capable to reduce metronida­zole. Th is system involves decarboxylation of malate bythe hydrogenosomal malic enzyme, reduction of NAD, andfurther transfer of electrons from NADH to ferredoxin byNAD: ferredoxin oxidoreductase. The acquisition of the fullanaero bic resistance therefore required elimination of theseact ivities in addition to that of PFOR .

133Heavy Burden of Colacium Epjbionts on Keratellacochlearis in Coastal Inlets in Aland, SW FinlandA. LAGUS and T. LINDHOLM 0

Department of Biology.Biocity, Abo AkademiUniversity, FIN-20520 Abo, Finland

The occurrence of epibiontic algae on rotifers was studiedduring the summer of 1994 in seven coastal inlets in Aland(northern Baltic Sea). Keratella cochlearis was often heavilyinfected by the euglenophyte Colacium sp., where as K. quad­rata and other rotifers were almost free from epibiont s. Epi­bionts occurred thr oughout the summer but heavy epibiontburden (>20 Colacium cells per individual of K. coch­learis) and high prevalences (up to 90-100%) occurred inthe middle of the summer. Low preval ences (less than 2%)were observed in one locality where K. cochlearis was abun­dant. The reason for this almost complete lack of epibionts onKeratella in one inlet, which was very similar to the nearb yones, is unknown. Epibionts occurred in both eutrophicand oligot roph ic conditions and at high and low abundancesof Keratella.

134Fine Structure of Mature Blepharisma lateritium CystsH. F. LARSEN1 and J. R. NILSSON2

' Priva te Laboratory, DK-5600 Faborg, Denmark and2Dept . Cell BioI. & Anatomy, Zoological Institute,Copenhagen Uni versity, DK-2100 Copenhagen 0,Denmark

Since isolation from nature in 1980, B. lateritium has beenkept at 5 °C where cysts are formed readily in aged cul­tures. The typical Blephar isma cysts are composed of a dis­tinct endocyst with a conical neck and a spherical exocyst .Both cyst walls react positi vely for carb ohydrate and muco­polysaccharide (PAS, Alcian blue). Fine structure of restingcysts (1% barbital buffered OS04) revealed the endocyst wallas thin densely packed, osmiophilic layers (extending from0.5-J.lm, dense "blobs") in which negat ive images of a fibrou smeshwork could be resolved. This structure wa s separatedfrom the encysted ciliate by a narrow amorphous, low den­sity zone which thickened at the bottom of the neck andformed the basis of a porous materi al, the plug. On the out­side of the endocyst, a corresponding meshwork of positi veimages of fine fibers borde red to and extended into the exo­cyst in a more loose configuration interrupted by islands ofdebris of bacteria and pigment granules. The boundary ofthe exocyst was composed of an orderly arrangement ofthe fibrous meshw ork. The substructure of the endocystand exocyst walls seemed to be identical, apa rt from the com­pactness of the fibro us meshwork and perhaps the embeddingmaterial.Below the corte x of the encysted ciliate, only occasionally wasa kinestosome, or microtubule, found . The compact cyto­plasm, devoid of vacuole s, contained numerous ribosomes,whorly membranous structures, mitochondria, pigment gran­ules, and lipid droplets, mostl y segregated in groups aroundthe compact macronucl eus and associated 2-J.lm micronuclei.Finan cial support from The Carlsberg Foundation is grate­fully acknowledged.

135A Tetrasporoblastic Microsporidium of Larvae of theCaddis fly Hydropsyche siltalai (Trichoptera)J. I. R. LARSSONDepartment of Z oology, Uni ver sity of Lund,5-22362 Lund, Sweden

Larvae of Hydr opsyche siltalai (Dohler, 1965 ) (Trichoptera,Hydropsychidae) in southern Sweden host a tetrasporoblas­tic microsporidium, producing pyriform, approximately2.1 x 2.5 - 3.7 urn great spores (living). All life cycle stageshave isolated nuclei. The exospore has distinct subdivi­sions, the polar filament is anisofilar with 4 - 5 coils, ar­rang ed in one layer of coils close to the spore wall in th eposterior half of the spore. The wide anterior coil measure sabout 100 nm, the narrow posterior coils about 70 nm indiameter. The polaroplast is divided into two sections; ante­riorly wide chambers, posteri orly closely arranged lamellae. Asphorophorous vesicle is produ ced at the beginning of thesporogo ny and the sporoblasts are released by rosette-likebudding. The episporonral space contains two kinds o f inclu­sions: fibrous or granular mater ial, and tubules of exosporematerial, which appear when the sporoblasts are individua­lized.

The microsporidium is new to science. It is temporarily placedin the badly known genus Gurleya Doflein, 1898, waiting fora better characterization of the genus based on a modern in­vestigation of the type-species G. tetraspora. The cytologicaldifferences tell that the species is not congeneric with Gurleyadorisae Larsson, 1995, another microsporidium of caddis flylarvae.

136Definition of Standard Culturing Conditions forSpirostomum ambiguum as the Basis for a NewFreshwater Ciliate Protozoan MicrobiotestA. LE Du-DELEPIERRE ~-, G. PERSOONE* andC.-A. GROLIERE"""Laboratory for Biological Research AquaticPollution, University of Ghent, J. Plateaustraat 22,9000 Ghent, Belgium, ~- "Laboratoire de BiologieComparee des Protistes, URA CNRS 138, ComplexeScientifique des Cezeaux, 63177 Aubiere cedex, France

Despite the importance of protozoans in the recycling of det­rital and bacterial biomass in the aquatic environment only afew toxicity tests have been developed with unicellular organ­isms and their application is limited to date to a few labora­tories worldwide.In an attempt to fill this void, efforts are presently made inLABRAP to develop a new low cost microbiotest with a largeheterotrichous ciliate: Spirostomum ambiguum. This specieshas been selected as a promising candidate test species for: itsvery large size- its slow swimming, its ubiquitous characterand its high sensitivity to various categories of pollutants.As a prerequisite to the possibility of routine application ofsuch a new microbiotest, culturing conditions of this test spe­cies need to be determined and optimized. The experimentsperformed eventually revealed that the best results for cultur­ing and long term maintenance of Spirostomum ambiguum inthe laboratory were obtained with reconstituted Volvic wateras culturing medium, oat flakes as food with dry leaves pow­der as vegetal adjuvants, 20°C and darkness as temperatureand light conditions respectively and weekly renewal of med­ium and food. Under the former conditions healthy culturescould be maintained for months with average generation timeof the ciliates population between 5 and 6 days. Work is inprogress to optimize these culturing conditions, in parallel,research is also in progress on the development of an acutetoxicity test based on the Toxkit type microbiotests with ef­fect scoring after 1 and 24 hours exposure to toxicants (seeposter abstract).

137A New Low Cost Microbiotest with the FreshwaterCiliate Spirostomum ambiguum: Test Protocol andFirst ApplicationsA. LE Du-DELEPIERRE and G. PERSOONELaboratory for Biological Research in AquaticPollution, University of Ghent, J. Plateaustraat 22,9000 Ghent, Belgium

In order to compensate for the scarcity of toxicity tests withprotozoans which are a key group of biota in the rapid recon­version of detrital and bacterial biomass in aquatic ecosys-

Abstracts . 441

terns, a new microbiotest has been worked out with theheterotrichous ciliate Spirostomum ambiguum. In parallelwith the definition of suitable conditions for controlled cul­turing of the test species in laboratory conditions (see abstractoral presentation) a standard testing procedure has been ela­borated, based on the principles of the Toxkit microbiotestsdeveloped in the Laboratory for Biological research in Aqua­tic Pollution at the University of Ghent in Belgium. The Pro­toxkit is carried out in 4 x 6 polystyrene multiwells, in 5dilutions of the toxicant, in reconstituted freshwater (EPAmedium), and with 10 organisms per test well in 3 parallelsper toxicant concentration. The ciliates are not fed during thetest. Sublethal effects (deformities) are assessed after 1 hourand lethal effects after 24 hours exposure to the toxicant at25 DC and in darkness. So far assays have been carried outon a number of pure chemical compounds. Lack of standar­disation of the culturing procedure of the ciliates at thepresent time and hence substantial differences in the physio­logical condition of the ciliates are at the origin of low re­producibility of the assays at the present step of theinvestigations. Yet, these preliminary experiments have re­vealed a high sensitivity of Spirostomum ambiguum for che­micals (particularly some heavy metals). Moreover asignificant correlation (99% probability) was found betweenthe sublethal (1 hour) and lethal (24 hours) effect assessment,pointing to the promising potential of this new low cost mi­crobiotest for very rapid toxicity detection in cases of emer­gency as well as for routine applications.

138Ultrastructure and Functional Morphology of theLorica of Diaphanoeca grandis (Choanoflagellida)B. S. C. LEADBEATERSchool of Biological Sciences, University ofBirmingham, Edgbaston, Birmingham, B15 2TT

Diaphanoeca grandis is a marine inshore 10ricate choanofla­gellate with a large siliceous lorica. Apart from a precise anddistinctive arrangement of costae, two thirds of the lorica islined by an organic 'veil' made up of microfibrils. Access tothe protoplast for food particles is achieved between thecostae at the rear end of the lorica. Bacteria and other parti­cles adhere to the outer surface of the collar where they areingested by pseudopodia. As in other tectiform loricate chao­noflagellates, costal strips are accumulated at the top of thecollar during interphase. Following nuclear division the celldivides and the accumulated strips are bequeathed to the ju­venile protoplast. Stages in the formation of the new loricawill be illustrated.Possible functions of the lorica have been investigated. Thedensity of the whole cell; the protoplast alone; the lorica withand without veil have been measured using isopycnic fractio­nation techniques. The significance of a protoplast surround­ing itself with a siliceous lorica have been assessed in terms ofcell suspension in a water column, the effectiveness of creatingwater currents containing food particles and resistance to pre­dation.

442 . Abstract s

139Distribution of Loricate Choanoflagellates in IcelandicWatersB. S. C. LEADBEATERSchool of Biological Sciences, University ofBirmingham, Edgbaston, Birmingham B15 2TT

Nanoplankto n samples containing loricate choanoflage llateshave been collected from 91 stations situat ed alo ng 13 off­shore transects aro und Iceland. Th ree separate watermasses, namely North Atlantic and Irminger wa ters; coas t­al, and ArcticlPolar waters, were sampled.29 species were recorded, 25 of which are pelag ic and char­acteris tic of the open sea. Overall concentrations of lor icatechoanoflagellates var ied between 103 - 105 cells L - 1. On thebasis of qualitative distr ibuti on, species could be classifiedinto three groups namely: wid espread, being found at moststa tions: intermediate distribution , being found at about50% stations; and relatively ra re. The 6 species that weremost widespread were also usually present in the largest con­centratio ns at individual stations. Many species showed var­iations in lorica size and out of th ree Bicosta and tw oCalliacantha species. B. antennigera and C. natans showeda significant negative correla tion of lor ica size with wa tertemperatu re. Some species, in the inter mediate range of dis­tr ibut ion category, showe d a significant positive cor rela tion indist ribution.Possible environmental facto rs affecting loricate choa nofla­gellate distribution in the open ocea n will be discussed.

140The SF-assemblins: a new Family of Proteins FormingCross-striated Fibers of 2 nm Filaments in ProtistsK.-F. LECHTRECK, A. BREMERICH and M. MELKONIANBotanisches Institut, Universitat zu Koln , D-50 923Koln , Germany

Non-act in filaments represent a significant component of thecytoskeleton in many protist gro ups. Among these struc tu­rally and biochemically heterogeneous filaments 2 nm fila­ments forming cross -striated fibers are highly distinctive.They consist of a unique protein family here termed SF-assem­blins, which have been shown to form paracrystals of ~ 30nm periodicity in vitro in a number of diverse protists (diplo­monads , ciliates, green algae). SF-assemblin was first isolat edfrom the system I fibers of the green flagellate Spermatozopsissimilis and conta ins two principal structura l domains: an N­terminal nonhelical proline-rich 31 residue domain and an a­helical rod dom ain of 253 residues with a 29-residue repeatpattern based on four heptad s followed by a skip residue. SF­assemblin and the related p-giardin form a special segmentedcoiled-coil which can pack into 2 nm protofilament s. Severa lpro tofi laments are laterally aligned to form cross-str iated fi­bers. SF-assemblins and their cross-striated fibers have beenconse rved during evolutio n from Archezoa through to"Crown-group" organi sms. Several SF-assemblins have beencloned and the role of different domains for in vitro-reassem­bly of paracrysta ls has been investigated.

141Concanavalin A Receptors and the ChemosensoryBehaviour of Tetrahymena thermophilaV. LEICKDepartment of Medical Bioch emistry and Genet ics,Biochemistry Laboratory B, Th e Universi ty ofCo penhagen, Pan urn Institute Blegdamsvej 3,DK-2200 Copenhagen N, Denmark

The relat ionship between concanavalin A (ConA) receptorsand the chemosensory behaviour of the ciliated protozoanTetrahym ena thermophila was studied using the pept ide che­moa tt ractants proteose peptone and fibroblast growth facto r(FGF).Studies on the chem osensory behaviour in the semisolidmethylcellulose showed tha t 50 ug/rnl Con A selectively inter­fered with the persistent element of swimming behavi our,wh ich is a high intensity behavioural response induced in che­motactic, gradients by of FGF and proteose peptone. Alph a­methyl-D-mannose (alpha-mm) abolished the inhibitory ef­fect of ConA suggesting that mannose -containing ConA re­cept ors are involved in maintaining persistent swimmingbehaviour.In liquid s, where persistent swimming is less important forcellular locomotion ConA did not interfere with the chemo­sensory behaviour as measured by a two-phase assay for che­moattract ion.Studi es of the cellular locat ion of ConA receptors showed apreferent ial clustering at the cellular an terior end as observedwith labelling of living (and glutaraldehyde-fixed) cells withfluorescent concanavalin A (FITC-ConA, 100 ug/ml ). 50mMalpha -mm abolished the FITC-ConA labelling suggesting apreferent ial clustering of mann ose-containing receptors atthe an terior part of the plasma membrane and cilia.It is suggested that the anterior mann ose-contain ing recepto rsfor ConA are part of a chemosensory plasma membrane struc­tur e important for signal transduction includ ing persistentswimming.

142Mosaic Pigmentation in a Ciliate, Mesodinium sp.,in a Coastal InletT. LINDHOLM •Department of Biology.Biocity, Abo AkademiUniversity, FIN-2052 0 Abo, Finland

A small, pigmented ciliate of the genus Meso dinium was de­tected in thin layers above theanoxic bottom in the reed beltof a brackish coastal inlet in Aland, SW Finland. Th e ciliatehad tr ifurcated tentacles and it was less active and less fragilethan M. rubrum , which has bifurcat ed tentacles. M ost indi­viduals were relati vely dark green or blue-green in appea renceand all such cells showe d a strong red chlor ophyll a fluores­cence in blue light . However, in many samples, cells with amosaic pigmentation were observed. Th ese "hybrids"showed both red and orange fluorescence, ind icat ing thepresence of two kind s of plastids, prob abl y of cryptophyteaffinity. Starch was present in the plastids, indicating photo­synthetic activity. No feeding behavio ur was observed. Theretention of several types of plastid s (or symbionts) wou ldbe an unusual way of light ada ptation.

143Extensive Glycosilation of Cyst Wall-specificpolypeptides take Place during Morphogenesis of CystWall in the Hypotrichous Ciliate Histriculus cavicolaA. A. LOPEZ, C. VELASCO, C. ALBA, C. CARAVACA,A. TORRES and J. MARTINDepartamento de Microbiologia, Facultad de Ciencias,Universidad de C6rdoba, San Alberto Magno sin,14004-C6rdoba Spain

Cyst wall of H. cavicola shows many components, some ofwhich immune cross react, but the basis for these heterogene­ity remains unknown. Positive PAS staining suggested thatextensive glycosilation take place after synthesis of at leastsome of the cyst wall specific polypeptides. This hypothesisis tested below.Absence of labelling with lectins MAA, AAA and PHA-L sug­gest that no complex N-linked glycans exist in cyst wall poly­peptides. The other six lectins label preferentially medium­high molecular weight components, which is in accordancewith the expected distribution of glycosilated forms of cystwall polypeptides. Two different labelling patterns are recog­nizable, both distinct of those obtained with antibodies. Oneof them is developed indistinctly by lectins GNA, WGA orDSA and is prevented by predigestion with N-glycosidaseF, indicating the presence in those glycoproteins of N-linkedhybrid glycans. The second pattern of labelling is obtainedwith either PNA, ACA or SNA, which proof presence ofa-linked glycans in glycoproteins labelled by these lectins.When cyst wall components are deglycosilated, A) the num­ber of components in the medium-upper part of the gel is sig­nificantly reduced and its mobility increased, B) lectinlabelling in western blot is prevented and C) the number ofbands recognized by each specific antibody is drastically re­duced to one or a few bands located in the lowest part of thegel, where number, intensity and mobility of bands is alsoroughly maintained. These results suggest that the vast major­ity of medium-high molecular weight cyst wall componentsare glycosilated forms of the lower size polypeptides. Thisserves also to confirm the peptidic nature of epitopes recog­nized by two monoclonal and several other antibodies.

144Chemoattraction in Euplotes vannus Affects theMembrane Potential and the Walking PatternW. LUEKEN, C. STOCK and T. KRUPPELAG Zoophysiologie, FB Biologie/Chemie, UniversitatOsnabruck, D-49069 Osnabruck, Germany

In the marine hypotrich Euplotes vannus, the coupling of be­haviour to the freely fluctuating membrane potential was in­vestigated by use of a computerized locomotion evaluationsystem (Multiple Track System, JVP, D-64331 Weiterstadt,and WINTRACK, Fa. Vogel, Bildverarbeitungssysteme, D­91054 Erlangen) and a single-electrode voltage-clamp sys­tem (npi SEC 1L, H.-R. POLDER, D-71732 Tamm). Undis­turbed cells walk in combinations of arcs and straightsegments, such that the track covers an extended area ofthe substrate without substantial lateral dislocation of thecell (RICCI et aI., 1987). The speed in walking rhythmicallyfluctuates, from zero to about 1000 um/s, with wavelengthsof about 800 ms. A gradient of the supernatant of a bacteriasuspension attracts the cells, within a few mins, to the source,

Abstracts . 443

where series of backward movements with subsequentchanges of direction occur. The behavioural elements respon­sible for lateral dislocation are correlated with specific mem­brane potential shifts. Complementary mating types of E.vannus and a clone of the related marine species E. raikoviare applied as other sources for chemoattraction.

145High Level Expression of Heat Shock Protein 70in Toxoplasma gondii is Associated with MurineVirulenceR. E. LYONS and A. M. JOHNSONMolecular Parasitology Unit, Department of Cell andMolecular Biology, University of Technology, Sydney,Gore Hill, N.S.W. Australia, 2065

Heat shock proteins (HSP) have long been recognised as play­ing significant roles in host-parasite relationships, and arewell characterized in protozoan parasites such as Leishma­nia, Plasmodium and Trypanosoma. We have investigatedHSP expression in three mouse-virulent strains (RH,ToxoENT, and ToxoP) and three mouse-avirulent strains(Beverly, ME49 and Fukaya) of Toxoplasma gondii by per­forming Western Blot analyses using a monoclonal antibodyagainst HSP65 of Mycobacterium bovis and a polyclonal anti­serum against HSP70 of Plasmodium falciparum as primaryantibodies. We initially observed that murine macrophagesexpress HSP65 when infected with either virulent or aviru­lent strains, although at a much higher level in mice infectedwith avirulent strains. In order to exclude host contaminationof protein samples, tachyzoites were purified from host cellsby nuclepore filtration for all further experiments. Differen­tial HSP expression consistent with virulence was observedbetween strains, with high levels of a 70 kDa HSP (HSP70)only detected in virulent strains in vivo. This protein wasnot observed in virulent strains in the immunocompromisedmouse or in vitro, suggesting induction by immunologicalstresses. This protein was only poorly expressed in avirulentstrains. A protein at 65 kDa was observed in all strains in vivoand in vitro, suggesting a shared epitope with HSP70. Theseresults are consistent with the hypothesis that the inducedexpression of HSP70 in virulent strains of T. gondii by immu­nological stresses may provide protection for these strainsagainst cell damage associated with invasion of the host, al­lowing the virulent strains to persist as tachyzoites withoutthe requirement for the encystation observed in avirulentstrains.

146Comparison of PCR Fingerprintings (RAPD Method)and Phylogenetic Analysis of European, African andAmerican Colpodid SpeciesJ. M. MALPARTIDA, A. MARTIN-GONZALEZ andJ. C. GUTIERREZDepartamento de Microbiologia-Ill, Facultad deBiologia, Universidad Complutense (UCM), 28040Madrid, Spain

The most widely distributed and characteristic soil ciliates aremembers of colpodid group, such as the genus Colpoda. Themain problem concerning to research in most groups of soilciliates, like colpodids, is the poor taxonomic standards for a

444 . Abstracts

fast and accurate identification of them. Therefore, the bio­diversity of these microorganisms is not very well known.In this research work we try to introduc e a new methodol­ogy, which, on the other hand, is extensively used in micro­bial ecology, but it is still poorly used in ciliatology. Thismeth odology, to identify ciliates, involves to study DNA poly­morphisms based on PCR amplification of random DNAfragments with single short primers (8-1 0 mer long) of arbi­trary nucleotide sequence (RAPD). We have used colpodidspecies from different places of Europe, Africa and Ameri­ca. After their identificat ion, by using a morphometric andcorticorype analysis, their total DNA was isolated from eachpure culture and the RAPD method was applied. Seven differ­ent primers were used. Th e proportion of shared RAPD pro­ducts among isolates was calculated using the formulaproposed by Nei and Li (1979). Similar ity values were ob­tained using data pooled from all and each 7 primers, ana­lyzed using UPGMA procedure and dendrograms wereproduced from data. RAPD analysis shown a high degreeof both interspecific and intraspecific genetic diversity in col­pod id ciliates. However, some primers may be useful to iden­tify some Colpoda species. The geograph ic location is also avery import ant factor which introduce an importa nt degree ofdissimilarity among isolates of the some species. The phylo­genetic analysis of 16 colpodids strain is discussed.This study is into an european proj ect (I1\l'fAS-94-3747).

147Ciliated Protozoa in Activated Sludge Plants of Madrid(Spain). II: Their Indicator ValueM. MARTiN-CERECEDA, A. GUINEA, S. SERRANO,L. ARREGUI and J. SuAREz *Dept. Microb iologia, Fac. Biologia, U.eM.; "Canal deIsabel II, Madrid (Spain )

Ten activated sludge plant s located in the community ofMadr id (Spain) were sampled during a year in order to deter­mine the ciliated protozoa inhabiting the aeration tanks of theplants. The identified ciliates were gathered into the following4 keygroups according their behaviour within the system:stalked, crawling, swimming and swimming-crawling.Multivariate statistical procedures (correlation and factoranalysis) were employed to describe the relationships be­tween groups and part icular species, and the main physico­chemical and operational plant parameters. The correlat ionanalysis mainly relates the abundance of stalked ciliates tothe high volumetric load values and to the low effluentBOD values, while the abundance of swimming ciliates is as­sociated to the high effluent BOD values and to the low sludgeage. Through Factor analysis six factors derived by Varimaxrotat ion were selected which expla ined 85.9 % of the var iabil­ity of the process. The first factor, considered as the processcontrol factor, group s together the mass and volumetric load.The biological importance of this factor is represented by twospecies of ciliates, Vorticella striata and Aspidisca cicada. Thesecond factor, considered as the nitrification factor, is repre­sented by Vorticella convallaria and the ammoniacal-N con­centrat ion. The third and fourth factor s named as biologicalfactors, concern the association amon g some species of cili­ates (Epistylis spp. with Aspidisca cicada and Vorticella stria­ta with Acineria uncinata). The fifth factor, tha t relatesLitonotus lamella to the sludge volumetr ic index, is consid­ered as the sludge quality factor.

148Ciliated Protozoa Activated Sludge Plants of Madrid(Spain). I: Occurrence and AbundanceM. MARTiN-CERECEDA, B. RODRIGUEZ*, P. CALVO,I. CAMPOS & D. FERNANDEZ-GALIANODept . Microbiologia, Fac. Biologia, U.eM.; "Canal deIsabel II, Madrid (Spain)

In this study, four categor ies of ciliates were establ ished de­pending on their abundance (scarce, moderate, abundantand dominant ) in samples collected from the areation tanksof ten activated sludge plant s. The total frequency of occur­rence and the distr ibut ion within these four categories, hasbeen determined for each identified genus. Results indicatethat the highest frequencies correspond to Vorticella (93%)and Epistylis (85%), the dominant genera in the major ityof samples. Other frequent genera (Aspidisca 64 %, Lit ono­tus 37% and Acineria 34%) with density values ranging inmost cases among scarce to abundant, are also found in theseplants. The remain ing genera are represented with a morelimited frequency of occurrence (less than 20%); Lox oph yl­lum 1%, Cinetochi lum 2%, Op ercularia 2% and Trithigmo s­toma 3% are the genera which present the lowest values offrequency and abundance.Most of the ciliates found by us have been described as com­mon inhabitants of activated sludge plant s. Only one speciesof the genus Acineria (A. uncinata) and tow species of thegenus Pseudochilodonopsis (P. fluviatilis and P. similis ) havenot been reported as typical ciliates of this process , probablybecause they can be easily confused with Trachelophyllumpusillum and Chilodonella uncinata owing to their morpho­logical resemblance.

149Evolution of Biomass and Photosynthetic Activity ofPhytoplankton in a Stream Receiving Treated Waterfrom a Wastewater Stabilization PondE. MAssERET, C. AMBLARD and G. BOURDIERLabo ratoire de Biologie Comparee des Proti stes, URA­CNRS 1944, Univ. Blaise Pascal, 63177 AubiereCedex, France

The treated water of waste stabilization ponds may lead di­rectly or indirectly to the eutrophicat ion of the stream whichreceives it. This study was carried out on a rural district la­goon in Massif Central (France) dur ing an annual cycle(April 1993-Jun e 1994), in order to determine the subse­quent impacts on both the physicochemical characteristics,biomass and photosynthetic assimilation of phytoplanktonin the stream which receive the discharge. An increase inthe concentrations of phosphorus and nitrogen and that ofthe fract ion of dead and/or healthy particles has been ob­served below the stabilization pond. This was confirmedby both high conductivity and DOC. We have observed theincrease of ChI a concentration s and autotrophic activity inthe stream beyond the discharge. The seasonal developmentof phytoplankton is similar at the downstream and in the finaleffluent . These results demonstrated that the water thrownout by the stabilizatio n pond resulted in a modification ofphytoplanktonic dynamics in the stream. This was showneither by an elevation of the aut ochthonous microalgal bio­mass due to nutr itional enrichment or, directly by the algalbiomass provided by the discharge.

150Effect of Inoculation of Ciliates in the Rumen of SheepReceiving High Starch Diet on Total Bacteria andFermentation CharacteristicsF.MATHIEU1,2,3, J. BOHATIER3, J. SENAU0 3, M BEN SALAH3

and J. P. JOUANy11INRA, Station de Recherches sur la Nutrition desHerbivores, Centre de Recherches de Clermont­Ferrand - Theix, 63122 Saint Genes Champanelle,France.2SANTEL France & Belgique, Avenue des cypres,53950 Louverne, France.3Laboratoire de Biologie Comparee des Protistes, URACNRS 1944, Universite Blaise Pascal (Clermont­Ferrand II), 63177 Aubiere Cedex, France

Six adult male Texel wethers fitted with rumen canula wereused. Their rumens were defaunated during the first periodand then inoculated with the genera Entodinium, Epidi­nium, Eudiplodinium and Isotricha. They were fed twicedaily (9 hand 16 h) a diet composed of 600 g of choppedhay, 600 g of pelleted barley and 150 g of soybean meal.Total bacteria, redox potential, pH, ammonia nitrogen(NH3-N), volatile fatty acids (VFA) and gases where mea­sured at different times after feeding.Total bacteria population decreased in the presence of proto­zoa from 10 to 30%. NH3-N concentration was lower in therumen of defaunated animals especially after 3 h « 90 rng/l),and strongly increased in the presence of protozoa (+ 20 to50%). Total VFA, acetate propionate and butyrate concentra­tions increased in the same way: + 20 to 30%, + 18 to 25%,+ 13 to 19% and + 20 to 40% respectively. The presence ofprotozoa had no effect on valerate but decreases caproate. Infaunated animals, isoacids were higher than in defaunatedanimals during 2 h after feeding (IC4: + 20%; IC5:+ 30%) but there were no differences at the next times.These observations confirm the stimulant effect of protozoaon protein breakdown into NH3-N. The presence of protozoastabilized rumen fermentations by decreasing between andwithin subjects variations. In the presence of protozoa theproportion of carbon dioxyde decreased as methane in­creased 2 h after feeding, thus the CO zICH4 ratio decreased(defaunated: 2.9; faunated: 2.6). Hydrogen proportion wasvery low «0.2%) except 1 h after feeding: 0.8% in faunatedanimals against 0.3% in defaunated animals. Ciliates producehydrogen and methanogenic bacteria associated with the pro­tozoa use the hydrogen formed. Rumen pH (6.6 before feed­ing) rapidly decreased after feeding to reach a minimum at 3 hlater (5.8). The redox potential strongly decreased after theinoculation of protozoa according to the increase of free hy­drogen 1h after feeding. The changes in redox potential canhave a strong influence on microbial populations but it is dif­ficult to find the real causes.

Abstracts . 445

151Phytoplankton Extracellular Products and SubsequentUptake by Heterotrophic BacteriaN. MAURIN, C. AMBLARO et G. BOURDIERLaboratoire de Biologie Comparee des Protistes (URA­CNRS 1944), Univ. Clermont, 63177, Aubiere Cedex,France

Rates of carbon flow from phytoplankton to bacteria wereestimated in an oligomesotrophic lake: the lake Pavin. Sam­ples were collected from 1, 10 and 15 m from the end of Aprilto the end of November 1993. Particulate and exudate pro­ductions were calculated from NaH14C03incorporation. Thephytoplankton carbon excretion rate varied from 0.22 to 1.82mgC.m~3.h-\ representing about 8% of total carbon uptake,and depended primarily upon the rate of its photosyntheticassimilation. The molecular size distribution of the dissolvedorganic carbon, released from phytoplankton, and subse­quently used by heterotrophic bacteria, were determined.Then, the bulk of the released substances was of low molecu­lar weight « 1500 dalton) with a minor contribution of highmolecular compounds (> 1000 dalton). The size of these mo­lecules seems to be decisive for uptake as a carbon sourceutilized first by aquatic bacteria that preferentially assimi­lated the low molecular weight.

152Chemotherapy of Protozoan Parasitosis of Fish andGeneral Look-out on MicrosporidiaH. MEHLHORNDepartment of Zoology and Parasitology, Universitiesof Bochum and Dusseldorf, Germany

Ornamental and food fish (in fresh or salt water) are parasi­tized by many protozoans of the phyla Sarcomastigophora,Sporozoa, Ciliophora, Microsporidia and Myxozoa. In mostcases there is no or a non-convenient chemotherapy.In the present review the life cycles of the most importantparasites were demonstrated in light and electron micro­scopy and an update is given on the recent control measure­ments. Since Microsporidia are abundant in fish and are alsofound in immunocompromized persons a broad-spectrumdrug would be highly desirable. The recent progress is dis­cussed.

153Soil Development on a Reafforested Strip MiningHeap: Protozoa as Monitoring OrganismsR. MEISTERFELD, H. COENEN and M. HEISTERBAUMInstitute of Biology II (Zoology), RWTH, D-52056Aachen, Germany

As a result of open cast mining of brown coal heaps of over­burden (area up to 10 krn-, height up to 200 m) were depos­ited. To allow reafforestation, the almost sterile sandy moundwas covered with artificial soil made from 20% loess and80% sand and more than 10 million trees were planted after­wards. A small experimental area was amended with humusof a primary forest to stimulate succession. In order to moni­tor the development of soil biota we have studied soil respira­tion, microbial biomass, flagellates as well as naked and

446 . Abstracts

testate amoebae. During a sampling period in 1984 almost allthese parameters declined significantly from primary forestover forest humus on the mound to the artificial soil. In1992 we repeated the study with somewhat different re­sults. Only the organic horizon of the primary forest had asignificantly higher respiration than the soils on the heap. Mi­crobial biomass followed a similar trend with only small dif­ferences between the Ah horizon of the primary forest and thetop soil on the heap. During summer, microbial biomass es­timates for the soils on the heap were higher. Flagellates andnaked amoebae of the soils on the heap had similar or higherabundances than those of the Ah horizon of the primary for­est. Testate amoebae showed a different trend. Abundances inthe forest humus on the heap were only 7% of that of the Ahhorizon of the reference forest and the artificial soils had evenless individuals. Species numbers on the heap were only 25%of that of the primary forest.These results show that testate amoebae are more sensitiveindicators and far better suited to monitor the soil develop­ment than microbial activity and abundances of flagellatesor naked amoebae.

154The Microcystis aeruginosa Paradox through a TwoYear Study of the Coupling of the Physical ChemicalEnvironment with Several Biological Parameters in aHighly Eutrophic ReservoirM. MICHARD 1, L. ALEYA1 and M. CHARPIN2

ILaboratoire de Biologie des Organismes etEcosysternes, Institut des Sciences et Techniques del'environnement, Place Leclerc, Besancon 25030 cedex,France.2Laboratoire de Biologie Comparee des Protistes,URA-CNRS 1944, 63177 Aubiere cedex, France

Several environmental and biological parameters were stu­died in a highly eutrophic reservoir to elucidate the Microcys­tic aeruginosa paradox since this species invaded thesuperficial layers of the reservoir while it did not gain anycompetitive advantage over other algae except buoyancy.The hydrodynamic characteristics, the nitrogen and phos­phorus input-output balance, the thermal structure ofwaters, the chemical characteristics of water column indi­cated that all conditions were ideal to provoke the Microcys­tis development: NIP mass ratios of inputs into the reservoirwere close to 4, high residence time of waters and availablestability of water column, occurrence of thermal stratificationwith a hypolimnetic anoxia contributing to the release up­wards of bioavailable phosphorus. Microcystis was station­specific. The variations of NIP ratios are the deterministicfactors of the mass occurrence of Microcystis aeruginosa.The summer growth phases of this species started as soonas NIP levels decreased below 5. The Microcystis blooms re­sulted in a strong increase of protein and carbohydrate con­centrations per unit biovolumes. In optimal growthconditions, Microcystis and all phytoplankton species direc­ted their metabolism to carbohydrate synthesis allowing ade­quate buoyancy of Microcystis.

155The Cytoskeleton of Opalina ranarum:An Immunochemical ApproachJ.-P. MIGNOT and B. VIGUESBiologie des Protistes, URA 1944, Universite B. Pascal,63177 Aubiere Cedex, France

By electron microscopy we have shown that a microfibrillarnetwork extends along the cortex of Opalina ranarum. Wesupposed it contributes in the positioning of somatic kineto­somes during the growing phase. This network originatesfrom the falcular matrix which seems to be equivalent ofthe centrosome in metazoan cells. In order to characterizethe nature and the functions of the cortical cytoskeleton,we have tested by indirect immunofluorescence, some antibo­dies specific of pericentriolar proteins (anti CTR210, ob­tained from M. Bornens), calcium-binding proteins (anti22-23 kDa, prepared by Vigues from the ecto-endoplasmicboundary of Isotricha prostoma) and kinetodesmal fiber (afamily of 30-36 kDa proteins, purified by L. Sperling fromParamecium tetraurelia). All these antibodies decorate thecortex of O. ranarum and particularly longitudinal stripswhich growth from the falx. But, more detailed examinationof the preparations treated with either anti 22 - 23 kDa or antiCTR 210, reveals a narrow band inserted orthogonally be­tween the two fields of longitudinal rows. With anti-rootletantibody, this band disappears. This band probably corre­sponds to the falcular matrix, a zone where the kine tosomesare not linked together by compact desmoses. At higher mag­nification, the longitudinal strips appear as dashed lines ar­ranged in zig-zag, a pattern resembling that of densedesmoses as shown by electron microscopy. With CTR 210antibody, the longitudinal strips seem to be also discontin­ual. Immunoelectron microscopy is now required to specifywhat part of the connective fiber and perikinetosomal matrixis reactive to the antibodies. Though preliminary, these resultsindicate that the cortical microfibrillar network of O. ranar­um, includes some proteins immunologically related to thosethat organize the centrosome through the cell cycle and thecortical patterning during cell morphogenesis in some ciliates.

156Evolution of the Cytoskeleton in OpalinidsJ.-P. MIGNOTI and E-M. AFFA'A2

IURA 1944, Universite B. Pascal, 63177 AubiereCedex, France, 2Biologie, Universite de Yaounde,Cameroun

All opalinids bear two groups of cilia: an anterior field offalcular cilia which are arranged in a narrow and curvedband called the falx and somatic cilia, with kinetosomeslinked by fibrous interconnective material arranged in line­ar, more or less longitudinal rows, such as the kineties ofthe ciliates.The outer surface of the cell is folded and each fold is sup­ported by longitudinal rows of microtubules which are nu­cleated in the falcular matrix.An other cytoskeletal component is constituted by a microfi­brillar network including calcium binding proteins. This net­work is joined to the falcular matrix and grows along thesomatic kinetosomes. It might be an important morphoge­netic element. Its morphology and development evolve ac­cording to the shape of the cell and the number of nuclei.

In Opalina ranarum, which is a flat cell with many nuclei, themicrofibrillar skeleton constitutes a bi-dimensional networkonly localized in the cortex.In Protoopalina pseudonutti, which is a species with only twonuclei, the microfibrillar skeleton growths in the endoplasm,forming a large axial hank surrounding the two nuclei at cer­tain stages. A microfibrillar sheath covers the cytoplasmicface of the nuclei.Cepedea sudafricana, which has many nuclei, appears as anintermediate mode!. Indeed, this species also possesses an en­doplasmic fibrillar network, but it is more scattered and doesnot interfere with the nuclear envelope.

157Seasonal Interchangeability of two Baltic Dominants,Hyaline Helicostomella subuLata and AgglutinatedTintinnopsis LobiancoiE. MIKOLAJCZYK and A. WASIKNencki Institute of Experimental Biology, 3 PasteurStr., Warsaw, Poland

Tintinnid species composition and abundance were deter­mined in surface water samples taken from four sites inGdansk Bay, and along four cruise tracks from Gdansk Bayto Helsinki. Two species, hyaline Helicostomella subulataand agglutinated Tintinnopsis lobiancoi were dominant atboth inshore and offshore sites. Other species, Leprotintin­nus botnicus, and five Tintinnopsis species, T. beroidea, T.baltica, T. campanula, T. cylindrica, and T. fimbriata werefound only occasionally and did not significantly affect totaltintinnid abundance.Our results showed the pronounced seasonal succession be­tween Tintinnopsis lobiancoi and Helicostomella subulata.The interchangeability of these two species occurred veryquickly. In the Gulf of Gdansk T. lobiancoi numbers in­creased rapidly with the onset of summer, and attained max­imal numbers in mid July, before disappearing abruptly bylate July to early August depending on the station. Duringthis decline H. subulata individuals were noted, with maxi­mal numbers in early August declining to zero by mid Septem­ber. At the same time, H. subulata abundance near theSwedish coast and in the Gulf of Finland remained highand did not decline significantly before mid October. Our re­sults show that waters off the Swedish coast and in the Gulf ofFinland were richer in tintinnids than other Baltic regions.

158Conjugation in Stentor coeruLeus: Reexamination ofNuclear ProcessesA. MIYAKE and V. RIVOLADepartment of Molecular Cellular and Animal Biology,University of Camerino, 62032 Camerino, Italy

We examined whether Stentor coeruleus undergoes "herni­sexual conjugation" and other unconventional nuclear pro­cesses in conjugation which were recently discovered inanother heterotrich ciliate Blepharisma japonicum (Miyakeet al. 1991, Europ.]. Protisto!' 27, 179-200). In hemisexualconjugation each of the non-meiotic micronuclei directly de­velop into a macronucleus, while meiotic micronuclei pro-

Abstracts . 447

duce new micronuclei through meiosis and karyogamy. Itoccurs in particular cells which produce different matingtypes by asymmetrical cell division.In intraclonal conjugation of a strain of S. coeruleus isolatedin Munster, less than half of the micronuclei entered meiosis.The rest remained swollen and survived into the postkaryo­gamic phase like in non-meiotic micronuclei in Blepharis­mao They eventually disappeared: hemisexual conjugationdid not occur. However, the long persistence of these "non­meiotic micronuclei" suggests that they might develop intomacronuclei, if the synkaryon fails in producing macronu­clear anlagen as it is the case in hemisexual conjugation. An­other similarity to Blepharisma was the lack of postmeioticdivision: the second meiotic division produced gametic nuclei.

159Litonotus-EupLotes (Predator-Prey) System: Role ofMicrotubules and Actin Bundles in the Engulfment ofthe Prey and Identification of a Fraction of EupLotesCFF able to attract the Predator with high EfficiencyA. MORELLI and F. VERNIDipartimento di Scienze dell' Ambiente e del Territorio,Universita di Pisa, 56126 Pisa, Italy

Litonotus feeds on Euplotes which proved to be capable ofreleasing some proteic factor(s) attracting the predator (Mor­elli et a!. 1994,]. Euk. Microbio!. Abs 21). This new round ofexperiments was planned to deepen our understanding of twospecific steps of the process: 1) the role of microtubules andmicrofilaments; 2) the activity of the substance capable ofattracting the predator. By means of antitubulin (FITC)­labelled was showed that a basket of microtubules sur­rounded specifically the cytostome of Litonotus; the basketdidn't appear labelled upon nocodazole treatment and thetreatment also prevented the ingestion of the prey. The pre­sence of actin was pointed out by phalloidin (FITC)­labelled, in particular at the neck level. Treatment with cyto­calasin-B acted against the actin microfilaments preventingthe engulfment of the prey.The CFF (Cell Free Fluid) of Euplotes was separated into 5fractions by means of molecular filters. These fractions weretested in a T-maze in order to verify their capability of attract­ing the predator. The fraction inducing the attraction had amolecular weight of between 5000 and 10 000 daltons.The protein content in this fraction was evaluated in 0.5mg/ml, and Litonotus succeeded in perceiving a protein con­centration of 0.0007 mg/ml. This fraction of CCF acceleratedthe reestablishment of the tubular and filamentous structures.

448 . Abstracts

160Are Archamoebae True Archezoa? The PhylogeneticPosition of Pelomyxa sp., as Inferred from LargeSubunit Ribosomal RNA Se~uencing

L. MORINI and J.-P. M IGNOT-ILabo ra to ire de Biologie cellulaire 4, URA-CNRS1134, Batiment 444 , Universite Par is-Sud , 9140 5­Orsay cedex, France.2Labora to ire de Biologie Co mparee des Protistes,Universite Clerm ont-Ferr and 2, Les Cezeau x, Bat.Zoologie, 24, avenue des Landais, 63177 Aub iereCedex, France

The taxonomic classification of Archam oebae, as defined byCavalier-Smith (1993 ), clusters them with two other phyla(Microsporidia and Metamonada) in the kingdom Arche­zoa. From a phylogenetic point of view, based mainly on ri­bosomal RNA sequencing and analysis, Metamonada andMicrospor idia are localized at the basis of the eukaryot ictree, a fact which would imply the same status for Archam oe­bac. Recent molecular data from Hinkl e et al. (Nucl. Ac. Res.,465,22, 1994) question this position, as one species from theorder Phreatamo ebia (Phreatamoeba balamuthi), clearly di­verges relatively late among eukaryotes, i.e. close to thecrown of the tree. Here, we report part ial sequencing ofthe large subunit ribosomal RNA from Pelom yxa sp., a spe­cies which is classified in the other Archamoebae order (Mas­tigamoebida). The phylogenetic analysis shows that thedivergence of Pelom yxa occurs just after the Eugienozoagroup, close to Physarum polycephalum. These results sug­gest that , as for Phreatamoeba, mitochondria and Golgi ap­paratus were secondar ily lost, and clearly support the ideathat Archamoebae should be disjointed from the Archezoakingdom.

161Glutathione Reductase of Plasmodium falciparum ­a Possible Target for Ch emotherapy of MalariaS. MOLLER!, K. BECKER2, B. BERGMANN!, R. H.SCHIRMER2 and R. D. WALTER I'Bemhard Nocht Institute for Tropical Medicine,Bernh ard-Nocht-Str. 74, 20359 Hamburg, Germany,21nstitute of Biochemistr y II, Heidelberg University, 1mN euenheimer Feld 328, 69120 H eidelberg, Germany

Malaria represents one of the greatest threa ts to human healthand welfare. Innate disorders leading to increased oxidativestress in human red blood cells confer prot ection from Plas­modium [alciparum infections, suggesting to study andmanipul ate the redox metabolism of parasitized erythrocytesfor therapeutic purposes. Gluta thione disulphide is one of themajor products in detoxificat ion pathways of reactive oxygenspecies and the flavoenzyme gluta thione reductase (GR) isresponsible to mainta in gluta thione in its active, reducedform, catalysing the reaction GSSG + NADPH + H + ->2GSH + NADP+. Here we report on the ident ificat ion ofthe GR gene from P. [alciparum. A 1.4 kb fragment of thegene was amplified by polymerase chain reaction using degen­erate oligo-nucleotides homologous to conserved regions ofknown GR sequences of other orga nisms. The deduced ami­no acid sequence revealed an overa ll identity of only 40% tothe human GR but most amino acids of known function are

identical. Southern blot ana lysis confirmed that the P. falci­parum GR is encoded by a single copy gene, as has been re­port ed for other GR genes. Notab le differences between thehuman and par asite proteins occur in the gluta thione-bindingpocket and at the inrersubunit contact area. These regions areof parti cular interest since they represent binding sites ofknown GR-inhibi tor s, like BCNO, offering the possibilityfor the development of new, parasite specific drugs. (Thiswork is supported by DFG and BMFT.)

162Selective Feeding of Paraphysomonas sp., anOmnivorous N anoflagellatc from Lake Constance,FRGH. MOLLERLimnological Institute, P.O. Box 5560, D-78434Kon stanz, FRG

Stock cultures of Paraphysomonas sp. (cell diameter: 10-1511m) from surface waters of Lake Constance were maint ainedwith either bacteria (wheat grain infusion) or cyanobacteria(Synechococcus sp.) as food. Prey organis ms used for experi­ments originated from the same lake: Spume//a sp., a smallheterotro phic nanoflagellate (cell diameter: 3 - 5 11m) and 3Synechococcus strains, which differed in shape and pig­ments. All cultures contai ned bacteria. Dur ing experiments,growth of prok aryotes was inhibited by penicillin and strep­tomycin (1 I1g ml" and 1.7 I1g ml r ' , respectively). In batchcultures, changes in predator and prey concentrations werestudied at 12 h intervals for 72 hours at 15 °C. Between 0and 48 h, growth rat es of Paraphysom onas ranged from0.55 to 0.81 d- 1• Growth was slowest when only bacteriawere present, intermediate with Synechococcus and fastestwhen Spume //a was offered. Ingestion rates were estimatedfrom the decrease of prey concentrations in experimenta lflasks compared to contro l flasks. In different experiments,8.7 - 20.5 bacter ia, 1.7- 3.4 Synechococcus and 0.7-1.1 Spu ­mel/a were ingested per Paraphysomonas and hour. Selectivefeeding was investigated by comparing clearance rates for si­multaneously offered prey items. For bacteria and rod -shapedcyanobacteria, clearance rates did not differ significantl y. Incontrast, the data clearly revealed selective feeding on coccoidcyano bacteria relative to bacteria as well as on Spume//a re­lative to bacteria.

163Organization of Heterotrophic PhagotrophicFlagellates and their Trophic StrategiesA. P. M YLNIKOVGroup of Protozoology, Institute for Biology of InlandWaters, Borok, Russia 152 742

Heterot rophic flagellates (HF) are colourless protists andhave some adaptations for uptaking food particles (phago­troph y). As a result of ultrastru ctural studies of 36 speciesthe corre lation between the tro phic behaviour of HF, peculia­rities of the consumed food and cell organization especia llythat of feeding apparatus (rA) and activity of flagella wasestablished. HF may be classified into some group s: bacterio­trophs, predators, sessile, gliding and swimming forms, in­gesting prey as a whole or sucking it by means ofmyzocytosis. Ameboid flagellate (apusomonads, cercomo-

nads) capture bacteria by pseudopodia of different shape. Thestramenopiles iSpumella, Bicosoeca, Histiona ) possess lip­like protrusion with some microt ubular band s. Pedinellidsuse a peculiar FA formin g axo podes (tentacles) for filtratin gwater. Bodonids possess constant FA consisting of cytostome,cytopharynx and some supporting microtubular and fibrillarsystems. Colpodellids (spiromonads) and katablepharids mayattack large eukaryotic prey and suck its contents resemblingfeeding of the suctorians. As a rule the carn ivorous flagellatescontain large extrusomes (rrichocysts and teniobolocysts), thebacterivorous flagellates possess small ones (microtoxicysts,kinetocysts, discobolocysts). It may be suggested that the evo­lution of FA and other components of flagellate cells resultedin emergence large taxomonic groups such as colpodellids,katablepharids, bodo nids, pedinellids, cercomonads. Bico­soecids and choanoflagellates usually prefer to inhabit in oli­gosaprobic waters, bodonids and majority of predators prefermesosaprobic waters. Ameboid flagellates may live in watersof different saprobility. Now the information about the struc­ture of FA and other structures of the cell explains the distri­but ion and trophic strategy of HE

164Wild Type and kin241 Mutant of Parameciumtetraurelia: A Comparison of Gravikinetic andElectrophysiological PropertiesU. NAGEL and H. M ACHEMERArbeit sgruppe Zellulare Erregungsphys iologie,Fakultat fur Zoologie und Neurobiologie, Ruhr­Uni ver sitar Bochum, FRG

Mechanical stimulation of a Paramecium cell at the anter iorend causes depolarization whereas mechanostimulat ion at theposterior end leads to hyperpolarization. This differentiatedresponse is due to the graded distribution of two types of an­tagonizing mechanoreceptor channels along the longitudinalaxis of the cell. It is assumed that reception of the gravityvector depends on these mechanoreceptor channels.A monogenic mutation in Paramecium tetraurelia modifiesthe cell size and the corti cal patt ern. It is also suspected tochange the distribution of the mechanoreceptor channels .The analysis of swimmin g behaviour reveals clear differencesbetween wild type and mut ant in swimming velocity, orienta­tion in the field of gravity, sedimentation rate, and gravikin­esis. Electrophysiological investigations show no significantdifferences in the basic electric pro perties (membrane poten­tial, input resistance, capacity) of mutant and wild type.Possibly the mutat ion affects the propert ies of the mechano­receptor channels or their distribut ion on the cell surface.

165Salicylates are Active Protist FixativesR. N EVERAVICIUS(Private Biological Lab or atory) Gedimino pro 49-A, 18 ,Vilnius 2001 , Lithuani a

From the group of salicylates two com binations were used asfixat ives. They are: salicylic acid (HOC 6H4COOH) and acet­ylsalicylic acid (CH 3COOC6H4COOH)6- (Aspirin "Bayer") .These two combinations were used in such concentrat ions: 20mg and 40 mg/100 ml distilled water. Salicylic acid 20 mgsolution pH 3.0, 40 mg solution pH 2.7. Acetylsalicylic acid

Abstrac ts . 449

20 mg solution pH 3.1 40 mg solution pH 2.9. Acetylsalicylicacid fixes prot ists more stably. It depends upon acetic grou p init . But optimum concentration is 40 mg/100 ml distilledwater. Too large concentr at ions of salicylates give rise to mor­phologic deformations of prot ists and their cytolysis. Solu­tions of acetylsalicylic acid after a long time begin tohydrolyse. At the same time their activities decrease. For thisreason it is advisable to prepare new solutions every month.Different Paramecium representatives were fixed. Th e dropwith protists was put into the fixative. Some seconds laterall prot ists are fixed. So fixed protists make an impressionof a sturous vitality. They can stay morphologically un­changed for a very long time.First of all salicylates block the energy system of the protistand then denature proteins of the cytoplasm. 2-3 minut eslater the cells darken. This shows that proteins of the cyto­plasm are denatured. Salicylates denaturing proteins of a cellat the same time stabilize its morphologic structure.Fixed protists were stain ed with hydrogenous dye solution. Tostain, the following dyes were used: safranin, eosin, erythro­sin, malachite green, methyl green, Nile blue, Congo red andthiazolyl red. Paint concentr ation was chosen empirically.Protists were tinted 10- 20 minutes after fixing. Depend ingon the purpose of a paint both cytoplasm and nucleus arecoloured brightly. Then, the well known preparations preser­vation method was used (0 .5. Sarkisov, 1951 ). Out of fixedand stained surroundings of prot ists the surplus solution wastaken away. Then , 2 -3 drops of 20-30% of polyvinyl alcoholsolution were put in. Some time later the water vapours andthe preparation is left covered with the thin polyvinyl film.

166Breakdown of Butyrivibrio fibrisolvens by RumenCiliate ProtozoaC. J. N EWBOLD, F. M. M ciNTOSH and R. J. WALLACERowett Research Institute, Bucksburn, Aberdeen,AB29SB, Scotland, UK

Recycling of bacterial protein in the rumen decreases the flowof microbial protein from the rumen and often causes exces­sive ammonia production. Ciliate protozoa are responsiblefor over 90% of the bacterial pro tein turnover, yet informa­tion on the characteristics of bacteria which influence theirsusceptibility to breakd own by the protozoa is limited. When[14C] leucine-labelled Butyrivibrio fibrisolvens SHl3 wasincubated in rumen fluid, the rate of [14C] release(11.65 % - 1) suggested that the bacterium would not survivein the rumen . Bacterial breakd own was almost enti rely due tothe presence of ciliate proto zoa in the rumen fluid. If prior toits addition to rum en fluid, the bacterium was grow n over­night in the presence of a filter sterilized extract preparedfrom sonicated washed protozoa, bacterial breakdown wasfar lower (2.7% h-1) . No reduction in breakdown was ob­served if the bacterium was grown in the presence of an auto­claved protozoal extract. Th is is not a universal property ofrumen bacteria, the breakdown of Slenomonas ruminantiumZ108 in rumen fluid was not affected by prior growth in thepresence of either filter sterilized or autoclaved prot ozoal ex­tract. The reduct ion in breakdown of B. fibrisolvens, whenincubated with filter sterilized protozoal extract, was asso­ciated with an increased production of extracellular polysac-

450 . Abstracts

charide. Some rumen bacteria may be able to protect them­selves from protozoal predation and degradation byaltering the characteristics of their extracellular capsule.

167Suprageneric Classification of CryptomonadFlagellatesG. NOVARIN0 1 and I. A. N. LUCAS2

IDepartment of Zoology, The Natural HistoryMuseum, London SW7 SBD, U.K., 2UCNW School ofOcean Sciences, Menai Bridge, Gwynedd LLS9 SEY,U.K.

Recent interest in the classification of cryptomonad flagellateshas dealt mainly with their position within the general sys­tematic framework of the unicellular eukaryotes and theirkin, with relatively little attention paid to suprageneric clas­sification within the cryptomonads themselves. Traditionalclassification systems based on light microscopy are no long­er adequate for the needs of the group because they cannoteasily accommodate taxa described using electron micro­scopy.A new system divides the cryptomonads into 3 orders basedon the presence and position of the nucleomorph. This systemappears to be consistent with available data on small subunitrRNA sequences. Owing to the ambiregnal status of crypto­monads, it is a "dual" system in which suprageneric taxa arenamed according to both Codes of Nomenclature applicableto protists.

168Protists from a Sewage-Contaminated AquiferG. NOVARIN0 1, A. WARREN!, N. E. KINNER2 andR. W. HARVEy3IDepartment of Zoology, The Natural HistoryMuseum, London SW7 SBD, England, 2Department ofCivil Engineering, University of New Hampshire,Durham, NH, U.S.A., 3Water Resources Division, U.S.Geological Survey, Boulder, CO, U.S.A.

Several species of flagellates (genera Bodo, Cercomonas,Cryptaulax, Cyathomonas, Goniomonas, Spumella) havebeen identified in cultures from a plume of organic contam­ination (treated sewage effluent) within an aquifer on CapeCod, Massachusetts, U.S.A. Amoebae and numerous uniden­tifiable nanoflagellates have also been observed.As a rule, flagellates were associated with solid surfaces, orwere capable of temporary surface attachment, corroborat­ing earlier observations from in situ and column transportexperiments suggesting that protists in the Massachusettsaquifer have a high propensity for association with sedimentgrain surfaces. Based on the fact that cultures from the uncon­taminated part of the aquifer yielded only a few species ofprotists, it is hypothesized that the greater abundance andvariety of food sources in the contaminant plume is capableof supporting a greater number of protistan species.

169Cadmium Toxicity in a Marine PhytoflagellateOlisthodiscus luteusA. NOVILLO and G. FERNANDEZ-LEBORANSLaboratorio de Biologia General, Departamento deBiologia Animal I (Zoologia), Facultad de Biologia,Universidad Complutense, 28040 Madrid, Spain

The toxicity and bioaccumulation of cadmium has been stu­died using a marine phytoflagellate, Olisthodiscus luteus N.Carter, Rhaphidophyeceae (according to Chretiennot-Dinet,1990), provided by the Plymouth Marine Laboratory, U.K.Cadmium acetate was used as a toxicant, and the concentra­tions of cadmium tested were 10, 50 and 500 pg Cd.l-1•

Olisthodiscus luteus was grown in 4L Pyrex flasks contain­ing 3L of f/2 culture medium without silicate or chelators(f/2-Si), and kept at a constant temperature of 17 ± 1 DC,and a 12:12 light:dark (L:D) cycle. The response criteriawere: growth rate, abundance, biovolume, and chlorophylllevels, with special reference to the bioaccumulation of thetoxic ion in the flagellates. The number of cells was deter­mined in vivo using a camera Fouchs-Rosentall. The cellularvolume was calculated by approximation to a sphere. Theconcentration of cadmium was measured by atomic absorp­tion using a graphite furnace. Spectophotometric analysis ofchlorophyll was carried out according to the standard meth­od. Four sets of experiments were performed, with three re­plicas per set.Olisthodiscus luteus is able to bioaccumulate 0.57-56.42fg.cell", Cadmium uptake is dependent on the time of expo­sure and on the internal concentration of the metal, the toxiceffects causing a reduction of this process. Cadmium deter­mines a decrease of cellular volume and this response maybe explained as the metabolic cost of the detoxification pro­cess. In addition, changes in the levels of photosynthetic pig­ments, as well as in the growth rate, were observed. In spite ofthe high concentrations used, the cultures were able to sur­vive. This might be explained by the fact that a large propor­tion of the metal was not bioavailable, between 13% and91% of the nominal concentration remaining in soluble formor sorbing to test vessels. A process acclimatization in the or­ganisms was observed.

170A new Colopodid Ciliate from Mosses, Bryometopusmuscicola n. sp. (Ciliophora, Colopodea)J. L. OLMO and C. TELLEZDepartamento de Microbiologia III. Facultad deCiencias Bio16gicas de la Universidad Complutense deMadrid (Espana)

Bryometopus muscicola n. sp. was isolated from moss sam­ples collected at several localities in rocks partially sub­merged at the Guadarrama river (Spain). At the laboratory,the moss was soaked with distilled water following thenon-flooded petri dish method described by Foissner. The de­scription is based on living and silver impregnated specimens.The morphological characteristics of this species are the fol­lowing: the cell is elliptic and measures 63-78 urn in lengthand has 25-30 somatic kineties. The adoral organelles are33-40 in number. Each organelle is composed of 3 rowsof kinetosomes. The paroral is different than in other speciesof Bryometopus in that it is formed by dikinetids packed close

together in the region of the mouth and more space in theupper part . The nuclear apparatus consists of a sphericalmacronucleus surroun ded by 1-4 micronuclei.A compara tive stud y with other related species is made.

171Intestinal Microsporidiosis: Serological StudiesC. O MBROUCK*, T. VAN GOOL*'", Y. BENHAMOU ~- * * ,

A. DATRY*, I. D ESPORTES-LrvAGE* and M. GENTILlNI *"Uni te Inserm 3 13, Centre Hospit al ier Un iversita ire dela Pitie-Salpetriere, 91 Bd de I'H6p ital , 75634 Parisced ex 13, France , ~- *Department of MedicalMicrobiology, Acad emic M edica l Center, Un iversity ofAmsterd am, M eibergdreef 9, 1105 AZ, Amsterdam,The N etherlands, ,~ ~. ,~ Service d'Hepato-Gastroentero­logi e, Gro upe Hospitalier Pitie-Salpetriere, 47 Bd del'Hopital , 7565 1 Paris cedex 13, France

Objective: To determine whether an Immunoblot assay de­tecting microsporidia specific antibodies is useful for diagnos­ing intestinal microsporidiosis in AIDS patients.Design: Sera from four HlV-seropositive patien ts .iJ.1fectedwith Encephalitozoon intestinalis, ten Hl v-seroposirive pa­tients infected with Enterocytozoon bieneusi, eight HIV­seropositve subjects expressing no signs of clinical diseaseand fourteen blood dono rs were assayed for ant ibodies toE. intesti nalis.Results: Prominant, dark-staining band s in the region be­tween 50 and 60 kDa are present in sera from AIDS patientsconfirmed to be infected with E. intestinalis. Half of the serafrom AIDS patients with E. bieneusi and 60% of the sera frompeople with no history of microsporidia also react with thisgroup of prot eins.Conclusions: Microsporidiosis seems to be an endemic infec­tion and the Immunoblot assay appears to be a useful methodfor detecting potential carrie rs of microsporidiosis in immu­nocompetent people.

172Differential Responses of Stentor coeruleus to Ethanol,Diethyl Ether, Acetone and AcetonitrileA. O RTIZ, J. BURRIEL and H . CANTARINO,Dept. of Animal Biology II, Biological Sciences Faculty,Complutense Univer sity, Madrid

Although protozoa are useful organisms, they have not beenmuch used in the study of pesticides. Th is could be due to thehydrophobic nature of these xenobiotics. Many of them, suchas lindane, aldrin, parathion, simazine, etc., are hardl y solublein water. This means that it is impo ssible to add them directlyto experimental cultu res.The aim of this work is to establish the prior cond itions ne­cessary to perform laboratory studies on different pesticides.A comparative study was therefore performed, by subjectingStentor coeruleus to different concentra tions of ethan ol, ether,acetone and acetonitrile, with the aim of discovering whichconcentratio ns had to effect on the growth of cultu res.Clonal asynchronous cultures were used. Cells were grown at22 ± 2 °C under cond itions of circad ian rhythm . Cultureswere grow n ion polystyrene dishes (4 x 2.2 ern) containi ng5 ml of sterile Pringsheim's medium. Vahine Yeast was sup­plied as nourishment , in uniform quantities and at constant

Abstrac ts . 451

time intervals. Ehtanol, diethyl ether, acetone and acetoni­trile were used. The range of dosage was from 0.01 to 4%.Incubation time was 60 minutes at 22 °C. Incubat ion was per­formed in 7.5 x 1 cm sealed glass tubes, each cont aining 200Stentor in 3 ml medium.Survival curves were obtained for each compound. At lowdosages (0.01- 0.2%) a 100% survival rate was achievedfor each of the four toxins. At levels of dosage from 0.2 to0.5%, the rate of survival was 95% for acetone and ether,and 100% for ethanol and acetonitrile. For ether, the expo­nential fall of the curve appears between 0.5 and 1%, whilethe shoulder of the curve or thre shold dose continues at theselevels for ethanol. Acetone and acetonitrile remain at an in­termediate level. Stentor is more resistent to acetonitri le thanit is to acetone.

173The Influence of Nourishment on the Catalasic Activityof Stentor coeruleusA. ORTIZ, J. BURRIEL and H. CANTARINODept. of An imal Biolog y II, Biological Sciences Faculty,Complutense University, Madrid

Resistance mechani sms against pesticides and ionizing radia­tion present a series of similarities. Th ese include the produc­tion of superoxide rad icals, hydroxyl, hydrogen perox ide, etc.Both processes involve antioxidant enzymes. In earlier stu­dies, we investigated the response of Stentor coeruleus to rays(C060) and a pesticide (paraquat).The aim of this study is to quantify the catalasi c activity ofStentor, establishing the relation between this and its pre­viously obtained radiological and chemical-biological para­meters.Clonal asynchrono us cultures were used. The Stentor strainwas supplied by the Jaime Ferran Institute, CSIC Madrid,and has been cultured in our facilities since 1982. Cells weregrow th at 22 ± 2 °C. Stentor was fed in two different ways: a)Vahine Yeast in uniform quantities and at constant time inter­vals, and b) mixed "a d libitum" feeding. Each sample in­cluded 1,300 fasting cells and had a final volume of 0.5ml. Cells were disrupted in a Vibracell sonifier dur ing 40sec., following the add ition of a phosphate buffer (50 mM,pH = 7). Catalasic activity and pro tein evaluation were car­ried out using spectrophotometry, following adapt ed Aebiand Lowry techniques, respectively, in Ultraspec III appara­tus.The results obtained to date have shown that the quant ity in gof protein per Stentor is 0.133 for those fed with yeast, and0.160 for mixed feed. Catalasic activity (I.U.) per Stentor is0.0179 for yeast feed and 0.0207 for mixed feed. Neverthe­less, if the units of catalasic activity per g of protein are com­pared, values are found to be higher in tho se cultures thatwere fed with yeast. These result s show that Stentor culturedwith mixed food generate an increase of protein synthesis,although this does not correspond to an increase of enzymesynthesis parallel to that of catalasic activity.

452 . Abstracts

174Paraquat Toxicity in ProtozoansA. ORTIZ, J. BURRIEL and H. CANTARINODept. of Animal Biology II, Biological Sciences Faculty,Complutense University, Madrid

As they are unicellular organisms, protozoa species greatlysimplify the observation of chemical-biological response.This is because each cell is affected as a unit, and is unin­fluenced by adjacent cells. Cell recovery is also an indepen­dent process. Stentor coeruleus is particularly useful, as itis possible to study the influence of paraquat on cell divi­sion, survival and recovery in the same organism. This studyshows the survival curves and kinetic response of Stentor toparaquat. Clonal asynchronous cultures were used. The Sten­tor coeruleus strain was supplied by the Jaime Ferran Insti­tute, CSIC Madrid, and has been cultured in our facilitiessince 1982. Cells were grown at 22 ± 2 DC under conditionsof circadian rhythm. Cultures were grown in polystyrenedishes containing 5 ml of sterile Pringsheim's medium. Va­hine Yeast was supplied as nourishment, in uniform quanti­ties and at constant time intervals. Ten cell samples of 200Stentor cells in 3 ml of medium were exposed to doses ofparaquat at 22 ± 2°C in 7.5 x 1 em glass stoppered tubes.Paraquat (1,1 dimethyl-4,4-bipyridinium dichloride) was in­corporated by treatment with Pared agrocros incubating Sten­tor at 60 minute intervals, administering doses of from 0.16and 5 ppm.The doses/survival curve had a well-defined threshold, indi­cating that a limited number of interactions are necessaryto kill cells. The LDso was 3.5 ppm. LD30 stood at 4-4.5ppm. Dq ranged from 2.5 to 3 ppm.The survival and reproductive rate curves for Stentor treatedwith doses from 0.16 to 1 ppm were obtained. Cells wereexamined immediately after incubation, after which theywere placed in dishes for continuous observation over a 10day period. The curves show: - A decrease in survival ratioduring the first days after incubation. - A gradual increasein reproductive ability after 5 - 7 days following applicationof the toxin.

175Macronuclear Changes in Gamma Irradiated StentorcoeruleusA. OTIZ, J. BURRIEL and H. CANTARINODept. of Animal Biology II, Biological Sciences Faculty,Complutense University, Madrid

We reported in previous papers that Stentor coeruleus showeda high level of resistance when exposed to gamma radiation.This study examines alterations of the nucleo-cytoplasmic ra­tio, as well as some induced abnormalities in the macronu­clear bead in Stentor irradiated with 60Co gamma rays.Clonal asynchronous cultures were used. The Stentor coeru­leus stain was supplied by the Jaime Ferran Institute, CSICMadrid, and has been cultured in our facilities since 1982.Culture takes place in sterile Pringsheim's medium at22 ±2 DC, at circadian rhythm and constant spectral distribu­tion, energy levels. Chlorogonium, which was also cultured inPringsheim's medium, was used as nourishment together withagar. Stentor was irradiated with 6OCo gamma rays 42 emfrom source, at a rate of dosage of 300 Gy/hour. Ten cell sam­ples (100 Stentor in 4 ml of medium) were exposed inpolystyrene stoppered tubes at room temperature of approxi-

mately 22°C for each dose. Stentor were fixed on albuminizedslides with ethanol-ether (1:2) for 30 minutes. Staining wasperformed using a modified Grosso triple stain. Nucleic acidswere studied using the methods of Feulgen and Brachet.The survival rate at lower doses (1,450-1,650 Gy) was 80­90%, and no changes were found in macronuclear nodes. Fol­lowing a high dose of gamma radiation (2,000 Gy) a certainnumber of specimens disintegrated before completion of theirradiation process (40% survival). The remaining cellsshowed certain macronuclear abnormalities:- An increase in the nuclear - cytoplasm ratio. - The fusion ofmacronuclear nodes. This appears at doses of 2,050 Gy, andincreases at higher dosages. - RNA is detected within macro­nuclear nodes (2,450 Gy). - This radio response behaviour byStentor coeruleus may be considered to be a survival strategy.

176Trypanasoma cruzi-Host Cell Interaction: AnOverview on the Receptor-ligand Recognition Systemsand the Modulation of Host Cell Gene Expression andSynthetic Activity by the PathogenA. OUAISSI l, R. FERNANDEZ-GOMEZ!, B. LAMKHIOUED2,

D. SOIZIe3 and O. BILLAUT-MuLOTl

lInserm U 415, 2Inserm U 167, Institut Pasteur, 'Rue duPro Calmette, Lille, France; 3Inserm U 156,Cite Hospitaliere, Place de Verdun, 59045, Lille,France

The infection of mammalian cells by Trypanosoma cruzi in­volves certain parasite and host cell glycoconjugates such asglycolipides and glycoproteins. Other studies have shown thatsialic acid could play a role in the recognition processes.Further exciting experiments have revealed that cell Iyso­somes fused very early with the T. cruzi-containing vacuolesthus providing the membranes required to form the parasiteparasitophorous vacuole, therefore suggesting that some sig­nal(s) is transmitted from the parasite to the host cell intra­cellular compartment resulting in clustering and fusion ofIysosomes at the site of invasion.Another important aspect of the parasite-host relationship isthe idea that the presence of amastigotes in the cytoplasm ofthe host cell might interfere with its normal physiologicalfunctions. This has led to a large number of observationsshowing that, indeed, the parasite acts on host cell gene ex­pression, induces functional disturbance in the generation ofsecond messengers and modifies the synthetic activity of thehost cell.On the basis of the above data, we have recently studied, forthe first time, the T. cruzi infection of astrocytes and reportedthat the parasite induced cell dysfunction. Our data suggestthat impairment of astrocytes may playa role in the patho­genesis of Chagas' disease particularly in children duringthe acute phase of T. cruzi infection.

177Penetration of Eimeria magna Sporozoites in theIntestine of RabbitsM. PAKANDL1 and P. COUDERT2

lInstitute of Parasitology, ASCR, Ceske Budejovice,CZ 37005, 2Laboratoire de Pathologie du Lapin,INRA, Monnaie, F 37380

Five-weeks old rabbits were inoculated with 7 x 10 7 sporo­cysts directly in the duodenum as described earlier (Parasi­tol. Res. 79: 593-598). The animals were euthanized after5, 10, 15, 20, 30, 45 min and after 1, 2, 4, 6 and 12hours. The tissue samples from duodenum and ileum wereprocessed for histology and transmission electron microscopy.Sporozoites occurred in the villous epithelial cells as soon as10 min past inoculation (m.p.i.). In 45 m.p.i., the sporozoiteswere observed in intraepitheliallymphocytes (IEL) and latertheir number in IEL successively increased. The sporozoiteswere first recorded in Lamina propria of the duodenum sixhours past inoculation (p.i.). At the same time, the first spor­ozoites occurred in the ileum. They were present in entero­cytes as well as in IEL and rarely in Lamina propria. In12 h p.i., the sporozoites were almost exclusively in the ileum.Although meronts and gamonts of E. magna develop in theileum and eventually posterior part of jejunum, sporozoitesinitially enter the duodenal epithelium. After this, they pene­trate in Lamina propria and the following route of their mi­gration is unknown. Nevertheless, it seems be probable thatnonluminal or extraintestinal traffic is employed.

178Macronuclear DNA Analysis of Vegetative Cells andResting Cyst of Colpoda infiata, Using Pulsed Fieldand Standard ElectrophoresisG. PALACIOS, A. MARTjN-GONZA~EZ and J. C. GUTIEEREZDepartamento de Microbiologia-III, Facultad deBiologia, Universidad Complutense, 28040 Madrid,Spain

During ciliate encystment the macronuclear system undergoesdrastic ultrastructural and molecular changes. Very little isknown on the presumable molecular changes that the macro­nuclear system can undergo during the resting cyst formation.In this study we try to find out somewhat more about thesepossible changes, studying the macronuclear DNA (Ma­DNA) from vegetative cells and resting cyst of the ciliate Col­poda inflata. The pulsed field electrophoresis (CHEF) shows aheterogeneous population bands between 200 and 2000 Kb,for the macronuclear vegetative genome, and between 180and 2000 Kb for the macronuclear resting cyst genome.The Southern hybridization with a 188 rDNA (555 pb)probe, which was obtained by PCR from C. inflata DNA,shows that this gene is located in a 200 Kb chromosomalband (CHEF) of vegetative cells. Both, CHEF with pulsetimes of 20, 40, 60 and 90 s and the effect of partial Bal31 digestion on mobility of C. inflata rDNA, have shown thatrDNA molecules are mainly linear molecules. The standardelectrophoresis analysis, after digestion with restriction en­zymes (MspI and BgII), on both vegetative and cystic Ma­rDNA, has shown big differences between them. A possiblemethylation of this ribosomal gene (18 S), at macronuclearlevel in cystic DNA, has been detected. Furthermore, fromthis analysis we can infer that Colpoda inflata rDNA is prob-

Abstracts . 453

ably organized as tandem repeats in linear molecules. We dis­cuss all these results within framework of the ciliateencystment process.This research work is supported by a grant from DGICYTproject PB-0076.

179Macronuclear Chromatin Study in Vegetative Cells andResting Cyst of Colpoda infiata: Chromatin Spreadingand Standard Transmission Electron MicroscopyG. PALACIOS, A. MARTjN-GONZA~EZ and J. C. GUITERREZDepartamento de Microbiologia-III. Facultad deBiologia, Universidad Complutense (UCM).28040 Madrid, Spain

It is known that during ciliate encystment drastic changes atmacronuclear level are observed (Gutierrez, 1985; Gutierrezand Perez-Silva, 1983; Martin-Gonzalez et al., 1992; Matsu­saka and Kimura, 1981; Walker and Maugel, 1980). In thiswork we study the macronuclear (Ma) chromatin changesduring encystment of Colpoda inflata, using both; spreadingchromatin method (Trendelenburg and Puvion-Dutilleul,1987) and standard transmission electron microscopy(TEM). Macronuclei were isolated from vegetative, precysticand resting cells. Like any eukaryotic cell the dispersed chro­matin is uniformly organized in nucleosomal chains. Replica­tion bubbles are observed only in active chromatin ofvegetative cells. For the second time in a ciliate macronucleusreport (Sergejeva and Bovyleva, 1988), a very special structur­al organization of Ma-chrornatin; hexagonal or polygonal la­mellaes in both vegetative and resting cyst, has been detected.These structures are more abundant in resting cysts. Thesepolygonal forms could represent a condensed transcription­ally inactive state of the Ma-chromatin. The incubation in0.1 mM borate buffer showed that this hexagonal formscan be completely decondensed into nucleosomal chromatinfibres. The standard TEM study has confirmed once again theexistence of a Ma-chrornatin condensation during encystmentin this ciliate, together with Ma morphological changes, anextrusion body formation and a nucleolar condensation ina only nucleolar mass. All these structural Ma changes areindicating that the mature resting cyst macronucleus is tran­scriptionally inactive. These results are discussed and theirphysiological and genetic implications are expressed.This research work is supported by a grant from DGICYTproject PB-0076. We thank Dr. M. F.Trendelenburg (Heidel­berg) for his valuable assistance in the spreading chromatinmethod.

180Early Origin of Foraminifera as suggested by SSUrDNA SequencesJ. PAWLOWSKI, I. BOLIVAR, J. F. FAHRINI1 and M. GOUy2IDepartment of Animal Biology and Zoology,University of Geneva, CH-1224 Chene-Bougeries,Switzerland, 2Laboratoire de Biometric, UniversiteClaude Bernard, Lyon, F-69622 Villeurbanne Cedex,France

Complete small-subunit ribosomal DNA (SSU rDNA)sequences of three species of foraminifera, Ammonia bee­carii, Allogromia sp. and Trochammina sp., were obtained

454 . Abstracts

from cloned PCR products. The sequences are unusually long,ranging from 2864 to 3341 bp and contain several small in­sertions. The obtained sequences were compared to SSUrDNA sequences of 26 other eukaryotic taxa in order to es­tablish the phylogenetic position of foraminifera. Based onneighbor-joining analysis, the foraminiferal lineage origi­nated early, close to the base of the eukaryotic tree. The for­aminifera diverged after diplomonads and trichomonads, butbefore euglenoids, trypanosomes and other later divergingtaxa, with relatively high (85%) score of bootstrap analysisperformed to evaluate the accuracy of the branchings. Theearly origin of foraminifera contrasts with several highlyevolved characters that these organisms possess and withthe apparent lack of foraminiferal tests in the Precambrianfossil record.

181Neospora caninum: Primary Structure of ITSl, ITS2and the 5.88 rRNA GeneS. PAYNE and J. ELLISDepartment of Cell and Molecular Biology, Universityof Technology Sydney, Gore Hill, NSW, Australia

N. caninum is a cyst-forming coccidian parasite which is nowknown to cause hind-limb paralysis in dogs and neonatalmortality in cattle and other livestock. At the molecular le­vel, the DNA of Neospora has not been extensively stu­died. A phylogenetic analysis of 185 rDNA sequences fromN. caninum with other coccidia showed a close evolutionaryrelationship with Toxoplasma. The study presented here de­scribes the DNA sequence of the transcribed spacer region ofthe rDNA of N. caninum which includes the 5.85 rRNA gene.The structure of this region of the rDNA is that typicallyfound in eukaryotic cells and bears sequence similarity tohomologous DNA of Toxoplasma.The sequence data generated has been used to develop a spe­cies-specific PCR test for N. caninum, which will prove valu­able in epidemiology studies on neosporosis.

182A Multivariate Analysis of Morphological Variation inUronema (Ciliophora, Scuticociliatida)B. PEREZ-UZ and A. WARRENDepartment of Zoology, The Natural HistoryMuseum, London SW7 SBD

Fourteen clones belonging to four species of the genus Urone­ma and one clone from Parauronema isolated from differentgeographical locations have been studied morphometrically.Linear measurements of taxonomic value were taken usinga program to record cartesian locations on images grabbedin a computer from video recordings of silver nitrate and sil­ver carbonate stained organisms. Data analyses on the vari­ables were conducted with the multivariate techniquesANaYA and canonical variates analysis (CYA). On the basisof 9 or 13 attributes, depending on the staining techniqueused, it was possible to discriminate all species morphometri­cally. Wide intraspecific morphometrical variability was ob­served in the species U. marinum; from nine clones studied inthis species, three distinguishable morphotypes were appar-

ent. These results also provided information on which char­acters are most useful for discriminating species on amorphometrical basis.

183Morphogenesis of Gymnozoum viviparum MEUNIER,1910 and G. sympagicum PETZ et al. 1995,Cyrtophorid Ciliates from Antarctic Sea IceW. PETzl, W. SONG2 and N. WILBERT3lInstitute of Zoology, University of Salzburg, A-S020Salzburg, Austria, 2College of Fisheries, OceanUniversity of Qingdao, 266003 Qingdao, P. R. China,3Institute of Zoology, University of Bonn,D-S311S Bonn, Germany

In mid-body, the morphogenesis commences with the intraki­netal proliferation of densely spaced basal bodies in the 4 spir­al kineties and in longitudinal rows n-7 to n. At first, onlythese 8 longitudinal rows divide giving rise to the daugh­ter's oral ciliature, i.e. one anlage each separates at the poster­ior end of a proter's kinety (very likely homologous to thecyrtophorid circumoral kinety) and one each at the anteriorportion of an opisthe's row (honologous to preoral kinety).The new adoral ciliature then forms a circle. Basal bodiesare subsequently proliferating in all other parental somatickineties which divide in mid-body. The posteriormost 8 ador­al rows ("preoral kinety") of the opisthe rotate 180 0 onto theother side of the new mouth then lying close to the "circu­moral" kinety. The opisthe's somatic kineties thereby shiftto their specific positions, e.g. the 4 spiral kineties encirclethe new cytostome. The daughter's main body axis is inclinedabout 45 0 to that of the proter now. The parental oral and atleast most of the somatic ciliature is not renewed during fis­sion. The morphogenesis of Gymnozoum (a junior synonymis Spiroprorodon) is rather similar to that of Pithites voraxand related species (Dsnoux 1976) which further corrobo­rates the close relationship of Gymnozoum and cyrtophorids(PETZ et al. 1995). Supported by Osterreichische Forschungs­gemeinschaft, Deutsche Forschungsgemeinschaft andDeutscher Akademischer Austauschdienst.

184Food Selectivity in the Hypotrich Ciliate Stylonychiamytilus - An Experimental Approach to QuantifyOmnivoryG. PFISTERInstitute of Limnology, Austrian Academy of Sciences,A-S310 Mondsee, Austria

In many limnological studies of the last decades, the trophicrole of ciliates in the microbial food webs has been consideredas either bacterivore, herbivore or predatory. In general singlespecies have been assigned to only one trophic level. Earlypublications have already pointed to omnivorous feeding inmany different groups of ciliates. However, the knowledgeon the quantitative aspects of ciliate food preferences is veryscarce up to now. The aim of this study was to quantify foodselectivity with the help of different fluorescently labeled po­tential prey organisms offered to the omnivorous ciliate Sty­lonychia mytilus. Different live-stained fluorescent ciliateswere offered as living prey. In addition, different fluores­cently labeled bacteria and algae, and inert fluorescent parti-

cles (FP) were offered in feeding experiments and evaluatedwith the help of epifluorescence microscopy. The results ofthis study showed a selective ingestion of different ciliatesas well as a clear selection between different algae. Bacteriaand FP were ingested, but negatively selected. There was avariability in food preference of different-sized cells of Stylo­nychia mytilus. Feeding rates were highest during the firsttwenty minutes of the experiments and reached a plateauafter 60 min of feeding. Food concentration was shown tohave strong effects on ciliate ingestion rates. Capture effi­ciency for ciliate prey was 70% at the beginning and declinedto a value of about 30% after 30 min. Further studies have toshow whether this method would be applicable to investigatecomnivorous feeding of protozoans in field experiments.

185Investigation of two Bacterial Infections in Ciliatesfrom Brackish Water by Light and ElectronMicroscopy, PCR, and in situ HybridisationL. PLATT-RoHLOFF+, E. BAIER-, H.-D. GORTz'fand K. HAUSMANN++Institute of Zoology, Dept. of Protozoology, FreeUniversity of Berlin, Germany and "Institute ofBiology, Dept. of Zoology, University of Stuttgart,Germany

During a search for endosymbiotic associations in protistsfrom marine and brackish water samples from the NorthSea coast, the cultivation of two strains of ciliates containingendosymbiotic bacteria was achieved. In a population ofParamecium caudatum, cells were found with gram-negativeshort bacterial rods in their macronuclei. The infection isstable and still present after one year of laboratory cultiva­tion. Another symbiotic association was found in a strainof a hypotrich ciliate that is preliminarily assigned to thegenus Uroleptopsis Kahl, 1932. Ciliates from this populationcontain gram-negative, short, rod-like bacteria in their cyto­plasm. The two endocytobiotic associations are described bylight and electron microscopy. Besides, the results of cultiva­tion of the host cells with antibiotics are presented. In order tomake an attempt at a phylogenetic classification of the endo­nucleobionts, part of the 16S rRNA-gene of these bacteriawas selectively amplified by the polymerase chain reaction(PCR) to enable us to partially sequence this gene. Besides,in situ hybridisation is performed using two labeled oligonu­cleotide probes, one of them specific for eubacteria, the otherderived from the specific PCR product. By this, the specificityof the PCR product is verified and the eubacterial nature ofthe symbionts is ascertained.

186Fine Particulate Detritus as a Potential Food Source forBacterivorous CiliatesT. POSCHInstitute of Limnology, Austrian Academy of Sciences,A-5310 Mondsee, Gaisberg 116, Austria

Several studies during the last few decades showed that sea­water and freshwater contain a special kind of detritus (deadorganic material) consisting of sub-micrometre particles(0.2-1 11m) and dead particles in the size range of larger bac­teria « 10 11m). The aim of this study was to investigate if so-

Abstracts . 455

called bacterivorous ciliates ingest dead organic particles andhow important this detritus is as a food source in comparisonto bacteria. Knowing that ciliates show a selective feedingbehaviour we looked for a special model type of detritus.Various autochthonous and allochthonous material (green al­gae, diatoms, zooplankton; macrophytes, leaves) was stainedby the fluorescent dye DTAF and artifically destroyed. Thisfluorescently labelled detritus (FLD) in the size range of0.2-2.3 11m was fed to the bacterivorous ciliate Dexiostomacampylum. Even at low food concentrations of 1-9 . 10 5 par­ticles per ml an ingestion of FLD was observed. Usually therewas no preference for a certain type of detritus but for a de­fined size class of particles. The ingestion rates of our experi­ments with detritus as a food source were similar to thepublished ingestion rates with bacteria as food.This research suggests that this kind of detritus could be apotential food source apart from bacteria for bacterivorousciliates. Open questions are the nutritive value of these par­ticles and the assimilation of this material by ciliates.

187Structural Characterization and Expression of the ~­

tubulin Gene Family in the Antarctic Ciliate EuplotesfocardiiS. PUCCIARELLI and C. MICELIDipartimento di Biologia Moleculare, Cellulare eAnimale, Universita di Camerino, 62032, Camerino(MC), Italy

To contribute to a better understanding of the molecular basisof microtubule cold stability, we studied the tubulin gene fa­mily in the Antarctic ciliate Euplotes focardii, a speciesadapted to environmental temperatures in the range from- 1.8 to + 2 "C, The identification of one ce-and three ~-tubu­

lin genes (named ~j, ~2 and ~3) have been previously describedtogether with the structure of the ~1 coding region (Miceli etal.]. Euk. Microbiol. 41,420-427,1994). Now the completesequences of the ~2 and ~3 genes have been determined to­gether with that of another ~-tubulin gene, named ~4' Thesesequences contain most of the structural divergencies earlierfound in the ~1 sequence with respect to the other known ~­tubulin sequences, thus supporting the hypothesis that mod­ifications of the tubulin primary structure are specifically cor­related with cell adaptation to cold. In particular, the ~3 and~4 genes showed additional unique substitutions, all clusteredin the conserved portion adjacent to the variable carboxy­terminal region. This region also appeared unique for the re­placement of three residues of glutamic acid, usually repre­senting sites of glutamylation (Glu434, Glu 438, Glu 441) , byresidues of aspartic acid and for the presence of a Ser442 re­sidue located in the motif Asp-Asp-Glu-Asp-Ser-Glu that re­presents a potential phosphorylation site for the enzymecasein-kinase I. The occurrence of glutamylation and phos­phorylation as posttranslational modifications of E. focardiitubulins was analysed by the use of specific antibodies. Clu­tamylation, that is a posttranslational modification detectedat both IX and ~ tubulin in a large variety of ciliates, includingother Euplotes species, was detected only at two cc-tubulinisoforms and phosphorylation, that has not been previouslyreported in ciliates, at two ~-tubulin isoforms. Financial sup­port was provided by the ENEA"PNRA"programm project.

456 . Abstracts

188Adhesion of Bacteria to the Surface of TermiteFlagellatesR . RADEK and J. ROSELInstitute of Zoology, Free University of Berlin, Berlin,Germany

Many of the trichomonad, hyperma stigote and oxymonadflagellates which inhabit the paunch of lower termite s bearectob iot ic bacteria on their surface. Spirochetes and rod-likebacteria may attach to the host cell by their tips (see e.g. [oe­nia ann ectens, M icrorh opal odina multinucleatum ), and therods may also adhere along their body sides (see e.g. Deues­covina glabra). At the attachment sites of]. annectens elec­tron dense material supports the plasmamembrane. Often,lectin/carbohydrate-interactions are involved in cell-cell ad­hesion. Thus, we used f1uorescently labelled and colloidal,gold labelled lectins in order to characterize the carbohydratecomposition of the glycocalyx . The lectins Con A, WGA andSBA bind to the surface of]. annectens only in regions sur­rounding the attached bacteria . The att achment sites them­selves are not marked. The plasmamembrane of D. glabradoes not interact with Con A at all, but the attached bacter iado. Feeding the termit es with filter paper soaked in a Penicil­lin/Streptom ycin solution removes most of the ectobi onts.While preformed attachm ent sites remain on the surface of] . annectens, nothing is reminiscent of the former cell con­tact s in D. glabra .

189Int ernalization and Recycling of Sphingolipids inParamecium primaureliaP. RAMOINO, F. BELTRAME*, A. DIASPRO* and M . FATO'"Istituto di Zoologia, Uni versita di Genova, 16126Genova, Italia, and *D ipa rt imento di Informatica,Sistemistica e Telematica, Universita di Genova, 16145Genova, Italia

Pagano and colleagues (I) developed fluorescent ceramideanalogues, N- [7-(4-nitrobenzo-2-oxa-1, 3-diazole}]-6-amino­ca-proyl-D-erythro-sphingosine (C6- NBD-Cer) and N-[5­(5,7-dimethylboron dipyrromethene difluoride}-l-penta­no yl]-D-erythro-sphingosine (CrDMB-Cer), which are vitalstain s for the Golgi apparatus and useful tools for studyingthe sorting and tra nsport of sphingolipids along the secretorypathway in mammalian cells. In our research, we have usedthese molecules together with confocal laser scanning opticalmicroscopy and digital image processing, to stud y lipid trafficin living Paramecium primaurelia . At low temperature, C6­

NBD-Cer and Cs-DMB-Cer are transferred directly to plas­ma membrane of paramecia without phagocytic uptakeand food vacuole formation; the endoplasmic reticulumand the macronuclear envelope become fluorescently la­beled. Upon warming the cells to 25 °C, part of these lipidsis rap idly internalized, but only few intracellular organellesexhibit a bright fluorescence emission. With increas ing timeat 25 °C, the phago -lysosomal system vesicles, cell membraneand cilia also become visibly labeled . The utilization of anaugmented reality digital system allows for a better analysisof the biologica l processes.(1) Rosenw ald A.G. and Pagano R.E., 1993 , Adv. Lipid Res.,26 : 101-118.

190Physical Mapping of the Genome of LeishmaniaC. RAVEL,P. WINCKER, P. BASTIEN, C. BLAINEAU, M. PAGESand J. P. DEDETGenome de s Parasites, La boratoire de Parasitologie,Faculte de Medecine, Montpellier

Despite the development of molecular studies on the genomeof Leishmania, many questions remain unanswered as regardsthe genetics of this organism. From the resolution of the mo­lecular karyotypes by pulsed field electrophoresis, we haveendea voured to etabli sh the general structure of this gen­ome. About 100 chromosome-specific "anonymous" DNAmarke rs isolated from plasmid libraries have been mapped,as well as 30 genes probe s. Also, several polymorphic micro­satellite loci have been characterized. This has allowed thedefinition of 29 physical linkage groups in the genome of1. infantum. The significant inter-strain polymorphisms ofthe molecular karyotypes could be accounted for by exten ­sive size variations between specifically identified homolo­gous chromosomes, as has been observed in otherprotozoa. Long-range restriction mapping of the smallestchrom osomes of 1. infantum showed that these size varia­tion s were mainly located in one of both subtelomeric regionsof the chromosomes, where tandemly repeated sequences ap­pear to be involved. Despite the chrom osoma l size variations,the general structure of the genome appeared remarkably con­served among all species examined, suggesting the scarcity oflarge interchromosomal rearrangements during the evolutionof this genus . Long-range restriction maps have now beenconstructed for 5 chromosomes ranging from 350 to 600kb, using up to six rare -cutting enzymes. The constructionof YAC libraries is in progress, directed towards the cloningand characterization of specific coding regions or other se­quences of interest like those involved in chromosome in­stab ility. The ease of transfe ction in Trypanosomatidsopens perspectives for the testing of the mechanisms and func­tional significance of chromosomal instabilit y in this par asite.

191Specific Affinity of Foraminifera Epip hytic onPosidonia oceanica (L) DelileT. RInES, H . SALVADO, J. M . AMIGO, M. Rus andM . P. GRACIALaboratori de Protozoologia, Department de BiologiaAnimal, Universitat de Barcelona, 08028 Barcelona

An study of foraminifera from six meadows of Posidoniaoceanica from Cap de Creus (1\TW Mediterraneum) from 2to 20 m deep has been carried out.In the faunistic analysis, a population of 28042 individualsbelonging to 23 families, 34 genera and 93 species, was de­termined, which reflected significat ivcly the posidonicalousbiocoenosis.A cluster analysis was made with 69 species once rejectedtho se being sporadic in the cluster we obtained five mainbranches (A, B, C, D, and E). Branch A grouped those speciesconstant but at percentages under 1% except for some specieslike Nubecularia lucifuga (in some cases reached 9.1%) orPeneroplis perturus (3.3 '10) . Branch B is constituted by un­usual species whose common characteristic is being foundbetween 10 and 40 m deep. Branch C is an homogenousgroup formed by abundant species which are typical from

Posidonia meadows (Blanc-Vernet, 1984) like Elphidium ma­eellum, Cibicides lobatulus, C. refulgens, Discorbis obtusaand Planorbulina mediterranensis. Branch E is composedby four species of genus Quinqueloculina typical from depthsbetween 10 and 30 m. Finally, group E is mostly formed byspecies of the family Miliolidae whose number of representedspecies is the highest of the different families found (30 spe­cies).

192The Simulation of Behavior of Ciliates and theComplexity of LifeN. RICCIDipartimento Scienze dell'Ambiente e del Territorio,Pisa-Italy

In spite of the positive trend followed by computer sciencesand of the optimistic expectations about their role in life si­mulations, the successful study of the behavior of ciliates andthe successive use of the appropriate software to simulate itled us to more realistic considerations. The behavior of cili­ates has been described exhaustively from both the qualitativeand the quantitative points of view by means of the ethogram(Ricci, Anim. Behav. '90). The second element is representedby a simulation program (the program CREECILI, reprodu­cing the CREEping of CILIates), capable of generating simu­lated tracks for a certain species, undistinguishable from thetrue ones. Thus we concluded that the phenomenon behavior(at first analyzed by its elementary components and by theirrelative relationships and later on reproduced by CREECILI)was "known", according to Aristoteles (Metaphysics, I, 57).A more careful consideration, however, led us to the followingconclusion: once the behavior is described as an ethogram, itcan be fed into CREECILI, which simulates (a) geometricallyperfect, but (b) adaptively meaningless tracks. There is nopossibility either to analyze the micro-stimuli continuouslyexciting each ciliate or to appreciate the microchanges inits physiological state: in other words, even the simplest mod­el of life (the behavior of ciliates) is far too complex to bepredictable: an important conclusion, to avoid ingenous ex­pectations and dangerous mistakes in the field of computersimulations of complex phenomena such as life.

193Use of Synthetic Wastewater in an Experimental Studyof the Microfauna in Activated SludgeM. RIDS, H. SALVADO, J. M. AMIGO and M. P. GRACIALaboratori de Protozoologia, Department de BiologiaAnimal, Universitat de Barcelona, 08028 Barcelona

Most of the changes observed in the composition of microor­ganism populations in sewage treatment plants are usuallyattributed to changes in affluent composition but this pointhas not always been proved. Thus, the use of a synthetic was­tewater may help in solving this problem, so we have com­pared the effect on the protozoan and small metazoanactivated sludge communities, of three different syntheticwastewaters whose composition is constant (we have calledthem: A-Lacoste et al., 1993, B-Gokcay et al., 1991 and C­a modification of B). The study has been carried out in experi­mental plants with a batch operation-type, a daily cyle of 24h, and supplementarily in C-water with a daily cycle of 8 h.

Abstracts . 457

A-water had to be rejected since it quickly acidified. In allcases, specific diversity decreased along the study in watersBand C, both in a similar way in the beginning but moreclearly in B-water at the end of the experience. Density ofciliates is also reduced along the study (24 h cycle) but alwaysranged between 106-105 indiv.lL, which can be related bothto the high retention times used and the competition of me­tazoan communities. Nevertheless, it has been observed thatciliate communities can be steady maintained in concentra­tions of about 10 6 indiv.lL for a period of 70 days, workingwith a 8 h daily cycle (experiment held only with C-water).The results obtained show that the use of a synthetic waste­water can be useful for the experimental study of ciliate po­pulation dynamics, simulating an urban sewage water.

194Abundance Measures in the Real WorldD. MeL. ROBERTS l, A. ROGERSON2 and J. ECCLESTON­PARRY3

lDepartment of Zoology, The Natural HistoryMuseum, Cromwell Road, London SW7 SBD, UK;2Universitiy Marine Station, Millport, Isle of Cumbrae,KA28 OEG, UK; 3School of Biological Sciences,University of Lancester, Bailrigg, Lancester LA1 4YQ,UK

Microbes occur in all wet habitats. The physical nature ofthese various habitats makes the direct comparison of num­ber of individuals recorded inappropriate. For instance, con­sider marine sand; protists live in the interstitial water and thegrain-size differences between two sites might mean that a2 x 5 ern core contains 20% more water in site a than siteb. Should we expect there to be 20% more individuals pre­sent in site a, all other things being equal? It is very wellknown that there is a greater abundance and a greater varietyof organisms associated with surfaces in aquatic systems.Many of these organisms are not attached to the surface,but merely associated with it. Is it more appropriate to mea­sure their abundance as individuals cm-2 or cm-3?

An attempt to resolve these issues will be presented in an ef­fort to define the major components of the problem, divorcedfrom the practicality of how such measurements might actu­ally be taken. There are strong indications that abundancemeasures have to be fractal in nature, measuring individualscrn ", where f is not an integer, but between 2 and 3.

195Computerised Access to Protistan NamesD. MeL. ROBERTS and G. NOVARINODepartment of Zoology, The Natural HistoryMuseum, Cromwell Road, London SW7 SBD, UK

Those who work with well-known taxonomic groups, such asthe flowering plants, the insects or the terrestrial vertebrates,often begin studies on ecology or biodiversity with a checklist.Within the microscopical world checklists exist for few phyla.Within the protista, the ciliates have the benefit of the pub­lication of comprehensive textbooks. For most other phyla,construction of a checklist means working through a vast ar­ray of primary literature which is only possible in a large well­found library.

458 . Abstracts

~very species name is defined or modified in a publication. Ast~me passes, th~ concept of what is being represented is re­fined. T.hus t~e. mform~tion necessary to keep track of namesand their posinon, t~elr synonyms etc., is a bibliographic re­c.ord. However, consider the problem of building a synonymylist for species a; a record links name a with name b; now it isnece~sary to check whe~her there are recorded synonyms forspeCl~s b, and.so on. This process only stops when there is nonew mforma.tion recovered. This type of search is called re­cursive and IS normally beyond the capabilities of flat-fieldor relational databases.Constraints on the design of a workable database to recoverspecies names and relationships will be described.

196Will the Chaperonin CCT be Involved in theFunctional Role of Tubulins in Ciliates?C. RODRIGUES-POUSADA, H. SOARES, L. CYRNE,A. C. CARDOSO, C. CASALOU and P. GUERREIROLaborat6rio de Genetica Molecular, InstitutoGulbenkian de Ciencia, Apartado 14,2781 OeirasCodex, Portugal

One of most attractive subjects in the field of microtubule(Mt) biogenesis is related to the biosynthesis of distinct func­tional Mt structures and the mechanisms responsible for theirfunctional diversity. Ciliates have been useful eukaryotic~odels t.o study: such i~teresting questions since they exhibithighly differentiated microtubular networks in combinationwit~ reduced genetic diversity concerning the tubulin genefamily, In Tetrahymena, for example all Mt arrays are builtfrom the products ~f a. single cx- and two P-tubulin genes[1]. The PIT1 gene IS highly expressed during cilia recoveryas compared to the PTT2 gene and its expression is coordi­nated with the o-tubulin gene [1]. However in Tetrahymenatu~ulin hetero~en~ity is generated by processes of post-trans­latlO~al,modlflcatlOns [~]. More recently, the discovery thattubulI? IS one of the major substrates of the cytoplasmic cha­p.e~o,nm CCT .(chaperonin-containing-TCP1) [3] rises the pos­sibiliry .ofthe mvolvement of this chaperonin in the biogenesisregulation of functional specific Mt arrays. Unlike homooli­gomer!c chaperonins. CCT comprises, in mammalians, atleast eight dIffere?t ,subunits (CCTcx, CCTILCCTs) and ap­pears to have limited range of physiological substrates[?, 4]. In th!s con,text we are interested in establishing a func­ti.o.nal relationship between the CCT and the biogenesis ofciliary Mts or other functionally specific Mt arrays. We havealready cloned two genes encoding the orthologues of themouse CCTy and CCTTj designated as TpCCTy [5] andTpCCTTj, respectively. We found that TpCCTy gene is coor­d!~at.ely express~d wi~h tubul!n genes during all stages of re­ciliation and conjugation studied. The characterization of theTet:ahY':lena C~T complex was done by raising polyclonalantibodies agamst carboxy-terminal aminoacid sequencededuced from the primary structure of TpCCTy. The Tetra­hymena CCT has been purified by ATP-agarose chromatogra­phy. Western blot analysis of the purified protein fractionsrevealed that Tetrahymena exponentially growing cells pos­sess a complex of about 700 kDa that contains theTpCCTy. We were able to show that the CCTcxsubunit is alsopresent in the referred complex using a rabbit polyclonal anti­serum against this subunit (a kind gift ofW. Welch, USA). Theresults will be discussed.

(1) Soares et al., (1991) Eur. J. Biochem., 197,291-299; (2)Penque et al. (1991), Eur. J. Biochem., 195,487-494; (3)Kubota et aI., (1994) Curro BioI., 4, 89-99; (4) Kim et al.(1994) TIBS, 19, 543-548; (5) Soares et al. (1994) J. BioI.Chern., 18,29299-29307

197Growth and Conjugation Studies of Tetrahymena inCerophyl MediumA. RON'~ and S. T. CHRISTENSEN***Department of Anatomy, The Hebrew University­Hadassah Medical School; Jerusalem 91120 Israel'~. '~Depart~entof Anatomy and Cell Biolog;, 'Carnpusvej 55, DK-5230, Odense M, Denmark

Tetrahymena thermophila BIII-1868 (mating type B-3 and~-7) were grown i? cerophyl medium CM (Pesciotta & Sa­tir, 1985, J. Cell SCI. 78: 23). Cells transferred to this mediumat an initial density of about 2000 cells/ml cease to multiplyafter ca. 30 h at a density of ca. 5 x 104 cells/ml. Thus CMcontains all the essential nutrients (e.g. amino acids, vitaminsand nu.cleosides) to s.upport cell multiplication, althoughabout five more do~blmgs can be achieved when using pro­teos: peptone medIUm, PP, or our best standard syntheticm~~lUm, SSM (Szablewski et aI., J. Protozool. 38: 62). ByI~llxmg equal num~ers o,fcompeten~ mating types during sta­ti<;>nary phase conjugation occurs in the cerophyl mediumwithout any pre-starvation. In this report we have investi­~ated cer.tain aspects of cell proliferation and conjugationin CM. FIrstly, we wanted to see whether CM, besides nutri­ent C<,lI~Il?onents, contains molecules which act as signals forcell dl.vlslOn: We have previously shown that T. thermophila inSSM m ~olllcal flasks require autocrine signal molecules forcell survival and proliferation in low density cultures « 1000cells/ml) the amount of these signals become too dilute in themedium, and the cells rapidly die. Since the supplementationof CM to these cuitures (> 0.1 % of CM) rescues the cells fromdying and promote proliferation, we suggest that CM con­tains compounds which somehow mimic or substitute forthe cell-pr~duced compound either by representing thecells' o~n signal I?olecules ?r by interfering with messengersystem mvolved in their signal transduction mechanisms.Furt~ermor~, w~ looked for protein synthesis changes inCM m conjugatmg as well as in vegetative cells: a 30 minpulse label of 35S-methionine of T. thermophila in CMYielded mor: than a 20-fold increase in the efficiency of la­belled protems as compared to PP medium. Three specificpolypeptides are synthesized in the late stationary of conju­gating cultures, but not in log or early stationary cells. Thecalculate.d MW or these polypeptides, which could hardlybe seen m cells conjugating in TRIS/HCl, are 42-44 kDa,26-27 kDa and 14.5 -15.5 kDa. All three could be involvedin the process of conjugation.

198Different Approaches to Determine the Real Nature ofEpixenosomesG. ROSATI, A. GIAMBELLUCA, P. LENZI, V. FRANCO,R. ANNA and C. BANDI'-Dip. Scienze Ambiente e Terrirorio, via A. Volta 4,56126, Pisa, 1st. Patologia Generale Veterinaria, viaCeloria 10, 20133 Milano, Italy

In the attempt to determine the nature of epixenosomes twodifferent technical approaches were used. At first in situ hy­bridization at the ultrastructural level was performed with thefollowing probes: 1) rDNA from Euplotes; 2) rDNA fromPisum sativum; 3) oligonucleotide from Phaseolus 18SrRNA; 4) synthetic oligonucleotide for detecting E. coli16S rRNA; 5) universal oligonucleotide for prokaryotic16S rRNA. A positive response was always obtained wheneukaryotic probes were used. On the contrary when prokar­yiotic probes were used no preferential labeling could befound on the epixenosomes. A positive response was also ob­tained when the gene encoding for \1 tubulin of Euplotes wasused. These results strongly suggest a eukaryotic nature of theepixenosomes and validate the hypothesis that they containtubulin. To better characterize these organisms DNA prepara­tion(s) were obtained from E. itoi devoid of epixenosomesand E. itoi harboring the symbionts. Following PCR amplifi­cation using as primers two conserved portions of the geneencoding for the small subunit rRNA in eukaryotes, two dif­ferent electrophoretic patterns were obtained. Further assayswith both eukaryotic and prokaryotic primers are now incourse.

199First Description in Myxidium leei of a CellularStructure Intervening in the Pathogenesis of Sparusaurata in AquacultureN. SAKITIl, G. KABRE1, V. TARER3 and A. MARQUES3

lUniversite Nationale du Benin, Laboratoire deZoologie, COTONOU, Republique du Benin.lLaboratoire de Zoologie, EA.S.T., Universite deOuagadougou, Burkina Faso. 3Laboratoire deParasitologie et Immunologie, Universite MontpellierII, 34095 Montpellier CEDEX, France

While working on the identification and on the action ofMyxidium leei Diamant et aI., 1994, a pathogenic histozoicmyxosporidia of Sparids (Sea bream) found in aquaculture,as structure leading to the multiplication of this myxospori­dia on its host was observed.Dispored pansporoblasts were scattered in the gut mucosa,their size reaching 20 urn, When old, they include two sporesfully constituated, as well as two cellular elements unidenti­fied to this day. Each element is constituated by a small cell.The cytoplasm of the enveloped cell (1.59 ± 0.90 urn dia­meter) has many ribosomes, its nucleus is dense and oftenover one urn (1.08 ± 0.84 um).The cytoplasm of the enveloping cell (4.90 ± 2.99 urn dia­meter) is rich in endoplasmic reticulum, it has a nucleus of1.47 ± 0.84 urn in diameter and a large nucleous reachinga diameter of 1 urn.

Abstracts . 459

The myxosporidium, first settled in the digestive tract, pro­gressively invades all the mucosa and induces the lethalpathology of these fishes. When appearing in a batch, the fishis stressed and dies.The observation of these forms could explain how thismyxosporidium progressively invades the digestive tract;the pathogenicity must be linked to the particular invadingcapacity of these bicellular forms released on the spot.The lack of reaction of the host, which in other Myxosporidialeads to cyst formation, seems to be the determining elementof the parasitosis progression and of its pathogenic compe­tence.

200Parastrongylidium pyriformis n. sp. (Ciliophora,Hypotrichida) from Activated Sludge SewageTreatment PlantsH. SALVADO, M. RIUS, M. P. GRACIA and J. M. AMIGOLaboratori de Protozoologia, Department de BiologiaAnimal, Universitat de Barcelona, 08028 Barcelona

A new species of hypotrich, Parastrongylidium pyriformiswas recollected in samples of activated sludge from a sewagetreatment plant near Barcelona. Fresh samples were observedin the facility and for the description techniques of silver stainand scanning electron microscopy were used.The morphology of this species is quite similar to Parastron­gylidium martinis'), despite observing clear differences. Theorganism in vivo shows a pyriform sharp shape in anteriorextreme and a round black pigmented posterior region.The size in vivo is about 60 x 30 urn. The adoral zone ofmembranelles occupies 30-35% of the body length andshows 18-23 (most usually 19-22) membranelles and theparoral zone of membranelles is constituted by double kin­eties, The somatic infraciliature is similar to that describedfor P. martini but with a higher number of kineties. It shouldbe stressed that in young P. pyriformis a dorsal line of pairs ofkinetosomes can be observed as in P. martini. In any case, theinclusion of this new morphotype in the genus Parastrongy­lidium will lead to an addition of new characters to descrip­tion of the genus.This new species of hypotrich has been found in 40% of theobserved samples but has never found to be above 15% of thetotal concentration of ciliates. The ecological exigences ofParastrongylidium pyriformis have been studied.(1) Fleury A. & Fryd-Vcrsavel G. (1984). Protistologica TXX:525-546.

201Cloning and Characterization of Macronuclear H4Histone Genes in Blepharisma japonicumM. SALVINI1 and R. BATISTONI1, IScuola NormaleSuperiore, Pisa; lDipartimento di Fisiologia eBiochimica, Universita di Pisa, Italy

A PCR based approach utilizing degenerated primers has beenused to amplify and clone macronuclear H4 histone genes inBlepharisma japonicum. The design of the two primers wasobtained comparing the published sequences of H4 histonegenes of Tetrahymena'; Oxytricha2, Euplotest in the regioncoding the COOH and NH2 protein termini, with the addi­tion of a partial nucleotide sequence, deduced by a sequencing

460 . Abstracts

of B. japonicum, H4 specific protein". A th ird oligonucle o­tide, based on a region in between the other two, sharing iden­tical nucleotide sequence in all above genera , has been util izedto identify the cloned PCR produ cts, by means of Southernblot hybridization. Two selected clones have been se­quenced. The two sequences, 264 long, show a close similar­ity to the H4 genes of Tetrahym ena and Oxytricha, (64 % and75% respectively). The tw o inserts in B. japonicum differfrom each other by about 9% at nucleotid e level, suggestingthat at least two H4 genes may be present in the macronucleusof this species. Prelim inary results, obta ined from Southernblot hybridizat ions, also indicate that some copies of H4genes colonize the B. [aponicum genome. An analysis ofthe deduced aminoacidic sequence shows that the two H4cloned genes are different in thr ee aminoacidic positions.However, these differences do not involve a very evolution­ary conserved H4 protein region , where two variants, differ­ing for a serine insertion, were previously identified in B.[aponicum, by a direct protein sequencing", Since in Sacchar­omyces cerevisiae the corresponding region of the H4 histoneis retained to have a direct impl ication in the mechanism forsilencing mating type genes", we are further investigating forthe H4 genes organizat ion in B. [apo nicum with the aim ofgain ing an insight into the mechan isms of the mating typedeterminati on in this ciliate.1 Bannon G.A. et al. N.A .R., 12, 196 1, 1984. 2-3 Harp erD.S. and jahn c .i ., GEN E 75, 93, 1989 and P.N. A.S.USA, 86, 3252, 1989.4 Bini E. et , aI., International Congressof Protozoology-Berlin, July 1993. 5 Kaine P.S. et aI., Cell, 55,27 , 1988.

202Cytoplasmic Microtubular Array and MeiosisInduction in Blepharisma japonicumG. SANTANGELO, P. BRUNO and S. N AVAR)Dipartimento di Scienze dell' Ambiente e del Territorio .Via Volta 4, 1-56100 PISA, Italy

The evidence of the universal role of cdc2 kinase and cyclineas matur ation promoting fact ors of both meiosis and mitosisproc esses increased the int erest of protozoologists in the studyof meiosis induction mechanism s in ciliates. In order to exam­ine the role of cytoplasmic microtubular system in the meiosisactivation process, conjugating pairs of Blepharisma [aponi­cum, in activation pha se, were treated with an antitubulinpolymerization agent (Noco dazo le), at low concentrations(1 ug/rnl). The clear-cut delay in the progress of meiosis intreated pairs suggested that nocodazole may reversibly affectthe induction pro cess. To identify specific effects of nocoda­zole on Blepharisma pairs in meiot ic activation phase, thecytoplasmic microtubular array was examined by means ofanti-tubuline indirect immunofl uorescence and electron mi­croscop y. The first technique allowed us to decorate the maincytoplasmic microtubular structures of Blepharisma but , inthese structures, no nocodazole-indu ced dam age wasfound. EM only revealed peculiar differences between thecell union region microtubular arrays of treated and un­tr eated pairs.

203Morphogenetic Study in Platyopbyra spumacola Kahl,1927 (Ciliophora, Colpodida)I C. SANTOS and C. F. BARDELEZoologisches Institut der Universitat Tubingen, Auf derMorgenstelle 28 , D- 72 076 Tiibingen, Germany

Th e morphogenetic process in the colpodid ciliate P. spuma ­cola occurs in free-swimming individuals. Th e dividing cellswere prepared for Protargol impregnation acco rding to Foiss­ner (1991). Standard SEM and Cryo-SEM techn iques werealso applied, sometimes with previous deciliation followingthe Rosenbaum & Carlson (1969) procedure. Th e mo rpho­genesis lasts about 12 hours. First, new kinetosomes areformed within the parent al dikinctids. The oral primordiumoriginates in 9 right lateral soma tic kineties in the posteriorth ird of the cell above the contractic vacuole pore. Th eopisthe's paroral is composed of a single row of paired ci­lia, long parental cilia and new short ones. They came succes­sively from the parental files and aligned themselves in aperpendi cular position. Posteriorly, about 12 ado ral orga­nelles begin to differentiate. Some new soma tic dikenetids ro­tat e and the new short anterio r cilia now lie to the left of thelong parental cilia. Or al reorgani zation in the proter sta rtswith resorption of the anterior kinetosome in the dik inetidsof the paroral and the postoral pseudomembrane. At theadora l organelles the 6 parent al kinetosomes give place to9. Next, the micronucleus begins to divide and the cleavagefurro w starts to develop. Th e opisthe's paroral no w has 2rows of cilia. A new row has proliferated in front of theold one. In the proter the anterior kinetosomes of the paroraland of the postoral pseud omembrane begin to renew. Somekinetosomes at the adoral organelles are absorbed. Conti nu­ously, the ora l primordium of the opisthe rot ate s to the left.The new short cilia of the opisthe's ora l structu res lengthenand the macronucleus starts to divide. In the latter stagesthe proter's oral ciliature is renewed. At th is point theopisthe's oral ciliature is complete and lies parallel to the lon­gitudinal axis of the cell, on the left side of the cell relati ve tothe proter's oral ciliature. It is only after the cell division thatthe opisthe reorganises its somatic dikinetids to build the post­oral pseudomembrane.

204Protists as Sources of Essential (n-3) PolyunsaturatedFatty Acids for Predator DevelopmentJ. R. SARGE/\,'T, M. V. BELL and R. J. H ENDERSONNERC Unit of Aquatic Biochemistry, Department ofBiological and Molecul ar Science s, Uni versity ofStirling, Stirling FK9 4LA , U.K.

Th e membrane phosphoglycerides of visual and neura l tissuesof vertebrate animals are unu sually rich in doco sahexaenoicacid, 22:6(n-3), often present as di-22:6 (n-3) molecular spe­cies. Some vertebrates, e.g. rats, convert the precursor essen­tial fat ty acid linolenic acid, 18:3(n-3), to 22 :6(n-3)sufficiently fast for normal visual and neural development.In mean , particularly during prenatal development, the situa­tion is less certain. In other verte bra tes, e.g. marine fish, theconversion of 18:3(n-3) to 22 :6(n-3) is negligible and a dietaryinta ke of 22:6 (n-3) is essentia l. Mar ine animals including fishare rich in (n-3) polyunsaturat ed fatt y acids (PUFA), espe­cially 22:6(n-3), that or iginate in phototrophic and hetero-

troph ic proti sts at the base of the food web. Among photo­troph ic proti sts, marine diatoms (Bacillariophyta) are richin 20:5(n-3) and C16PUFA but have little 22 :6(n-3) and no18:5(n-3); dinofla gellates (Dinomastigota) and members ofthe Prymnesiophyta , including Isochrysis galbana, Phaeocys­tis poucheti and coccolith ophore Emiliania buxle yi, are richin 22:6(n-3) and 18:5 (n-3) but have relatively little 20:5 (n-3)and no C16PUFA. Heterot rophi c l abyrinthulomycota suchas Scbizochy trium are also rich in 22:6(n-3) but have no18:5(n-3) or C16PUFA. All these prot ists biosynthesise22:6(n-3) de novo although it is possible they also convert18:3(n-3) ingested in Cyanobacteria to 22:6(n-3). Marinephototrophic protists rich in 22:6(n-3) are flagellated andphototactic. Their 18:5(n-3) is concentrated in thylakoid gly­colipids whereas their 22:6(n-3) is concentrated in extra thy­lakoid di-22:6(n-3) phosphatidylcholine. Such findings arediscussed in relation to the evolution of (n-3) PUFA biosynth ­esis and (n-3) PUFAfunctions in protists, and the roles of (n-3)PUFA in vertebrate developm ent.

205To xicity Assessment of Xenobiotics by Bio-assaysPerformed with Protozoa and Mammalian Cell LinesM. P. SAUVANT(*), D. PEPIN(*), C. A. GROLIERE( ~- * ) and].BOHATIER( **)(*)UFR Pharmacie, Lab. H ydrologie & H ygiene, BP38 ,63001 Clermont-Ferrand, France. (**) URA CNRS1944, Lab. Biologie Comparee des Protistes, ComplexeScientifique des Cezeaux, 63 177 Aubiere, France

In toxicology and ecotoxicology, simple handling assays andsensitive models are required for the development of alterna­tive methods. Various models, such as algae, bacteria , protist,crustacea, fish, have been mentioned; established cell lines arealso frequently used for the determination of the acute toxi­city of air, soil and water pollut ants. Moreover, few studieshave compared the performation of these models to othe rsand to in vivo data.After a brief restatement of the main and recent works per­formed with the most commonly used protozoan Tetrahyme­na pyriformis Gl, the discussion is focused on the potential ofprot ists as toxicological too l compared to the potential ofmammalian cell lines, applied for the toxilogical stud y of xe­nobiot ics and for the ability of both models used as predictivemodel for health risk assessment.A specific example is provided with the comparison of theperformances of Tetrahymena pyriformis GL model and ofthe murin e L-929 established cell line for the toxicologicalscreening study of organic and inorganic xenobiotics. Goodcorrelations are observed between the results obtained withboth models. Moreover, Tetrahym ena pyriformis Gl is moresensitive to organ ic substances than l-929 cells, and l -929cells are more sensitive to inorganic substa nces than the pro­tozoa model. Both models, included in a battery of assays,complemented one another in screening study. The relativeefficiency and the limitat ion of both models are related totheir specific behaviours and to the differences of experimen­tal protocols performed. Well-defined exper imental condi­tions and standardized assays are required, specially forTetrahymena pyriformis GL assays.

Abstracts . 461

206Cytotoxicity and Intracellular Distribution ofAluminium in Tetrahymena pyriformis GLM . P. SAUVANT( *), D. PEPIN(*),]. BOHATIER( **) andC. A. GROLIERE( *~.)(*)UFR Pharmacie, Lab. H ydrologie & H ygiene , BP38,63 001 Clermont-Ferrand, France. (**) URA CNRS1944, Lab. Biologie Comparee des Protistes, ComplexeScientifique des Cezeaux, 63 177 Aubiere, France

The potential tox icity of aluminium (AI) was firstly men­tioned about 1970, when substantial accumulation of Al inhuman brain cells has been established in patients with hac­modialysis encephalopath y or more recently, suggested withAlzheimer disease. To date, attention is focused on Al indrinking water and residues, which could be generated dur­ing water purification with AI coagulants salts.The aim of this study is to investigate the cytotoxicity and theintracellular a distribution of 4 AI-salts (chloride, sulphate,nitrate and oxide ), with out and with 3 chelators (humicacids, citric acid and EDTA), to the ciliated protozoan Tetra­hym ena pyriformis GL. The IC50 toxicological index is cal­culated according to the Generation Time Assay.Addition of Al to culture medium affected Tetrahym ena pyr­iformis GL growth rate. Mo rphological abnormalites are ob­served with 2-3 hours and are connected to the phagocytosisof AI-particles. Tetrahymena pyriform is Gl is very sensitiveto the 3 soluble AI-salts (chloride, nitrate, sulphate) (IC50about 10 ug/ml] and less sensitive to AI-oxide (IC50 = 495ug/ml). Moreover, Tetrahymena pyriformis Gl concentra tedrapidly insoluble AI-parti cles.The cytotoxiciry of AI is modified by the presence of chela­to rs: increased with humic acids and decreased with criticacid and EDTA. The quantification of Al in Tetrahymena pyr­iformis Gl and in the culture medium by ICP allowed the AI­bioavailabilit y to be investigated. The increase of AI intracel­lular amount is significan tly correlated with the increase ofthe toxicity of Al administered currently with chelato rs.

207Analysis of SSU rRNA of Myxidium lieberkuehniBrings Evidence that Myxozoa are Metazoa Relatedwith BilateriaM. SCHLEGELl,]. LOM2, A. STECHMANN3, D. Bernhard",D. LEIPE\ 1. DYKOvA2 and M.SOGIN4

' Zoologisches Institut, Un iversitat Leipzig, 04103Leipzig, FRG; 2Institute of Parasitology ASCR, 37005Ceske Bude jovice , Czech Republic; 3ZoologischesInst itut, Uni versit at Tubingen , FRG; "Center ofM olecular Evolution , M arine Biological Laboratory,Wo od s Hole, Mass. 02 54 3, USA

Sporogo nic plasmodia of Myxidium lieberku ehni (Myxo ­sporea) were collected from the urinary bladder of pike(Esox lucius). The DNA isolated from trophozoites that werepooled from different host individuals was used for ampl ifi­cation and sequencing of the 16S-1ike rRNA coding region.Standard amplification techniques were used which yieldedDNA sequences of 2084 and 2085 nucleotides, respec­tively. They were found to have a GtC contents of 47%. Phy­logenetic comparisons of the two Myxidium sequences werecarried out, using the neighbour-joining, parsimony and max-

462 . Abstracts

imum likelihood methods. They consistently grouped withthe bilaterian metazoans (bootstrap value 100%) and less sig­nificant therein with the nematodes (60% bootstrap). Thusthe phylogenetic analysis of Myxidium 16S-like rDNA indi­cates that myxozoans have their origin within the metazoansand are closely related with the bilaterians. They are neitherrelated to the cnidarians nor to the alveolate protists. Sincemyxozoans seem to have appeared in metazoan phylogenylater than cnidarians, they probably must have undergoneconsiderable reductions during their evolution. At present,our data do not allow to say whether myxozoa are the sistergroup to all bilaterians or whether they branched off laterwith the nematodes.The two DNA sequences obtained differ in 51 sites, or 2%,which may indicate the presence of two morphologically in­distiguishable cryptic species, or that Myxidium consists ofseveral genetically independent lineages which accumulatednumerous point mutations in the SSU rRNA genes.

208Redescription of Hemistasia phaeocysticola comb.nov., a Member of the BodoninaE. SCHNEPF!, M. ELBRACHTER2, A. MARTIN3 andI. BALZER3

ILehrstuhl fur Zellenlehre der Universitat Heidelberg,2Biologische Anstalt Helgoland, WattenmeerstationList/Sylt, 31. Zoologisches Institut, UniversitatG6ttingen, Berliner Str. 28, 37073 G6ttingen, Germany

A new member of the group of Bodonina has been isolatedfrom water of the North Sea, at ListiSylt, Germany. The body­shape of this opportunistic parasite is metabolically variable,mostly pyriform to cylindrical. From a subapical flagellarpocket, two large flagella arise, one of them being a trailingflagellum supporting the cell during feeding. Mastigonemesare missing, but paraxial bundles at the basis of the flagellaare present. The oral apparatus is reinforced by tubulin rodsand, after feeding, a protuberant food vacuole appears in theposterior part of the cell. Further characteristics of the speciesare the presence of a kinetoplast and a peripheral lacuna. Thenucleus is located in the anterior part of the cell and containscondensed chromatin. Numerous trichocysts are concen­trated to a peripheral lateral battery. Cell division in Hemista­sia phaeocysticloa is preceded by an attachment of the cellapex to the substratum and by subsequent rapid clockwisemovements followed by the retraction of the flagella and as­suming of a spheroidal, cyst-like shape. About one hour later,two daughter cells are formed, one of them carrying the foodvacuole. The principal source of its alimentation seems to becytoplasm of decomposing cells, although it is also observedto attack living diatoms (Coscinodiscus walesii) and dinofla­gellates (Gonyaulax polyedra) in culture. Optimal growth hasbeen observed at low temperatures and light intensities, con­ditions perhaps resembling the natural habitat of this organ­Ism.

209Length and Restriction Site Polymorphisms Detected inthe Ribosomal DNA Internal Transcribed SpacerRegions of Different Leishmania SpeciesG. SCHONIAN, C. SCHWEYNOCH, K. ZLATEVA, F. DEUTSCH,L. OSKAM and W. PRESBERInstitute of Microbiology and Hygiene, Charite,Humboldt-University Berlin, Germany

The internal transcribed spacer (ITS) lying between the nucle­ar ssu and isu sequences and flanking the 5.8S gene was foundto be highly variable in many bacteria, fungi and protozoa.The ITS regions of L. donovani complex, L. major, L. tropi­ca, L. mexicana, L. amazonensis, and L. braziliensis complexamplified by PCR differed in size. Restriction site polymorph­isms could be detected by digesting these amplified ITS re­gions with different restriction enzymes. Specific RFLPpatterns were obtained for all Leishmania species tested.The patterns of the members of L. donovani complex(L. donouani, L. infantum, L. chagasi) as well as of the mem­bers of the L. braziliensis complex (L. brasiliensis, L. guya­nensis, L. panamensis) were, however, identical. Lengthpolymorphisms could be also shown in the ITS region of var­ious L. tropica strains from different geographical locations.By amplifying this DNA region two fragments of different sizewere obtained in some L. tropica strains whereas other strainshad only the upper or only the lower PCR band. This differ­entiation of L. tropica strains was supported by the restrictionanalysis of the amplified ITS regions. The reason for the highvariability of the internal transcribed spacer region within thespecies L. tropica remains to be established.This PCR method may prove useful for the species identifica­tion in Leishmania isolates as well as for phylogenetic andtaxonomic studies within this genus.

210New Trends in Chemotherapy on Human and AnimalBlood ParasitesJ. SCHREVEL, V. MILLERIOUX, V. SINOU, F. FRAPPIER,R. SANTUS and P. GRELLIERMuseum National d'Histoire Naturelle, 75231 ParisCedex

The tropical or infections diseases, due to parasitic protozoa,represent one of the major cause of mortality in the world.The absence of efficient vaccines against malaria and the ab­sence or the toxicity of drugs for the African and Americantrypanosomiasis, the emergence of chemoresistance enlight­en the necessity to propose new antiparasitic strategies basedon the identification of molecular targets which are eitherspecific or sufficiently different from the host molecules inorder to synthesize new structural analogs.The development of intraerythrocytic stages of Plasmodiumfalciparum needs exo and endogenous lipids to synthesize thenumerous membranes during the schizogony and to differ­entiate merozoites. Inhibitors of the HMG-CoA reductaseand inhibitors of the isoprenoid metabolism are efficientinhibitors in vitro. Similar results are obtained in vitro withBabesia divergens.Among antimicrotubular drugs, the taxol derivatives are effi­cient inhibitors of the in vitro development of P. [alciparum,B. divergens as well as epimastigote forms of Trypanosomacruzi. Taxotere is 4 to 10 times more active than taxol and

the ICso agai nst these parasites are lower than th ose requ iredfor cancer therapy. As pulses of few hours are able to kill P.[alciparum and due to the goo d partitio n of Taxotere in ery­th rocytes and in high density lipoprot eins, the taxoid deriva­tives sho uld represent pot ent ial antiparasitic dru gs.Th e targetting of drugs to intraerythrocytic stages of P. falci­parum could be realized by high density lipoproteins loadedwith new photosensitizers, for example pheophorbide deriva­tives. After the photodynam ic reac tio n, these probes are ableto era dica te the P. [alciparum and B. diverge ns infected redblood cells (RBC) without hemolysis or appa rent modi fica­tions of the healthy RBC. Th is stra tegy could open newtre nds for producing blood fractions safe for tran sfusion.

211Gamma Tubulin: the Gene, Protein and SubcellularLocalisation in Trypanosoma brucei and other ProtistsV. SCOIT and T. SHERWINSchool of Biological Science s, University ofM anchester, Manchester, M1 39PT, United Kingdom

Gamma tubulin, the most recent ly discove red mem ber of thetu bul in super-family, has since been shown to be present inmany euka ryotic systems from fungi to hum ans. Its locat ionat the microtubule orga nising cen tres of these organ isms hasled to a prop osal th at it plays some role in microtubule nu­cleation .Th e cytoskeleton of T. brucei is compose d of four distinctmicrotubule networks: the flagellar axoneme, the basal-b odycom plex, the sub-pellicular array and the intranuclear mioticspindle. The axoneme is subtended from a typical basal body,however no organ ising centre has yet been found for the sub­pellicular or mitotic microtubul es.Using degenerate aligonucleotide pri mers we have isolated agenomic clone from T. brucei which contains a full-lengthg~mma tubulin gene. Th is has been sequenced and is pre ­dicted to encode a protein of 447 amino acids which exhibitsthe highest degree of hom ology with gamma tubulins fromhuman and Xenopus laevis (67.2% amino acid identity)and only 57.7% identity with P. [alciparum gamma tubu­lin. N~rthern blot anal ysis detects a major transcript ofapproximately 2.2Kb, plus a larger minor transcript (ap ­~roximately 3.6Kb), in poly A+-selected RNA from a procyc­he culture. PCR analysis of first-stand eDNA from differentlife cycle stages suggests the gene is tr anscribed in procyclic,slender and stumpy life forms of th is par asite.A fusion protein comprising the almos t full-length gammatubulin gene product (amino acids 8- 447) plus an amino­terminal histidine tag has been expressed and puri fied fromE. coli, and used to raise polyclon al and monoclon al ant ibo­dies. Th ese ant ibodies have been used to define the intracel­lular locat ion of gamma tubulin in T. brucei and otherproti sts.

Abstr act s . 463

212Candida-Host InteractionsJ.-M. SENET and R. ROBERTFac ulte de Pharmacie, 16 bd Da viers 49100 Angers,Fra nce

Candida albicans and a few other species are usuall y enco un­tered as commensa ls on epithelial surfa ces of humans andanima ls. Sett lement of such micro -organi sms is relat ed tomore or less specific ad herence mechan isms. Th e transitionfro m a commensa lism to a candida I infection involves mod­ificatio ns of both the host and the fungus. Principally we aredea ling with the impai rment of imm une defenses togethe rwith inflammatory reactions balanced by fungal phenotypicmod ifications such as filarnent ati on which in turn increasesthe capacities of adhesion and protease secretion. Wh ateverthese modifications are, host-par asite relationships imply amulti-step colonization procedure.Fixation of the fungus to epithelium seems largely med iatedby lectin-like mannoproteins, which are located on the fungalcell wall and interact with fucose or N-acetyl-glucosamin re­sidues of epithelial surfaces . Salivary components and mucinswere demonstrated to intervene in the se interact ions forwhich electros ta tic and hydrophobic forces are involved. Dis­tinctly, fixa tion of fungal cells to endo thelium seems to be dueto protein-protein interactions. Complement receptor-likemolecules (CR3, CR2) were described on the surface of theouter layer of the cell wa ll and mediate fixation to endo the­li~ 1 c.ells and al~o.to the ext ra-cellular matrix. More preciselybinding t? laminin, collagen, fibronectin, entactin, has beencha rac ten zed and funga l ligands were identified. A, socalled, multifunctional adhesio n of Candida albican s(~FA-C) is responsible for the fixa tion of the yeas t (mainlyfilamentous forms) to fibrinogen, lam inin , C3d and also toplastic surfa ces. Th is ad hesin is co mpose d of three main com­ponents of > 200,68 and 60 kDa and reacts as a tru e receptorwith a dissociat ion consta nt of 0.5 nM .Injected t~rough the caudal vein in mice, yeasts are qu icklycoated With platelets thus forming big aggregates lead ingto rapid elimination from the blood stream. In vitro studiesshowed that resting platelets can link, through the integringp Ilb IlIa, to a 45 kDa mannoprot ein belonging to the fungalsurface. The fixation lead s to activating platelets which thentight en the linkage by extern alizing the thrornbospondin; th isa,dhesin is involved in the direct binding of thrombin pre-ac­tiva ted platelets to the fungus. Within plasma, fibrinogen andcomplement participate in the platel et fixation to Candidacells. Alto.gether the se interact ions are likely to play an impor­tant role m the pathogenic processes of candidias is.

213Ultrastructural Organization of the Resting Cysts ofBursaria ovata Beers 1952G. SERGEJEVA and A. SAMOSI-IKINLab?ratory of Cytology of Unic ellul ar Organisms,Institute of Cytol ogy of Acad emy of Science of Russia,St . Petersburg, 194064

Previously, the restin g cysts of Bursaria ovata Beers 1952 werestudied by light micr oscopy only, and the lack of the"bridges" connecting the ecto - and endocyst layers of the cyst

464 . Abstracts

wall was described as the attribute of this species. Bur our EMstudy showed the presence of " bridges" in the cyst wall of B.ova ta . Th is discrepancy between light and EM data can beexplained by the very peculiar organization of the meso­cyst. The mesocyst of B. ovata represents a very intricateclosed system of numerous spreading and bending mem­brane-like structures of different thickness and specific ar­rangement. The "bridges", represented by dense stacks ofthe endocyst membrane-like structures, go through the meso­cyst space to the ectocyst layer of the cyst wall and so, at thelevel of the light microscope, can be masked by the mesocyststructures. Therefore, the "bridges" cannot be a species char­acteristic, as they are present in both Bursaria.The cytoplasm of the B. ova ta cyst shows many featur es in theEM, similar to B. truncatella (Sergejeva et al., 1993): thefolded cell surface , preservat ion of subpellicular flat al­veoles, epiplasm, kinetosomes and clustering of mitochon­dria in the endoplasm, a multitude of autophagic andsecretion vacuoles, numerous ribosomes and polyribo­somes, tightly arranged; the ultr astructure of mitochondriais significantly altered in comparison with that of activecells. Macronuclear (Ma) organization is altered in restingcells as compared to active cells of B. ouata: the nuclear mem­brane becomes folded and a large number of small chro matinbodies develop regular outlines, suggesting liquid crystal andcrystal orga nization in the Ma of B. ova ta as demonstrated bychromatin spreading (Sergejeva & Bob yleva, 1988), freeze­fracture and thin sections (Livolant et al., 1993) in B. trunca­tella. The chromatin bodies of the Ma of B. ovata do not formcentres of condensation and the nucleoli do not fuse in largestruc tures as observed in B. truncatella. The specific feature ofthe resting cyst of B. ova ta is the presence of numerous peri­nucleolar bodies. The micronuclei of both species have a si­milar ultrastructure in cysts. Th e data will be discussed interms of phylogenesis, taxonomy and ecology of Bu rsaria.Supported by Russian Basic Research Foundation (Pr. 93­04-21803).

214Biology of Bursaria truncatella O.EM. and B. ovataBeers 1952 under Laboratory Culture ConditionsN. BOLsHAKovA, E. LANINA and G. SERGEJEVALaboratory of Cytology of Unic ellular Organisms,Institute of Catology of Acad emy of Science of Ru ssia,St . Petersburg, 194064

Recently, most data accumulated dur ing more than 200 yearsof Bursaria research history have been summarized in themonograph of W. Foissner (1993). But many questions ofbiology and taxonomy of Bursaria rest to be elucidated. Sinceciliates of the genus Bursaria generally occur in nature dur ingshort periods of spring and autum n, many questions of theirbiology can be elucidated by using of laboratory cultures.From 1970 cultures are maint ained in the Laboratory of Cy­tology of Unicellular Organisms, originated from more thantwenty natural populations of Bursaria, collected in differentseasons and in different regions of the former Soviet Union. Inlaboratory condit ions the life time of stocks and strains variedfrom some days to six years for B. truncatella and two yearsfor B. ova ta . After lengthy cultivation of both species, we con­cluded that B. ouata is not the "large form" or season "race"of B. truncatella, but represents a real distinct species of thegenus Bursaria. Our arguments are follow: We never observedmorphological transformat ion of the one species on the other

and vice versa; every species preserves specific behaviour atdifferent stages of the life cycle, independently of the stockorigin and culture conditions. The species clearly differ byrelation to temperature of cultivatio n and food preference.The conditions leading to conjugation of B. trun catella donot provoke the sexual process of B. ova ta . The laboratoryconditions stimulating B. ova ta to conjugate are uncertain,but the morphology of the young exconjugants differs fromthat of B. truncatella. The fine structure of B. ova ta restingcysts differ from that of B. truncatella (Sergejeva and Samosh­kin, see this volume); the voluminous mucous coa t, seen inyoung cysts of B. ova ta, is a temporary structure, disapp ear­ing in aged cysts. B. ova ta differs from B. trun catella by moredeveloped anterior and posterior transversal fibrils of somatickinetosomes. By modified Laemmli's method of electroph or­esis qualitative and quant itative differences in patterns of pro­toplasmic soluble proteins in the resting cysts of the same agebetween B. truncatella and B. ovata were revealed. The datawill be discussed in terms of phylogenesis of Bursaria speciesand problems of using these protists in basic and applied biol­ogy. Supported by Russian Basic Research Foundation (Pro­ject: 94-04-21803).

215Cholesterol-Lecithin Liposomcs and pH of MediumModulates the Cytoplasmic Streaming in ParameciumbursariaJ. SIKORA, H. FABCZAK and S. FABCZAKDepartment of Cell Biolo gy, Nencki Institute ofExperimental Biology, 02-093 Warszawa, Poland

Cyto plasmic streaming velocity (csv) in Param ecium bu rsariadepends on the concentration of solid particle s in the mediumin dose dependent manner, while there is evidence that not onthe food vacuole formation rate (fvfr). In order to elucidatewhether cell membrane is involved in modulation of cytoplas­mic streaming, effect of cholestero l-lecithin liposomes and pHchange of medium was investigated. Incubation of par ameciain medium containing cholesterol-lecithin liposome s mark­edly changes the fvfr and vcs. Incorporation of foreign phos­pholipids into plasma membr ane increase fvfr reaching 2-foldvalue after 2 h, while csv remain decreased during first 2 h. Inthe range of pH between 6- 8 the increase of csv take place.However the nature of the relat ion between the surface of thecell and the mechani sm cont rolling the velocity of streamingremain obscure, these findings confirm supposition that plas­ma membrane in P. bursaria may play an essential role in re­ception and transdu ction of stimuli which affects thepropulsion and/or cont rol the mechanism of intracellularflow.

216Stru ctural and Ultrastructural Obs ervations of CiliateUrceolaria sp. (Urceolariidae-Peritrichia), Epib iont ofthe Starfi shI. D. da SILVA NETOLab. de Protistologia, Dep. de Zoologia, Instituto deBiologia/Setor de Microscopia Electronica do Institutode Microbiologia, Universidade Federal do Rio deJaneiro, Rio de Janeiro-Bresil

With a disc-like shape, Urceolaria sp, (length: 55 um-d iarn: 65urn) was found fixed on the Endoplopatiria stellifera sta rfishin Baia de Guana bara (Rio de Janeiro). In the front part, buc­cal cilia tu re, an approximately 400 ° spira l, is formed from astichodyade paroral and forms a three par allel-row ciliatedkinetosomes polykinety. Th e right paroral kinetosomes havelined postciliary microtu bules and the left kinetosomes have aposteriorly directed dense fiber. Onl y the external polykinetykinetosomes have postciliary fiber s, and the kinetosornes arelinked together by fibrillar structures. In the posterio r part theconcave ad hesive disc has three ciliary wreaths, separated byectoplasmic ridges. The interna l wrea th - also the widest - iscomposed of severa l ten-kinetosomes rows . From each one ofthem, a per iodic kinetodesmal fiber and fibrillar networks gotoward the straight and the curve d dent icles, gathered by fi­brillar structures of the skeletal ring. In the front wrea th, thekinetosomes are lined in pa irs in a zig-zag formation. Th ekinetosomes of the most externa l wreath are lined with nospecific arrangement . The micronucleus is located in a depres­sion of the macronucleus extremity. The cytoplasm is rich inmitochondria with tubular cristae, in dense vesicles and indigestive vacuoles. CNP q.300256/93-0

217Evidences for the Existence of a Functional MicrobialFood Web within Arctic Sea-ice BiotaT. SIME-Nga ndo and C. AMBLARDCNRS-URA 1944 , Biologie Comparee des Protistes,Universite Blaise Pascal, 631 77 Aubiere Cedex, France

Sea-ice annually covers about 14 x 10 6 km2 in the northernhemisphere and 20 x 10 6 km 2 in the southe rn hemisphere. Itis now recognized that aqueous interstices within sea-ice de­velop a diverse, abundant, dynamic, and highly productivemicro bial com munities. However, biologists have never hadthe opportuni ty to investigat e the comm unity character isticsof heterot rop hic proti sts in the arc tic sea-ice. As a first step inevaluat ing an hypoth esis on the existence of a functio nal mi­cro bial food web in the arc tic sea-ice, phagotrophic pro tistswere exam ined for evidence of their response to the springaccumulat ion of their potential prey (bacteria and algae),at the northern (Resolute Passage, Canadian Arct ic Archipe­lago, 74 "N , 95 OW) and southern (Saroma-Ko Lagoon, Hok­kaido, Japan, 44 "N, 144 °E) limits of the ann ual sea-ice in theArct ic. Most of the orga nisms were conce ntr ated in the ice­wa ter interface whe re ab unda nces were several orders of mag­nitud e higher than in the plankto n. At Resolute Passage, pro­tozoan communities were dominated by sapro pelic-typespecies which were active and appare ntly endemic to theice-biota. This contrasts with observa tio ns at the southernlimit of sea-ice in the Arctic where ice seems to serve as atemporary habitat for planktonic communities, similar to ob­serva tions from Antarc tic. Th e seasonal increase in nanopro-

Abstracts . 465

tozoa and microp rotozoa succeeded to the biomassaccum ulation of their potent ial prey (i.e. bacteria and al­gae, respectively), implying the existe nce of a functional mi­crobial food web within arctic sea-ice biota. Heterotrophicnanoflagellate biomass greatly exceeded (30 - 60 x ) bacterialbiomass. Coupled with similar potential growth rates, thissuggests the utiliz at ion of addition al (non-bacter ial) fooditems by ice-brine flagellat es. Above findings indicat e thatprotozoan may rep resent one of the basic levels of the icefood chain, partly because of the known limitat ion of bacter­ial production in polar conditions.

218The Imprint of M icrozooplankton Communities on theEcology of the Lower St. Lawrence Estuary, Quebec(Can ada)T. SIME-NGANDOCNRS-URA 1944, " Biologie Comp aree des Protistes",Universite Blaise Pascal, 631 77 Aubiere Cedex, France

Th e Lower St. Lawrence Estuary (LSLE, 48 °60'N, 68 °20'W)is characterized by high nutr ient supp ly and high standingstocks of large-size metazoan zooplankton (copepods, eu­phausiids, and fish larvae) which imp ly an efficient trophictransfer from the lower to the higher trophic levels. In con­trast, the annual pri mary production in the LSLE is lowerthan those of similar estuar ies, partl y due to the large flushingrates and strong stra tification duri ng the spring freshet whichare thought to delay the phytoplankton bloom unti l earlysummer, for a short-t ime period (i.e. June-July). Further­more, the phytoplankton bloom in the LSLE is made up ofsmall size diato ms, likely and inadequate prey source forthe dominant large metazoans. As a first step in evalua tinga hypoth esis on the major ro le of microzooplank ton astrophic link in the LSLE, ciliate biomass (CilC) and microzoo­plankton grazing activit y were examined fro m May toSeptember 1992, in relat ion to bacterial (BactC), phytoplank­ton (PhytoC) , and particulate organic (POC) car bon bio­masses. Findings were follows: (1) CilC averaged 22, 8,and 4 % of BactC, PhytoC, and POC, respectively. (2) Com­pared to bacteria, ciliates were able to respond more rapidlyto an increase in phytoplankton biomass, and were underhigher predation pressure. (3) Ciliate community was domi­nated by individuals with sizes of up to 15 times larger thanthat of the blooming diatoms. (4) Direct observations re­vealed pro tozoan ind ividuals with up to 10 blooming algalcells in their body wa ll. (5) Fina lly, microzooplankton assem­blages (at the depth of chloro phyll max imum in Jul y) graze d,on average, 54 ± 21% of algal production, a value which canbe enhanced by up to 60 % in the abse nce of metazoan zoo­plankto n. Above findings suggest that herbivory by microzoo­plan kton can enhance trophi c transfer from the microbi alfood web to higher trophic levels in the LSLE, and may ex­plain the apparent paradox of low annual algal productionand large standing stock of metazoan zooplankton in this en­viron ment.

466 . Abstracts

219Analysis of rRNA Gene Sequences shows a CommonFlagellate 'Genus' to be PolyphyleticM. A. SLEIGH, J. RICE, S. M. TONG, C. D. O'CONNOR,I. G. GILES and P. H. BURKILL*School of Biological Sciences, University ofSouthampton, Southampton S016 7PX, and"Plymouth Marine Laboratory, Prospect Place, WestHoe, Plymouth, PLl 3DH, U.K.

Colourless chrysophytes that bear silica scales are placed inthe genus Paraphysomonas; they are very common marineand freshwater heterotrophic flagellates. Four species of Para­physomonas have been isolated from the estuarine Southamp­ton Water, cultured and identified from scale ultrastructure.Comparison of partial nucleotide sequence of their 18SrDNAs indicates that three smaller species, P. butcheri, P. for­aminifera and P. imperforate are closely related to one an­other, while the larger P. vestita is only distantly related tothem. Phylogenetic comparison of almost complete 18SrDNA sequences of P. vestita and P. foraminifera with se­quences from the EMBL and GenBank databases places P.foraminifera near the green scaly synurophytes, while P. ves­tita is well separated from these and, among the small numberof chrysophytes yet sequenced, forms the oldest branch. Para­physomonas is shown by this analysis to be polyphyletic.Since P. vestita is the type species of this genus, the smallerspecies listed above must be placed in a new genus when ul­trastructural data is available to support generic diagnosesthat distinguish Parapbysornonas from the new genus.

220Trypanothione MetabolismK. SMITH and A. H. FAIRLAMBDepartment of Medical Parasitology, London School ofHygiene and Tropical Medicine, UK, WC1E 7HT

Trypanothione, Nl, N8-bis(glutathionyl)spermidine, is un­iquely found in trypanosomatids. A related glutathione-poly­amine analogue Nl, N9-bistglutathionyllaminopropyl­cadaverine has also been detected in Trypanosoma cruzi epi­mastigotes. Trypanothione and its biosynthetic precursor,monoglutathionylspermidine, are maintained in the reducedstate by the NADPH dependent enzyme, trypanothione re­ductase (TR). TR and the isofunctional mammalian glu­tathione reductase· are structurally and mechanisticallysimilar. The critical difference, however, is that these enzymeshave mutually exclusive substrate specificities. Currently, TRis our principal target for the design of new trypanocidaldrugs.As a polyamine-containing analogue of glutathione, trypa­nothione has subsumed many of the protective roles of thistripeptide including; maintenance of thiol-redox, defenceagainst oxidative stress and heavy metals. Moreover, a trypa­nothione-S-transferase exhibiting peroxidase activity hasbeen purified from Crithidia fasciculata. Although it is notclear why trypanosomatids make trypanothione, understand­ing why may provide the best chemotherapeutic target of all.

221Changes in Leishmania major and L. turanicaComposition in Zoonotic Cutaneous LeishmaniasisFoci with Different Endemicity of TurkmenistanM. V. STRELKOVA", L. N. ELISEEV*, E. N. PONIROVSKY'~",

P. I. EROKHIN'~'~ and D. A. EVANS*'~*

*Martsinovsky Institute of Medical Parasitology andTropical Medicine, Moscow 119830, Russia,*'~Institute of Zoology, Ashkhabat 744000,Turkmenistan, **"London School Hygiene andTropical Medicine, London WC1E 7HT

285 Leishmania isolates from Rhombomys opimus obtainedfor 1992-94 in Turkmenia ZCL foci with different endemi­city: hyper-, meso-, hypoendemic. Using polyacrylamide gelelectrophoresis in 8 enzymes: PGI, PGM, 6PGD, ME,MDH, G6PGD, ALAT, 56 isolates were identified as L. ma­jor, 217 as L. turanica and 12 isolates as mixture of L. ma­jor + L. turanica. L. major is pathogenic for humanswhereas L. turanica is not pathogenic. Two types of naturalfoci with typical seasonality of R. opimus infection by differ­ent Leishmania can be distinguished. The first type, L. tura­nica predominate throughout the year and for several years,whereas L. major circulate at very low density (1-5%). Hu­man ZCL in such foci is registered occasionally (hypoendemicactivity). The second type, the proportion of L. major in hostpopulation increases during the transmission season (May­September) whereas L. turanica predominate during the restof the year. The infection rate of L. major may vary in Sep­tember from 20% to 100% and decreased by spring to 1-5%and less. Such features of epizootic process are typical for bothmeso- and hyperendemic foci. Differences between these fociare associated with regularity of L. major accumulation inR. opimus up to 50% and more by the end of the transmis­sion period. In hyperendemic foci such a situation is regularlyobserved each year; in mesoendemic foci, L. major accumula­tion up to 50% can observed for several years, but in certainyears it is not registered.

222Frenkelia spp. in the Populations of their Final andIntermediate HostsM. SVOBODovA, J. VOTYPKA and P. VORgEK'~Dept. Parasitol., "Dept. Zool., Charles University,Faculty of Science, Vinicna 7, 12844 Prague, CzechRepublic

Frenkelia spp. (Apicomplexa: Sarcocystidae) are dixenouscoccidian parasites of buzzard (Buteo buteo) and its rodentprey. Environmentally resistant, infective oocysts are ex­creted in buzzards' faeces (final host), while large tissue cystsdevelop in the brain of rodents (intermediate hosts). The pre­valence of F. glareoli and F. microti was studied in South Mor­avia, Czech Rep., in 1993-94. Buzzard faecal samples werecollected on nests with youngs, rodents (Microtus arvalis,Apodemus spp., Clethrionomys glareolus) were snap­trapped. We found sign. difference of Frenkelia sp. sporo­cysts prevalence in faecal samples from nests in 1993(57%) and 1994 (90%), which was due to different diet ofbuzzards (M. arvalis 24% and 52%, resp.). Diet compositionalso influences the prevalence in the final host during one sea­son: in 1993 the diet of early broods differed significantlyfrom that of late ones, and the prevalence in these 2 groups

was 72% and 41 %, resp.; however, no such differences werefound in 1994. The prevalence increases with age of nestlings,75% of faecal samples being positive before fledging. Theprevalence of F. microti in M. arvalis collected on nestsand snap-trapped differed significantly (8.5% and 2.3%,resp.), suggesting the increased susceptibility to predationis the mechani sm for effective maintenance of the parasitein host populations.

223Agglutination of Leishmania Promastigotes by MidgutLectins of Phlebotomine SandfliesM. SVOBODovA, L. PALANovA and P. VOLF

Department of Parasitology, Charles University,Faculty of Science, Vinicna 7, 12844 Prague 2, CzechRepublic

Lectin activities present in the gut of females of various sand­fly species agglutinated Leishmania parasites. Significant dif­ferences were found between agglutination of Leishmaniaproma stigotes of various species and strains. In general, high­er titres were observed in natural para site-vector comb ina­tions, e.g. in P. argent ipes and Le. donovani. Phlebotomushalepensis and Lutzom yia longipalpis gut extracts exhibitedthe highest agglutination titres, while Lu. carmelinoi the low­est. Lectin activity depended on sex and physiological state ofthe fly.High activities were found in sandfly females only.Theactivity was increased in females which took blood and themagnitude of lectin response to blood feeding differed in spe­cies-specific manner. This could be an important factor of in­teraction specificity between Leishmania and its vector.Antibodies directed against lect in activity of Lu. longipalpiswere prepared in rabbits and used for lectin localizati on onsections embedded in LR White; in unfed females the lectinwas present on microvilli and epithelial surface. Midgutlectin of Lu. longipalp is was isolated using HPLC and char­acterized using electrophoretic and immunochemical meth­ods. It consisted of 60- 62 kDa subunits which reactedspecifically with rabbit antib odies in immunoblotting.

224Iron Acquisition by Tritrichomonas foetusJ. TACHEZYa, P. SUCHANa, J. KULDAa and J. SCHREVELbaDepart ment of Parasitolog y, Faculty of Science,Cha rles University, 12844 Prague, Czech Republic;bLa boratoire de Biologie Parasitaire et ChimiotherapieURA CNRS, Paris, France

Acquisition of iron from lactoferr in (Lf) and transferrin (Tf)by the para sitic protozoan Tritr ichom onas foetus has beenstudied in vitro. Specific, time dependent and saturable bind­ing of iodinated ligands to the outer membrane of T. foetus at4 °C was only demon strated for I25I-Lf. About 1.7 x 105binding sites of a single class with Kd ~ 3.6 11M was esti­mated by means of Scatchard analysis. Binding of Lf wasnot inhibited by an excess of Lf glycopeptides suggesting thatthe Lf-interactions with the binding site were independent 0

the sugar moieties. Internalizat ion of the bound Lf was ob­served at 37 °C. The cell-associated radioactivity after 30min. incubation of the parasite with 125I-Lf at 37 °C was

Abstracts . 467

about 3.5-fold higher than the amount bound at 4 "C. In con­trast to Lf, binding of 125I-Tf was non specific and did notdisplay saturable kinerics.The ability of the cells to remove and accumulate iron from Lfand Tf was examined by using the 59Fe-saturated proteins.The iron uptake 59Fe-Lf by incubatin g T. foetus 60 min at37 °C showed the classical satu rable curve with rapid slopein the 15 first min (380 pmol Fe per mg protein) and a secondslope giving a value 495 pmol. In contrast, 59Fe-Tf showed aregular uptake with 80, 180 and 577 pmol Fe per mg proteinat 15,30 and 60 min, respect ively. This uptake was correlatedto the acidification of the medium (pH 7.4 to 5.6 ), indicating arelease of iron from Tf and subsequent uptake of iron by thecells with a mechanism not related to a receptor-medi atedendocytosis. Treatment of trichomonads with 10 11M monen­sin, an agent that raise the pH of acidic compartments, de­creased iron accumul ation from Lf but increased ironaccumulation from Tf to 80% and 161 % of the amount ac­cumulated by untreated cells, respectively.These results indicate, that T. foetus possesses at least twodifferent mechanisms for iron acquisition (1) Lf-dependentwhich probably involves receptor-mediated endocytosi s. (2)Tf-dependent, which may depend on extracellular releaseof Fe from the ligand. Further studies are required to eluci­date mechanism s of iron uptake from these host proteins.

225The Morphology of two Suctorians of the GeneraAcineta and Pelagacineta, Epibionts on CopepodsM. L. TATO PORTO and G. FERNANDEZ-LEBORANSLaboratorio de Biologia General , Departamento deBiologia Animal I (Zoologfa), Facultad de Biologia,Un iversidad Complutense, 28040 Madrid, Spain

The suctorians were found in samples of copepods and mysidscollected on the continental shelf of Arcachon (France), at adepth of 80 m. The crustaceans were fixed in a 30% solutionof formalin in sea water. A total of 2345 copepods of differentgenera were observed, belonging mainly to Calanus helgolan­dicus and Cbiridius sp., in approx imately equal proportion.The epibionts were isolated using a steroscopic microscope,and stained with methyl green, after which photomicro­graphs were taken. 21 specimens of copepods showed epi­bionts:- A suctorian very similar to Pelagacineta euchaetae (Sewell,

1951 ) Curds 1985, located on the last abdominal segmentof the copepod. Th is suctorian had an elongated body(90 x 50 urn}, with retra ctile tentacles arranged in two fas­cicles, an elongated, C-shaped nucleus and an internal bud.The body was located on a thecostyle 257 11m in length.

- At the base of the ant ennae or on the last abdominal seg­ments, 1-2 specimens of a suctorian of the genus AcinetaEhrenberg, 1855 were found. They are very similar to A.karamani Hadzi, 1940, measuring 75 x 40 11m, with twowell developed actinoph ores, in which were inserted 2 fas­cicles of capitate tentacles. The body did not completely fillthe loricae, which had an average length of 132 11m andshowed transversal folds. In some suctorians there wereinternal buds.

Both suctorians were accompanied by apostomatids belong­ing to the genus Vampirophrya, Chatton & Lwoff, 1930.

468 . Abstracts

226Protozoa in Beechwood Soil Systems: Influence onMicroflora and Nutrient TurnoverD. TIETZE, M. BONKOWSKI, M. SCHAEFER and S. SCHEUII. Zoologische s Institu t, Abt. Okol ogie, Berlin er Str.28, D-3 70 73 Gbttingen

In a microcosm experiment with soil of four different beech­wood sites the influen ce of naked amoebae on microbi al bio­mass and nutrient turnover was studied. Th e beechwood sitesformed a gradient from basalt to limestone. After defaun at ion(chloroform fumigation) half of the materials were inoculatedwith Acanthamoeba sp. from laboratory cultures, the otherha lf was left without protozoa (contro l).During the experiment leaching of P043- , N03 - , NH4 + andproductivity of CO 2 were measured for 126 days at regularinte rvals. Density of amoebae, microbial biomass and micro­bial Oz-consumption were determin ed after 42 days and atthe end of the experiment.In genera l total microbial biomass increased in presence ofprotozoa, whereas their respira tory activity decreased.In con trast, presence of protozoa did not consistently effectnutrient leaching.

227New Intestinal Ciliate Genus (Ciliate: Paraisotrichidae)from the ElephantsO. TIMOSHENK0 1 and S. IMAI2

lIn stitute of Zoology, Kiev, 262601 Ukraine, 2Nippo nVeterinar y and Animal Scienc e University, 1-7-1 ,Kyon an-cho, Musashino-shi , Tok yo 180, Japan

Fecal samples collected from Asian (n = 5) and African(n = 4) elephants in Kiev, Moscow and Warsaw zoos andfixed in 10% formo l solutio n have been examined.As a result, thre e species belonging to the family Paraisotri­chidae, wh ich should be classified as new, have been recog­nized. Also new genus should be created for them. Round­shaped body with ter minal "spout" and somatic ciliaturein the anterior arch and posterior rows, as well as concretionvacuole in presence, are the ma in characteristics of the genus.New species mainly differ in size of the body and number ofposterior ciliary rows .The genus can be distinguished from the oth er paraisotrichidsby arrangement of soma tic ciliature, as well as terminal"spout" in the presence. One of the new species, besides con­cretion vacuole, possesses concrement granules in row poster­iorly. Presence of those, as well as nonuniform ciliature, closernew genus to the genus He/icozoster, which used to be oc­curred in both elephant species. Both genera seem to be ori­ginated within proboscides, whereas Paraisotricha andRhisotricha in perissodactyls, and Enterophrya in rodents.

228Soil Testaceans from the Hungarian CentralMountains] . T OROKDepartment of Systemat ic Zoolo gy and Ecology of theEotvos Lorand University, H -1445 Budapest Pf. 330

Testaceans are imp ortant components of soil faun a. In Hun­gary however, investiga tio n of the soil habitat was less inten­sive than that of the wa ter and Sphagnum ha bitats. Th e aim ofth is preliminary study was to get information about speciescomp osition of the testacean communities at different sam­pling sites .Samples were taken during 1994 at several areas in the North ­ern and Transdanubian Centra l Mountains. Collect ion re­ferred to fragmented litt er and raw humus layers , whichwere expected to be the richest in species. Identification tookplace after extracting empty shells by flotation. Results ofqualit ative investigations are presented, comparing the com­positions found at the different sites. Morphology and biome­trical characterization of new species for the Hungarian faunaare also described.

229Superoxyde Dismutase in TrichomonadsH . TOURNU, P. D ELGADO, J.-L. MOLAT, D. DIVE'; andE. VISCOGLIOSILaboratoire de Biologie Co mpa ree des Protistes, UR ACNRS 1944, Aubiere, France and "Inserm U42,Villeneuve d'Ascq, Fra nce

In the course of the study of the defensive mechanisms agai nstthe toxic products of O 2 reduction, the superoxide dismutase(SOD) is an essenti al enzyme for the surviva l of aeroto lerantorganisms. Th is enzyme has been examined in some details intrichomonads by biochemical techniques and is likely to con­tain iron. A molecular analysis of the SOD from the parasiteTrichomonas vagina/is has been car ried out with the aim ofusing this enzyme as phylogenetic indicator and as target forchemo therapeutic agents. Indeed , in the recent years, tricho­moniasis has emerged as the most common sexually trans­mitted disease of parasitic origin. Using degenerat eoligonu cleotide primers derived from regions highly con­served in prokar yotic an d euka ryot ic iron-containing se­quences, a genomic DNA fragme nt (400 pb) was amplifiedby the polymerase chain reaction . Th e fragment was usedto isolate iro n-contai ning SOD specific DNA and gDNAclones from T. vagina/is libraries. So far, sequenci ng identi­fied four distinct genes including a pseudogene explainedby mult iple dupl icat ions in th is multigenic family. Th e pri­mary structure of these trichomonad SODs showed the char­acter istics of an iron-containing type of SOD. Theevolutionary divergence among T. vaginalis SOD sequencesin terms of aminoacid changes were slight indicating that mul­tiple dupli cations occurre d very lat e in T. vaginalis. In a glo balphylogenetic tree, T. vaginalis is closely related to other pro­tozoa such as Leishmania donovani. Mo reover, our tree indi­cated a fundamental split between the protozoa whichemerged within the Eubac teria and the Archaebacteria whic hcom posed a mon ophyletic gro up. In conclusion, iron-contai n­ing SODs from parasitic proti sts such as T. vaginalis, L. do­nouani, Trypanosoma or Plasmodium may serve as targets for

chemotherapeutic agents since these SODs might be inhibitedby compounds which do not affect mammalian copper, zincor manganese-SODs.

230Optimization of Culturing Conditions of the CiliatedProtozoan Spirostomum teres in View of its Utilizationin Freshwater Pollution Microbiotests. First Approachto the Ciliate Sensitivity to some XenobioticsL. TWAGILIMANAl, C.-A. GROLIERE 1 and J. BOHATIER 1,2

lLaboratoire de Biologie Comparee des Protistes, URACNRS 1944, Universite Blaise Pascal/Clermont II63177 Aubiere Cedex France, 2Laboratoire de BiologieCellulaire, Faculte de Pharmacie, Universited' Auvergne/Clermont I, 24, Place Henri Dunant,63001 Clermont Ferrand Cedex, France

For determining the pollution degree in aquatic medium orproviding against the potential impact of a toxic effluentor a xenobiotic in natural waters, bioessays using differentaquatic organisms (Microalgae, Rotifers, Crustaceans, Mol­luscs, Fishes, .... ) are considered. Surprisingly, only a few spe­cies of ciliates have been proposed, principally Tetrahymenain axenic culturing.Our study concern optimization of culturing conditions ofheterotrichous ciliate Spirostomum teres in view of its utiliza­tion in microbiotests. This ciliate has been selected as suitablecandidate species for: its ubiquitous distribution, ease cultur­ing in holoxenic medium, large size for an unicellular (150­400 um) and its sensitivity to xenobiotics tested in preliminarystudies. For that purpose and on base of our first observations,we have tested different temperatures, different media, twotypes of food, and have considered the influence of lightand darkness.The shorter generation time, which is 36 h in exponentiallymultiplication phase, is obtained at 25°C in the darknessin Volvic mineral water with high bacterial population devel­oped on grains of wheat. In these conditions, it is possible toobtain a means of 2600 cells per ml up to 4 weeks. On theother hand, if a strain of A. aerogenes is given as the uniquebacterial food source, there is no improvement of the genera­tion time.Preliminary assays on the acute toxicity of xenobiotics haveshown a very high sensitivity of the ciliate to the Copper ionand to Thiram. We have respectively obtained 24 h LC 50 of37.2 ppb and 0.484 ppm. The work in progress on otherxenobiotics confirm this sensitivity to heavy metals andpesticides.These results open out new perspectives of using this ciliate incost effective freshwater pollution microbiotests.

231Intraclonal Polymorphism in the Antarctic CiliateEuplotes focardiiA. VALBONESI, F. ApONE and P. LUPORINIDepartment of Molecular Cellular and Animal Biology,University of Camerino, Camerino 62032, Italy

In Euplotes focardii, an Antarctic ciliate, a marked poly­morphism ensues in cultures with a cell density higher that100 - 200 cells/ml and fed with the green algae Dunaliella ter­tiolecta. "Normal" cells (ovoid in shape, with average dimen-

Abstracts . 469

sions of 72 x 54 urn and an argyrome of a "double" type)progressively transform into "giants" (nearly spherical inshape with dimensions that may reach 180 x 170 urn, andan argyrome of a "multiple" type). "Giant inducing sig­nals", either soluble or cell-anchored, were not identified.Yet, the acquisition of a "cannibalistic" behaviour leads tothe appearance of giants through a "macrostome" stage.This stage derives from normal cells, repleted with food, inwhich most of the digestive vacuoles occupy the body rightside. When division occurs, the fission furrow cannot deepensynchronously and symmetrically at either side of these cells.Instead, its development on the cell right side is delayed withrespect to the left side because of mechanical obstruction.Thus, an opisthe will appear in which the anterior left areais unusually extended and contains an increased number ofadoral membranelles (from 10-12 to 25 -30) and a widercytostome (the peristomial "V" widens from 15-16 0 toabout 30°). Such transformed cells may not only ingest morealgae, but may also start preying on sibs of smaller dimen­sions. Thus, soon they become giants and revert to thenormal stage (through 3-4 cell divisions) following exhaus­tion of small preys. This morphological plasticity and oppor­tunistic behavior shown by E. focardii imply an adaptivefunction in relation with the cell need of gaining the maxi­mum of food resources when these, as typically in Antarcti­ca, flourish periodically.

232Bonamia ostreae and Marteilia refringens: TheProtistan Threats of the Culture of the European FlatOyster Ostrea edulisP. VAN BANNINGNetherlands Institute for Fisheries Research RIVO­DLO, P.O.B. 68, 1970 AB IJmuiden, The Netherlands

Shellfish aquaculture of many countries has been confrontedwith serious infectious diseases. Epizootic outbreaks due toviruses, bacteria, protozoa, fungi, and metazoa have de­creased the aquaculture production of several commerciallyexploited shellfish stocks. For the aquaculture of molluscs,protistan pathogens have shown to be a most seriousthreat. In Europe, epizootic developments of two protistanparasites, Bonamia ostreae and Marteilia refringens, havecaused a serious breakdown in the production of theEuropean flat oyster, Ostrea edulis, over the last 30 years.The characteristics of both protistan pathogens are consid­ered, including their devastating impact on the stock sizeof O. edulis.

233Species Detection and Strain Identification ofTrichomonas vaginalis by using PCRS. VANAcovA, J. FLEGR and J. KULDADep. Parasitol., Charles Univ., Vinicna 7, Prague12844, Czech. Rep.

PCR techniques are being widely used in many epidemiologi­cal and taxonomical studies. We have developed a specificPCR assay, which can be used for a diagnosis of Trichomonasvaginalis infection. The specific primers TCTAATGTTT­GATGTAA and TCTTATTCTAGTTTTATTT were basedon a sequence of a repetitive DNA element (Mol. Biochem.

470 . Abstracts

Parasitol. 54, 247- 256,1992). The 540-bp product of ampli­fication was identified by agarose electro phoresis. The speci­ficity of the assay was confirmed by the amplification of DNAsamples originated from 30 strai ns of Trichomonas vaginalisof different geographi cal origin, 3 strai ns of Trichom onas gal­linae, 8 strains of Trichom onas tenax, 2 strains of Tetratricho­monas gallinarum, 4 strains of Pentatrichomonas hominis, 2strains of Trichomitus batrachorum , and 1 strain of Tritricho­mo nas foetus, T. suis, T. augusta and T. mobilensis. Two dis­tinct DNA fragments of a similar length were amplified in allT. vaginalis strains. No PCR-produ cts or different size-distri­butions of fragments were observed in non-T. vaginalis sam­ples assayed.For the discrimination between different stra ins of T. vaginaliswe used RAPD techn ique. In this technique either a singlerandom primer 10 nucleotides of length (Oberon, USA) ora single T. vaginalis specific primer was used for the amplifi­cation. Most of the primers provided a characteristic strainspecific pattern after the electroph oresis of the PCR products.

234Identification of the Gelation Factor ABP-120 fromEntamoeba histolyticaM . VARGAS, P. SANSONElTl and N. GUILLENUnite de Pathogenic Microbienne M oleculai re, InsermU 389, Pasteur Institute, 28, Rue du Dr. Roux 75724Paris, Cedex 15 , France

Entamoeba histolytica is the parasite responsible for amoe­biasis. This protozoan infects 10% of the world popul a­tion. Presently it is known that adhesion, phagocytosis,contact of troph ozoites with target cells, and tissue inva­sion, require the motile activity of the amoeba. During loco­motion E. histolytica forms a fronta l pseudopod. Studies onthe nonpath ogenic amoebae Dictyostelium discoideum haveshown that the growth and rearrangement of actin filamentsin the pseudopod is mediat ed by several actin binding pro­teins; one of them, ABP-120 is a dimer that cross-links F-ac­tin . To determine whether a protein similar to ABP-120functions in E. histolytica motility, we have identified amayor actin binding factor, then we have isolated and se­quenced the eDNA encoding this protein. The ORF predictedby the DNA sequence has 76% homology with the ABP-120gelation factor from D. discoideum and show an consensussite for actin binding and a potential calcium-binding loop.The biological activities of ABP-120 are presently under in­vestigation.

235Cloning of Cyst Wall Genes of the Hypotrich CiliateHistriculus cavicolaC. VELASCO, A. RODRIGUEZ, A. A. LOPEZ, C. ALBA,C. CARAVACA, A. T ORRES an d J. M ARTINDepart amento de Microbio logia, Facultad de Ciencias,Uni ver sidad de C6rdoba, San Alberto Magno sin,14004-C6rdoba, Spain

Starvation induces encystment in many hypotrich ciliates.Th is is accomplished by a rapid synthesis of a mult ilayeredcyst wall composed of glycoproteins, carbohydrates andother substances. This synthesis is preceded by transcriptionof specific genes, concomit ant with a shutdown of regular

rranscnpnon and translation in vegetative cells (Prescott,D.M ., Microbi ol. Rev., 58 ; 233-267, 1994 ) and followedby postranslat ional glycosilation (Lopez et aI., 8th EuropeanConference on Ciliate Biology). By this reason, beside the pos­sibility of synchronous induction, encystment is a good op­portunity to study transcriptional and posttran slationalregulation in hypotr ichous ciliates.In our study total DNA from the hypotrich ciliate H. cavicolawas used to contruct a genomic library in phage M13mp1 8 byconventional methods. Transfection of E. coli DHaF by elec­troporat ion give us a primary titre of the library of 5.106 pfuJmg. Screening of clones expressing cyst wall-specific se­quences is being made by using antibodies that specificallyrecognize pept idic epitopes of cyst wall components (Lopezet al. in prep. ).So far, we have isolated a clone carrying a 230 bp fragmentwhose sequence reveals a ~-sheet region, a consensus se­quence for binding of lipopolysaccharides and high hydroph i­lic and hydrophobic extremes. When used as a probe thissequence allowed the ident ification by Southern of the corre­sponding full size macronuclear DN A molecule. Purificationand isolation of other cloned sequences are in course by usingdifferent monoclonal and polyclonal antibodies from a panelraised against cyst wa ll components.

236Centrin-like Proteins: Functions in EntodiniomorphidCiliatesB. VIGUES and Ch. D AVIDLaboratoire de Biologie Comparee de s Protistes, URA1944 CN RS, Universite Blaise Pascal, 631 77 AubiereCedex, France

In entodiniomorphids, a group of Rumen ciliates which lack auniform ciliation, myonemes converge on the bases of ciliaryzones that can be retracted under stress conditions, which en­tails a transient immobil izat ion of the cell. MABs have beproduced that identify a 23 kDa Ca++-binding protei n by im­munoblot and immunoprecipitation and label myonemal fila­ments in immunoelectron microscopy. We have shownthrough biochemical, immunological and protein sequenceanalysis that the 23 kDa protein, termed "calmyonemin" ,is an analog of centrin. A detailed immunofluorescence studyperformed on Entodinium reveals that calmyonemin is pre­sent at several defined locat ions where it is likely to be in­volved in mot ility processes. The dynamics ofcalmyonemin-based cytoskeleton has been followed through­out cell division. Taken together, these results provide newdata on the diversity and functions of centrin-containingstructures in unicellular organisms.

237Actin of ParameciumE. VILLALOBO, C. DIAZ-R AMOS and A. T ORRESDep art amento de M icrobiolog ia, Universidad deSevilla, Espana

A 1.1 kb DNA fragment of Paramecium tetraurelia has beenampl ificated by PCR using a couple of degenerate primers foractin of ciliates.

DNA sequencing of the fragment showed that it contains twointrons. The first intron, 29 bp long, is located toward theamino terminus. The second one is 27 bp long and is placednear the carboxy terminal region. Both introns are A + Twithand contain Gff and NG flanking sequences.The fragment showed homology with all ciliate actins. Thehighest homology was found with Tetrahymena actin (73%and 84 % of similarity in nucleotide and amino acid se­quence, respectively). The highest divergence was found withthe actin of Euplotes (only 64% and 72% of similarity in nu­cleotide and amino acid sequence, respectively). The Parame­cium actin showed a clear divergence with conventionalactins (68% -64% of identity in nucleotide sequence). Inter­estingly there is a significant level of homology (63% identicalin nucleotide sequence) between our Paramecium actin andthe canine centractin.

238Costa Proteins of Trichomonads Represent a NewClass of Proteins Forming Striated Roots in ProtistsE. VISCOGLIOSI, G. BRUGEROLLE, A. TUFFERY, J.-L. MOLATand P. DELGADOLaboratoire de Biologic Comparee des Protistes, URACNRS 1944, Aubiere, France

In trichomonads, the costa, a broad striated root connectedthe basal bodies, has been described as consisting of bundlesof 2-4 nm filaments. Two major costa types can be distin­guished based on the band pattern although each type exhi­bits the same periodicity (42 nm). The production ofmonoclonal and polyclonal antibodies and the use of bio­chemical techniques revealed that costa proteins are com­posed of several major polypeptides with molecular weightdetected between 100 and 135 kDa. We showed that costaproteins composing the two costa types belong to the sameprotein family. The behavior of costa proteins towards chemi­cal agents, their resistance to chaotropic agents and their highmolecular weight differ from those found with other proteinscomposing other striated roots such as centrin, assemblin andgiardin. These antibodies were also used in order to followcell morphogenesis during division. The parental costa wasretained in one sister kinetid and the new costa was synthe­sized very early in the other one. Finally, a Trichomonas va­ginalis cDNA expression library in A ZAP II was screenedwith anti-costa antibodies. Two positive cDNA clones wereidentified. The approximately 1200 and 2200 bp inserts weresequenced. The preliminary analysis of the deduced amino­acid sequences do not show any significant similarity withthose of centrin, assemblin and giardin. In conclusion, ourresults indicated that costa proteins might represent a novelclass of striated root-forming proteins and the elucidationof the complete amino-acid sequences of costa proteinsshould verify this point.

Abstracts . 471

239Cell-to-cell Interactions of Importance for theDevelopment of Severe MalariaM. WAHLGRENMicrobiology and Tumour Biology Centre, KarolinskaInstitutet, Sweden

The capacity of Plasmodium falciparum infected erythrocytesto form rosettes has been found to be associated with the viru­lence of the parasite. In search of a parasite associated roset­ting ligand we found a set of clone, strain- or isolate specific,SDS-soluble, low molecular weight polypeptides of Mr22 000 - Mr 45 000 expressed at the surface, of the malariainfected erythrocytes. A Mr 35 000 polypeptide was strictlyassociated with rosetting of the Palo Alto strain. The ro­sette-associated polypeptides were stably expressed afterlong-term in vitro cultivation only if the parasite was conti­nously enriched for the rosetting phenotype. PfEMP1's of dif­ferent sizes were also detected.We have found the infected erythrocyte to bind either immu­noglobulin M or IgM and IgG which the parasite seems to useto form electron-dense fibrillae that mediate binding to hostcells. The ABO-blood group oligosaccharides ([cxgaINAc(l­3)~gal(1-3)cxfiuc] or [cxgal(1-3)~gal(1-3)cxfiuc]) were in inhibi­tion experiments also found to be involved as rosetting recep­tors and blood group a RBC formed smaller and weakerrosettes than the blood group A, B, or AB RBC. The bloodgroup a phenotype has in epidemiological studies been impli­cated to protect against cerebral malaria. In conclusion, roset­ting seems to be mediated by low- to medium sized molecularweight polypeptides and immunoglobulins on the surface ofthe infected erythrocyte and the ABO blood group sugars onthe uninfected counterpart.

240Molecular Epidemiology of Pneumocystis cariniiInfectionA. E. WAKEFIELDMolecular Infectious Diseases Group, Department ofPaediatrics, Institute of Molecular Medicine, JohnRadcliffe Hospital, Oxford, UK

Molecular techniques have been employed to further the un­derstanding of the epidemiology of infection with the oppor­tunistic pathogen Pneumocystis carinii, an organism whichcannot be propagated by in vitro culture. The DNA se­quences of two independent loci have been used to analyzegenetic divergence among isolates of P. carinii from the in­fected lungs of five different mammalian hosts. The resultssuggest that host species specificity occurs in P. cariniiinfection and indicate that P. carinii infection in man maynot be a zoonosis. Data from isolates of human-derived P.carinii indicate low levels of divergence among parasitesfrom Britain, USA, Brazil and Zimbabwe.The source of P. carinii infection in man and how it is acquiredremain unclear, although data from the rat model of P. cariniipneumonia suggest an airborne route. DNA amplification ofsamples of the air spora of rural Oxfordshire have demon­strated the presence of sequences identical to P. carinii DNA.

472 . Abstrac ts

241Effect s of Some Environmental Factors on Soil TestateAmoebae - Ecological, Morphological and GeneticInvestigationsM. WANNER, J. NAEHRING"' and R. MEISTERFELDInstitute of Biology II (Zoology), "Institute of Biology I(Botany), RWTH, D-5205 6 Aachen, Germany

Clonal cultures were used to test the influence of environmen­tal facto rs on growth and shell size of terrestrial testate amoe­bae. Food and temperature significantly influenced culturegrow th and shell size of all species investigated. Detailed ex­periments with Cyclopyxis kahli showed that cultures kept at17 °C and sparse food supp ly entered exponential growthphase later and had extended genera tion times as comparedto cultures kept at 21 °C and provided with food in surplu s,whereas shell size was largest at low temperature and highfood density. Significant interactions between different treat­ments occurred frequentl y, while adaptations to the treat­ments were not observed. Culture grow th and shell size ofall species investigated were highly variable under altered en­vironmental conditions. This fact points to new possibilitiesin bioind ication, but also reveals consequences for taxonomi­cal work based on shell characteristics. Th erefore a geneticapproach based on RAPD-PCR (Williams er aI., Nucl. AcidsRes. 18, 653 1-6535, 1990) was developed to complementecological and taxonomical data.

242Protists as Bioindicators in Soils and GroundwatersAffected by Sewage Sludges and EffluentsA. WARRENThe Natural Hi story M useum, Cromwell Ro ad ,London SW7 5BD, UK

Most sewage-treatment processes result in two productswhich could be harmful to public health and to the environ­ment; a liquid effluent and solid or semi-solid sludge. Thispaper documents the use of prot ists as bioindicators in twoenvironments affected by the disposal of such products;groundwater contaminated by treated sewage effluent andsoils amended by heavy-metal contaminated sewage sludge.Groundwater makes up over 95% of the world's availablefreshwater resource s, yet, because the subsurface environ­ment is remote and difficult to sample, comparatively littleis known about the biological processes that occur there. Re­cent evidence suggests that pro tists are widely distributed inthe subsurface alth ough almost nothing is known abo ut theiridentity or ecological role. Studies of an orga nically-contami ­nated sandy aqu ifer on Cape Cod, USA have show n that sub­surface protist popul ations may be used as indicato rs ofpollution . Sewage sludges are increasingly used as soil ferti­lizers. However, many contain heavy meta ls which may af­fect soil communities and/or enter the food chain. A simplebioassay of the growth and reproduction of the common soilciliate Colpoda steinii has been developed for monitor ing thebiological availability of heavy metals in soils.

243H yaline and Agglutinated Loricae of some TintinnidSpeciesA. WASIK*, E. MIKOLAJCZYK* and R. LIGOWSKI **"Nencki Institute of Experimenta l Biology, 3 Pasteur Str.,Warsaw, **University of I:.6dz, 12/16 Banacha Str., I:.6dz,PolandHyaline loricae of Cymatocylis affinislconvallaria, Helicosto­me lla sub ulata and agglutina ted loricae of Codone llopsisgaussi, Laackmanniella naviculaefera were examined.Scanning electro n microscopy revealed differences in Codo­nellopsis and Laackmanniella surface structures of an ann u­lar collar and a bowl, where particles are adhered. The borderbetween both parts is sharp. The bowl surface of C. affinis/convallaria as well as spira l and non-spiral parts of H. sub ­ulata are smooth.X-ray analysis of the loricae of all examined species revealedtheir non-mineral nature.The bowls of both species, Cd. gaussi and L. nauiculaefera areagglomerated with biogenic and non-biogenic particles. Frombiogenic particles, diatoms Fragilariopsis cylindrus and F.pseudo nana most frequently occur.

244Gravity Response of Paramecium caudatum Modifiedby Iron FeedingD. WATZKE and R. BRAuCKERArbeitsgruppe Zelluliire Erregungsphysiologie, Ruhr­Universita t Bochum, D-44780 Bochum, Germany

Paramecium shows a well known negative gravitactic andgravik inetic behaviour. We assume that gravikinesis is causedby the mechanical load of the cytop lasm acting on mecha­noreceptor channels of the plasma membrane. In the experi­ments presented the density of the cytoplasm was increased byiron-feeding. For determination of gravikinesis upward anddownward swimming rates, cell orientation and sedimenta­tion rates were analyzed using computer-aided video record ­ing. Iron-fed cells showed larger negative values ofgravikinesis than cont rol samples. For both gravikinesis de­pends on orientation angle. The relation between the gravi­kinesis-subcomponents in control samples is inverse iniron -fed cells. In addition the precision of graviorientationwas increased in iron-fed cells.

245The Control of Ciliate Abundance by MetazoanGrazingS. WICKHAMInstitut fur Okologie, Universitat Gre ifswald,Schwe denhagen 6, 18565 KlosterlHiddensee, Germ an y

Contro l of ciliate abundance has traditionally been thought tobe due to resource supply (botto m-up control) . However, inrecent years, there has been mounting evidence that predators(to p-down control) may also be importa nt. In many fresh­water systems the c1adoceran Daphnia is an important phy­toplankton grazer, capa ble of graz ing part icles in the sizeranges of both ciliates and ciliate prey. It has been shown thatwhen large Daphnia are abundant, they exert a strong preda­tion pressure on ciliates, and are capable of obtaining signifi­cant amounts of nut rition from them. There is also ample

evidence that calanoid copepods prey on ciliates, but there hasbeen considerably less work in cyclopoid copepods. Cyclo­poids, unlike calanoid copepods, are only raptorial feeders,but may also be important ciliate predators. However, cyclo­poid predation on ciliates appears to be a function of theavailability of alternative prey, as well as ciliate density. More­over, cyclopoids prey on other zooplankton such as Daphnia,potentially releasing ciliates from predation pressure througha trophic cascade. Ciliates appear to have few effective de­fenses against the generalist predator Daphnia, but may beable to reduce their vulnerability to at least cyclopoids.Thus, top-down control may be at least as important as bot­tom-up control in regulating ciliate abundance.

246Impact of Combined Sewer Overflows on the ProtistCommunity of a Small Urban Stream, PreliminaryResultsJ. WIDERA

Universitiit-GHS-Essen, Inst. f. Okologie, Abt,Hydrobiologie, D-45117 Essen, Germany

In urban areas the majority of small brooks is episodicallypolluted by combined sewer overflows (CSOs). During occa­sional stormwater events or thaw the discharge which exceedsthe capacity of the sewage system is dumped into runningwaters. These discharges consist of untreated domestic sew­age diluted by surface runoff. A lack of knowledge exists withregard to the ecological impact of discontinuous dischargeevents. Some of these questions are studied by protozoologi­cal means at a small urban stream in the eastern Ruhr-area(Northrhine-Westfalia, Germany). Population dynamics ofselected protozoan species and taxa of the Subphyla Mastigo­phora and Sarcodina and of the Phylum Ciliophora are ana­lysed. Several important physico-chemical parameters likeflow-through or oxygen-content are recorded continuouslyup- and downstream the CSO. In case of discharge eventswater-samples are taken automatically. Oxygen content val­ues of about 2 mg/l during discharge events were occasionallyfollowed by temporary anoxic conditions. The ammonia con­tent of the discharge reaches values of about 2 mg/I NH4+.Protozoological investigations are carried out with glass­slides exposed in situ. At least two samples from up- anddownstream the CSO are evaluated within a weeklyrhythm. The stochastic discharge of combined sewer yieldsshifts in the structure of the protist community. Long termeffects lead to different protozoan communities up- anddownstream the CSO. The number of taxa is quite similar,but cell-densities downstream are always higher than up­stream, the CSO. The share of vagile cells upstream theCSO is much higher and Sarcodina like Arcella discoidesor Actinophrys sol can be found with high steadiness whereasdownstream the sessile protists, predominantly peritrich cili­ates dominate the community. Short-term effects of dischargeevents mainly consist in a distinct increase of the number ofperitrich ciliates like Carchesium polypinum, Vorticella cam­panula or V. picta downstream the CSO. The initial phase ofthis increase can be detected some days after a dischargeevent. Some weeks after the event the share of peritrich cili­ates decreases again if no further discharge event occurs in themeantime. This shifting in the protist community can be in­terpreted as result of nutrient-input during stormwater dis­charges and subsequent consumption after transfer throughthe trophic level of bacteria to the heterotrophic protists.

Abstracts' 473

247The Influence of Previous Diet on the Rate of Ingestionof Inert Particles of Different Sizes by EuplotesS. A. WILKSDepartment of Biology, University of Southampton,Southampton S016 7PX, U.K.

Euplotes mutabilis, isolated from the sea near Southampton,is routinely cultured by feeding with the bacterium Vibrio na­triegens. It can also be grown on pure cultures of such algae asDunaliella tertiolecta, although on this diet it appears to growless vigorously than it does on bacteria. The average size ofcultured Vibrio is near to 111m (0.6 x I-211m), while Duna­liella is about 6 11m long.The clearance rates of Euplotes that had been cultured onthese two different types of food were measured using a seriesof fluorescent latex microspheres of sizes ranging from 0.6 to6.5 11m, each presented at a concentration of about 10 6 par­ticles ml-1for a period of 10 minutes after a starvation periodof 24 hours. Ciliates that had been cultured on bacteria in­gested maximum numbers of particles in the 0.6-3 11m sizerange. The highest values of clearance were around 100 nlciliate-1h-\ with these smaller particles, decreasing to 60­75 nl ciliate-1 h-1 with particles 5.7-6.5 11m in diameter.These clearance rates compare with a maximum value of1.2111 ciliate."! h-1recorded for this culture of Euplotes wheneating live bacteria.Ciliates that had been cultured on algae ingested similar num­bers of fluorescent particles of 1-3 11m and 5.7 - 6.5 11m sizes.In each case the clearance was about 35 nl ciliate."! h-1. In­vestigations are continuing to discover why these ciliates con­sistently had lower clearance rates than those cultured onbacteria although the ciliates that had been cultured on thedifferent diets were approximately the same size.

248Evidence for a Functional ~-adrenergic ReceptorSystem in ParameciumE. WYROBA

Nencki Institute of Experimental Biology,02-093 Warsaw, Poland

Endocytosis and ciliary movement in Paramecium have beenfound to be modulated by ~-adrenergic ligands and the ~­

adrenergic receptor sites were localized at the cell membraneusing the dansyl analogue of propranolol (DAPN). Phagocy­tosis, uptake of fluid phase and ciliary reversal were studied.P-blockers exerted a time- and dose-dependent inhibiting ef­fect on the process of phagocytosis and increased both the rateand extent of fluid phase endocytosis measured as a horse­radish peroxidase uptake. p-agonists enhanced phagocytic ac­tivity in a stereospecific manner and their effect was abolishedby beta-antagonists. However, the most potent stimulants ofParamecium phagocytosis were phorbol ester and forskolinwhich, on the contrary, diminished the fluid phase uptake.P-blockers significantly shortened both the continuous andperiodic ciliary reversal whereas forskolin and phorbol esterincreased a duration of these motor reactions. All these spe­cific physiological responses to ~-adrenergic ligands andfluorescent localization of their binding sites revealed an ex­istence of a functional ~-adrenergic receptor system in Para­mecium. This cell modulated by ~-adrenergic ligands provedto be an useful model to study uptake of photosensitizers usedin photodynamic therapy of tumors.

474 . Abstracts

249Microaggregates: Structure and Colonization in theElbe-EstuaryH. ZIMMERMANNInstitut fur Hydrobiologie und Fischereiwissenschaft,Universitat Hamburg, Zeiseweg 9, D-22765 Hamburg,Germany

Aggregates of detritus, living organisms and inorganic matterhave significance as microenvironments and transport agents.To get information about the significance of those aggregatesand their community in the Elbe-estuary, aggregates were iso­lated and observed with special models under the Iight- andepifluorescence microscope.Aggregates can be produced either by living plants and ani­mals especially as mucus feeding webs of zooplankton, orby the biologically-enhanced physical aggregation of smallerparticles. The aggregates containe enriched microbial com­munities. Microbial communities associated with aggregatesundergo complex successional changes in community struc­ture and function. In spring and autumn associated rotiferswere the main top down controlling organisms on the aggre­gate community. During summer aggregates and their com­munity are selective reduced by raptorial metazooplankton.

Author Index for AbstractsNumbers indicate the abstract numbers

250Simultaneous Estimation of Ingestion andAccumulation of Biomass by Bacterivorous MarineCiliates and Flagellates Using a Dual Radioactive­labelling TechniqueM. V. ZUBKOV and M. A. SLEIGHDepartment of Biology, University of Southampton,Southampton S016 7PX, U.K.

Live bacteria, quantitatively labelled with 3H-thymidine and14C-leucine, were fed to ciliates (Euplotes and Uronema) orflagellates (Pteridomonas and Paraph ysomonas ) in 4- to 8­hour grazing exper iments. Macromolecules labelled with3H are accumulated in the protozoa to a negligible extent,so the disappearance of 3H-labelled macromolecules indi­cates the rate of ingestion of bacteria. Between 16% (in someflagellates) and 60% (in some ciliates) of the 14C label is ac­cumul ated in macromolecules in the protozoa, giving a mea­sure of the conversion of bacterial biomass to protozoanbiomass by the protozoa concerned under the conditionsused. The extent of accumulation of 14C-labelled macromo­lecules in the protozoa depended upon the nutritional histor yof the predator. The method can be used to indicate thegrowth efficiency of the protozoa and the extent of nutrientregeneration that they produce. It is being evaluated for use infield studies.

Aescht, E. 1Affa'a , E-M. 156Alba, C. 143,235Albergoni, V. 116Albert, M. 18Aleya, L. 154Alix, A. J. P. 49Alphei, J. 23Amblard, C. 149, 151,217Ambroi se-Thomas, P. 2,50Amigo, J. M. 3,4,52, 191,

193,200Arnoros, G. C. 4Andreu, J. M. 5Anna, R. 198Apone, F. 231Arevalo, J. 65Arhets , P. 101Arnaud, J. 88Arregui, L. 5, 147

Bahri, S. 6Baier, E. 185Ball, S. J. 48Balossier, G. 21Balzer, I. 208Bandi, C. 198

Bafiuls, A.-L. 65Baranova, A. M. 8Barasoain, I. 5Bardele, C. E 203Bastien, P. 190Batistoni, R. 201Beck, C. 9Becker, B. 10Becker, K. 161Bell, M. V. 204Belmegenal, A. 50Beltrame, F. 189ben Hassine, O. K. 6ben Salah, M. 150Benbernou, N. 93Benhamou, Y. 171Benichou, j.-c. 53Benyahya, M. 11Berchtold, M. 12Berg, M. v. d. 104Berger, H. 13Bergmann , B. 161Bernard, C. 14, 15Bernhard , D. 207Berninger, D.-G. 16Berthold, A. 17Bhaud, Y. 18,92

Biderre, C. 19Billaut-Mulot, O. 176Blaineau, C. 190Bogaerts, P. 20Bohati er, J. 11, 20, 150,

205, 206, 230Bohrn, J. 94Bolivar, I. 180Bolshako va, N. 214Bonhomme, A. 21,93Bonhomme , P. 21Boni, S. 22Bonkowski, M. 23, 226Bonnet , J. L. 24Bott iroli, G. 56Bouchard, P. 25Bouffault, E 79Bourdier, G. 149, 151Branke, J. 12Braucker, R. 26,244Bremerich, A. 140Bricheux, G. 27Broers, C. A. M. 105Bronnvall, A. M. 28Brown , S. 29Bruchhaus, I. 30Brugerolle, G. 31, 55, 238

Brul, S. 104Bruno, P. 202Bulat , S. A. 32Burakowski, T. 124Burkill, P. H. 219Burricl , J. 172, 173, 174,

175Burzlaff, A. 33

Cales-Quist, D. 53Calvo, P. 148Camadro, J. M. 97Cameron, C. M . 34Campbell, C. D. 34Campos, I. 148Camus, D. 35, 88Canning, E. U. 111Cantarino, H. 172, 173,

174,1 75Caravaca, C. 143, 235Cardoso, A. C. 45, 196Casalou , C. 196Cavalier-Smith, T. 36Chao, E. E. 36Charpin, M. E 37, 154Chessa, M. G. 38