ABSTRACTS 13A

alanine and serine per 108 cells h-l, as well as 4-6 f.Lmolesarginine. Doubling arginine in the medium Hexamita led to areduction in glycogen storage, glucose, alanine and serine ca-tabolism. No regulatory mechanism was evident from availableinformation.

43Giardia intestinalis Needs Glucose to Complete Hs Life Cy-

cle. l.G. ZALITIS and D.S. CROSS, School of Biochemistryand Molecular General, University of New South Wales, Syd-ney, Australia.

Previous studies have shown lhal Giardia trophozoites andcysts contained glycogen equivalent to glucose of about 4 and7 f.Lmolesper 108cells, respectively. We have studied the originand utilisation of cyst glycogen. The yield, glycogen contentand viability of the cysts were dependent on glucose in theencystation medium. The optimal glucose concentration was 15mM or higher. Encystation of trophozoites in 1 mM glucoseresulted in a 80% decrease in cyst numbers, with low glycogencontent and abwater at 37°C. In normal cysts, incubated at 25or 37°C, glycogen decreased exponentially, tl/2 being 36 and20 hours, respectively. Cyst numbers remained constant till theglycogen had been depleted. Normal cysts started to swell orrupture in water after about 100 hours at 37°c. By adding 14Cglucose at 2-hour time intervals to encysting trophozoites, max-imum labeling of cyst glycogen occurred between 0-4 hours,decreasing to negligible after 12 hours. From these results, itcan be inferred lhal cyst glycogen was formed from glucose inencysting trophozoites. Giardia trophozoites grew well in lowglucose medium, as judged by cell number increase and JHthymidine incorporation. During growth in low glucose medi-um, trophozoite glycogen content progressively decreased toabout 10% of normal in 48 hours, when medium glucose wasundetectable. Prolonged culture in low-glucose medium had noeffect on trophozoites other than to decrease the rate of celldivision or encystation. Normal cysts obtained by exposing en-cysting trophozoites to 14C glucose had 90% of the label inglycogen. Incubation of cysts at 37°C resulted in a decrease in14C glycogen with a qualitative increase in 14CO2and acetate.Cysts appear to metabolise glycogen via glycolysis to maintaintheir cell integrity.

44The Cryptosporidium parvum Lactate Dehydrogenase Gene.

G. ZHU*, M.l. MARCHEWKA and l.S. KEITHLY, Wads-worth Center, New York State Department of Health, AlbanyNY.

Lactate dehydrogenase (LDH) and malate dehydrogenase(MDH) are two enzyme s present in archaebacteria, eubacteriaand eukaryotes. In eukaryotic cells, LDH is localized in cytosol,while MDH isoforms can co-exist in the cytosol and organelles,including rnitochondria, cWoroplasts and peroxisomes. TheLDH/MDH protein sequences are highly conserved and phy-logenetically related. Both LDH and MDH are 2-ketoacid:NAD(P) oxidoreductases, sharing similar tertiary structures.Despite all of these structural and functional sirnilarities, mostLDfl/MDH enzymes are highly specific to their substrates, i.e.,either lactate or malate, bul not both. The substrate specificityis correlated with the presence of a single arnino acid in thecatalytic síle: all MDH possess a positively charged arginine,whereas all LDH contain an uncharged residue, e.g., glutamineat the same position. Both LDH and MDH enzymatic activitieshave been reported for Cryptosporidium parvum. Prior to this

study, molecular characterization of these enzymes was lacking.Here we present both molecular and phylogenetic analyses ofa C. parvum LDH homologue (CpLDH1). The CpLDH1 geneencodes 330 amino acids, which are highly conserved with oth-er LDH/MDH, including thosefrom its apicomplexan relatives,Plasmodium falciparum and Toxoplasma gondii. The key resi-dne at the active site of CpLDH1 is a non-polar glycine, ratherlhali a charged arginine, strongly suggesting lhal lactate is thepreferred substrate for this enzyme. RT-PCR indicates the ex-pression of CpLDH1 occurs both in sporozoites and intracel-lular parasites. Interestingly, phylogenetic analyses suggest lhalall apicomplexan LDH, including CpLDH1, are a sister groupto both eubacterial and archaebacterial MDH. This (apicom-plexan LDH and bacterial MDH) clade is then a sister groupto other eukaryotic and prokaryotic LDH. The significance ofthis mixed lineage for apicomplexan LDH is not fully under-stood, blit warrants further investigation.

Czech Section Society of Protozoologists30th Annual Meeting

April 25-28, 2000(Abstracts 45-53)

45Immune Response of the Host to Infection with the Micro-

sporidian, Encephalitozoon cuniculi Levaditi, Nicolau etSchoen, 1923. P. BRAUNFUCHSOVA, l. SALAT, Institute ofParasitology, Academy of Sciences of the Czech Republic, Ces-ke Budejovice, Czech Republic.

In the present study, the immune response of immunocom-petent BALB/c mice and severe combined immunodeficient(SCID) mice to intraperitoneal infection with the rnicrosporid-ian, Encephalitozoon cuniculi was analyzed. The production ofthree cytokines, interferon gamma (IFN-gama), interleukin 10(IL-lO) and interleukin 12 (IL-12), was measured. High levelsof IFN-gama were detected in ex-vivo cultures of peritonealexudate cells (PEC) of BALB/c mice, a lower, bul earlier IFN-gama response was observed in PEC from SCID mice. Theearly IL-lO response was detected in ex-vivo cultures of sple-nocytes from BALB/c bul not from SCID rnice, explaining adelay in the IFN-gama response in BALB/c rnice. The higheramount of spores in peritoneal macrophages was observed dur-ing the infection of SCID mice lhal develop a lethal infection.The populations of natural killer (NK) cells of both rnice strainswere activated by the infection. Duration and rate of NK activ-ity differed between these strains. Production of IFN-gama cor-related with NK activity. Production of specific antibodies wasdemonstrated from the 9th day after infection. Cytotoxic T-lym-phocytes (CTLs) were generated in vitro by incubation of im-mune splenocytes with irradiated E. cuniculi spores. TheseCTLs were able to kill infected syngeneic macrophages.Theresults of this study support the theory of the essential role ofIFN-gama and specific CTL activity for host protection.

46Phylogenetic Position of Some Trichomonad Species Isolated

in Vitro from Different Animals. I. CEPICKA, V. HAMPL, l.KULDA and l. FLEGR, Department of Parasitology, Facultyof Science, Charles University, 128 44 Prague, Czech Republic.

Many trichomonad species described so far from differentanimals have been inadequately described and their taxonornicposition is doubtful. In a project aimed at taxonornic revision

,14A ABSTRACTS

and phylogenetic relationships of some parabasalid groups weexamined over 200 specimens from 90 species of marnmals,birds, reptiles, amphibians and snails and obtained 60 isolatesof trichomonads from faeces, rectum, caecum and oral cavityof 40 host species. Trichomonads belonged into 7 genera: Mon-ocercomonas (2 species), Hypotrichomonas (1 sp.), Trichomitus(3 sp.), Tritrichomonas (2 sp.), Trichomonas (2 sp.), Penta-trichomonas (1 sp.) and Tetratrichomonas (more than 10 spe-cies). The gene for 5.8S rRNA with the flanking areas ITSland ITS2 of 10 strains of Tetratrichomonas from different spe-cies of Bovidae, Suidae and Tayassuidae, ODe strain from anAfrican elephant and two strains of Trichomitus from differentlizards was amplified, c1oned, sequenced and ana!ysed usingMaximum parsimony and Neighbor-joining tree constructingmethods. Tetratrichomonads from ruminants and from Africanelephant created ODec1ade, bul differed morphologically fromT. buttreyi (the on]y well described Tetratrichomonas inhabitingruminants and pigs). Simi]arities within this c1ade varied from67.6% to 86.6%. As similarities between rour distinct speciesof the genus Trichomonas varied between 86% and 89%, thesetetratrichomonads probably belong to several separate species.Tetratrichomonas gallinarum formed the sister branch of thisc1ade. The Tetratrichomonas strain from Chelydra serpentinawhich was morphologica!ly similar to T. brumpti from landturtles formed another c1ade with T. limacis and T. prowazeki.

47Relationships of Flagellates of the Genus Monocercomonas

from Different Host Species. V. HAMPL, I. CEPICKA, J.TACHEZY and J. FLEGR, Department of Parasitology, Facultyof Science, Charles University, ]2844 Prague, Czech Republic.

Monocercomonas grassi is widely distributed genus of gutparasites from vertebrates and insects. Most species were de-scribed from reptiles, blit their taxonomic validity as well asthe phylogenetic position of this genus in the evo]ution of theorder Trichomonadida remains unc1ear. We studied the relation-ship of ODeisolate of Monocercomonas ruminantium from cowand 5 isolates of Monocercomonas sp. from reptiles (Varanusexanthematicus (VAR-I), Tropidophis melanurus (R]83), Na-trix sipedon (ATCC 502210), Python regius (PYR 1-1), Eu-meces sp. (EUMM». The sequences of 5.8S rRNA with theflanking areas ITSl and ITS2 (5.8S rRNA) of all strains weredetermined. On the basis of these sequences the phylogenetictree was constructed by the Neighbor-joining (NJ) and Maxi-mum parsimony (MP) method. In both trees the strains fromreptiles formed ODebranch with the bootstrap va]ue of 100. Therelatedness ofEUMM, PYR 1-1, VAR 1 was very high (99.4%identity). On the other band, the sequences of strains ATCCand R183 showed considerable differences from the previousstrains (97.2% and 93% identity, respectively) as well as fromeach other (92.4% identity) indicating lhal both may representdifferent species. The iso]ate of M. ruminantium was c1earlyseparated from the reptilian c1ade (85% identity). This isolateformed a monophy1etic group with reptilian monocercomonadsin the tree constructed by Mp, bul not in the NJ tree. In accor-dance with u]trastructural data as well as with the phylogenetictree based on the l6S rRNA both branches of monocercomon-ads (reptilian c1ade and M. ruminantium) formed sister taxonsto the genu s Tritrichomonas.

48Increased Prevalence of Latent Toxoplasmosis in Victims of

Traffic Accidents in Prague. J. HAVLlCEK*, P. KODYM **,

M. MALY ***, Z. SMAHEL**** and J. FLEGR*, *Depart-ment of Parasitology, Faculty of Science, Charles University,]28 44 Prague; **Nationa! Diagnostic Laboratory for Toxo-plasmosis, National Institute of Public Health, Prague; ***De-partment of Biostatistics, National. Institute of Public Health,Prague, and ****Department of Anthropology and Human Ge-netic, Faculty of. Science, Char]es University, Prague, CzechRepublic.

Toxoplasma gondii, coccidian parasite whose frequency inmail can reach 80% in some countries is known to influencethe behaviour of its host. This is usually considered to be anevolutionary adaptation of the parasite aimed to increase theprobability of its transmission from an intermediate host (anyendothermic vertebrate) to the definitive host (cat) by predation.In human the latent toxoplasmosis results into the changes inpersonality profile as well as into the increase of reaction timesmeasured in simple reaction time test. In modem society suchbehavioural changes can increase the probability lhal Toxo-plasma-infected mail falI victims of an traffic accident. In thisstudy we compared the preva!ence of latent toxoplasmosis in103 victims of traffic accidents (car drivers and pedestrians hitby a car) with lhal obtained in two epidemiological surveysamong inhabitants of the same area. We exc1uded from the anal-ysis the persons who: did not actively participate in the acci-dent, drank the a1cohol or were not residents of Prague. Durresults of age-stratified study suggest lhal the subjects with la-telit toxoplasmosis have 3.] times higher probability of the ac-cident than the uninfected contro]s (c.I.95 = 1.92-5.0]; Man-te]-Haenszel Chi-square = 25.219; P = 0.000). In the youngestage category (15-29 years) the relative risk of an accident forToxoplasma-infected subjects was 4.35 (C.I.95 = 2.14-8.81).Because of its high preva!ence in mailY developed countries,the latent toxoplasmosis might be in fact a very serious publichealth problem.

49Effect of Nisin Against the Cu]tures of Some Rumen Ciliates.

S. KlSIDAYOVA, A. LAUKOVA, and P. SIROKA, Academyof Sciences, Institute of Animal Physiology, Kosice, Slovakia.

The growth of the rumen ciliate Entodinium caudatum wasnot significantly affected by the nisin concentration of 0.01-0.4mg/ml during short-term (5 day) treatment in vitro. Long-term(30 days) treatment decreased the growth of Epidinium ecau-datum f. caudatum et ecaudatum at the ni sin dose of 0.1 mg/ml. However, the growth of Entodinium caudatum was not sig-nificantly influenced by the long-term (30 days) treatment at thenisin dose of 0.1 mg/ml. Nisin partly substituted gluten in thecultures as a source of amino acids. Inhibition of Gram-positivefacultative anaerobic bacterial population of the cultures (lac-tobacili, enterococci, staphylococci, and amylolytic streptococ-ci) was detected. The production of the rota! volatile fatty acidswas significantly increased during long-term treatment of bothcultures. The proportion of propionate was significantly in-creased (from 13 mol% to 30 mo]%) on the account of acetate(from 73 mol% to 61 mo]%).

50Opportunistic Properties of Brachiola algerae (syn. Nosema

algerae) (Microspora, Protista) Demonstrated in the SCIDMice. B. KOUDELA*,** and J. VAVRA**,***, *Departmentof Parasitology, University of Veterinary and PharmaceuticalSciences, Brno; **Institute of Parasitology, Academy of Sci-ences of the Czech Republic, 37005 Ceske Budejovice; ***De-

ABSTRACTS 15A

partment of Parasitology, Faculty of Science, Charles Univer-sity, 128 44 Prague, Czech Republic.

The human isolate of Brachiola algerae (Visvesvara et al.,I.Euk. Microbiol. 46, WS, 1999) was inoculated intraperitone-ally, intramuscularly and subcutanously into Severe CombinedImmunodeficient Mice. Spores were also applied to the eye sur-face and were fed to the mice host. No signs of disease werenoted up to 60 days p.i., when the animals were autopsied andtheir organs examined by histology, using staining methods andCa1cofluor M2R spore labeling. It was found lhal the micros-poridium developed in the liver and peritoneal macrophages ofthe immunodeficient mouse host, bul auly after the ocular ad-ministration of spores. The infection of liver was severe andwas manifested as hepatosplenomegaly and multifocal miliarynecroses and granulomas containing parasites. No microspori-dia were found in any other tissues. The identity of the parasitewas confirmed by TEM which revealed characteristic tubulov-esicular "secretory materials" on the plasma membrane of alldevelopmental stages of B. algerae except sporoblasts andspores. Our observation is the first one demonstrating the op-portunistic capability of Brachiola algerae to grow in mam-malian viscera, far from the síle of spore application. Eye canevidently serve as portal of entry for microsporidia into a mam-malian organism. It is hypothesized lhal the physico-chemicalmilieu of the conjunctiva and comea helped to adapt the orig-inally "poikilothermic microsporidian" to the conditions withinthe homoiothermic organism.

51Trichomonads in Oral Cavity and Respiratory Tract of Hu-

mans: a New Species of Pathogenic Tetratrichomonas? K. KU-TlSOV A, I. KULDA and I. TACHEZY, Department of Para-sitology, Faculty of Science, Charles University, 12844 Prague,Czech Republic.

Trichomonas tenaxis regarded as a harmless comensal of hu-man oral cavity. Although cases of pulmonary trichomoniasishave been described in patients with lung abscess, bronchiec-tasis or cancer disease, pathogenicity of T. tenax remains con-troversial. In addition, occasional infections of the respiratorytract of infants by genitourinary parasite Trichomonas vaginalishave been reported. The aim of present study was to distinguishT. tenax strains isolated from human oral cavity and bronchi aswell as from other trichomonad species by two methods ofDNA analysis: [1] PCR method based on fandum amplifiedpolymorphic DNA (RAPD), and [2] nuc1eotide sequencing ofITSI-5.8rRNA-ITS2 region. Axenic strains of trichomonadsisolated from oral cavity (5 strains) and from bronchi (5 strains)was a kind gift of Prof. I. Teras, Tallin, Estonia. The RAPDmethod using 9 fandum primers (UBC, University of BritishColumbia, Vancouver, Canada) provided complex pattems ofamplified DNA fragments which divided Estonian strains intotwo distinct groups: "bronchial group" which consists of 5bronchial and 2 oral strains, and "oral group" which wasformed by remaining 3 oral strains. Both "bronchial" and "oralgroup" were distinct from T. vaginalis, Pentatrichomonas hom-irtisand Tritrichomonas foetus. Comparison of nuc1eotide se-quences encoding ITSI-5.8rRNA-ITS2 region (approximately370 bp) showed consistent differences between strains of"bronchial" and "oral" groups. Three nuc1eotide substitutionsand two deletions were observed in ITSI, two in 5.8rRNA andtwo in ITS2 region. These results together with RAPD analysissuggest lhal the "oral" and the "bronchial" groups representdifferent trichomonad populations or subspecies. Surprisingly,comparison of the sequences between Estonian strains and T.

tenax Hs-4:NIH (ATTC 30207) showed auly 66 % similarity.Further analysis inc1uding sequences of T. vaginalis, Tricho-monas gallinae, Tetratrichomonas gallinarum, P. hominis, T.foetus and Tritrichomonas mobilensis showed, lhal the Estonianstrains are c1osely related to T. gallinarum. The sequence sim-ilarity between Estonian strains and T. gallinarum was 99 %and 95 % for bronchial and oral strains, respectively, whilecomparison with other species showed >70 % similarity. Mor-phological studies by means of light microscopy (protargolstaining), and by electron microscopy confirmed lhal all Esto-nian strains belong to the genus Tetratrichomonas: the organ-isms possessed 4 anterior flagella, the recurrent flagellum ex-tending as a free posterior flagellum behind the undulatingmembrane, and discoid parabasal aparatus. Our results suggestlhal the Estonian strains represent a new species of the genusTetratrichomonas which can inhabit oral cavity and bronchi ofhumans. Future work will be focused to confirm presence oftetratrichomonads in cases of human respiratory tract infections.

52Heterogenity of alfa- and beta-tubulin in microtubular sys-

tems of Giardia intestinalis. P. TESAROV A and E. NOHYN-KOV A, Department of Tropical Medicine, 1st Faculty of Med-icine, Charles University, Studnickova 7, 128 00 Prague 2,Czech Republic.

It is well known that Giardia intestinalis, a bi-nuc1eated fla-gellate, possesses several cytoskeletal systems based on micro-tubules. Of these, flagellar axonemes and basal bodies representpermanent structures persisting during whole cell cyc1e, whileoccurrence of median body, adhesive disc and mitotic spindleis limited to certain phases of the cyc1e. We used a set of 15monoc1onal antibodies specific to different N- and C-terrninaldomains of alfa- and beta-tubulin, tyrosinated, acetylated andglutamylated alfa-tubulin, phosphoserine and phosphotyrosineto investigate occurrence and distribution of tubulin isoformsand posttranslational modifications in permanent and cell-cyc1edependent microtubular structures. Two-dimensional gel anal-ysis revealed at least ten isoforms of alfa- and three isoformsof beta-tubulin. Immunoblotting showed that all of alfa-tubulinisoforms were posttranslationaly modified with different pattemof tyrosination, acetylation, glutamylation and phosphorylation,and indicated phosphorylation of dominant beta-tubulin iso-formo Immunofluorescence staining showed different distribu-tion of posttranslational modifications within Giardia cytoskel-eton. Immunogold labeling revealed lhal tyrosinated tubulinwas present on surface of outer doublets of axonemal micro-tubules, whereas no label was detected in central pair micro-tubules. In addition, flagella were decorated with antibodies rec-ognizing the very C-terminal end of both, alfa- and beta-tubu-liTI,which, in contrast, did not label cell-cyc1e dependent struc-tures, namely mitotic spindle, median body and adhesive disk.In these, N-terminal ends of alfa- and beta-tubulin subunitswere detected with domain-specific antibodies. Moreover, acet-ylation was the auly posttranslational modification found intheir microtubules. Our results indicate lhal microtubular sys-tems of Giardia contain a significant amount of tubulin iso-forms differing in posttranslational modifications as well as insubcellular localization which could reflect their specific, yetunknown functions.

53Species Composition and Vectors of Avian Trypanosomes-

Molecular Evidence. I. VOTYPKA*,**, M. SVOBODOVA**,

16A ABSTRACTS

P. VOLF** and J. LUKES, *Institute of Parasitology, CzechAcademy of Sciences, 37005 Ceske Budejovice; **Departmentof Parasitology, Faculty of Science, Charles University, 128 44Prague, Czech Republic.

About 100 species of avian trypanosomes (Kinetoplastida)have been named so far, however, most of them are consideredto be synonyms. It remains unclear if each trypanosome speciesparasitizes a single host species or if it occurs in a wide spec-trum of hosts. Mosquitoes, blackfiies, hippoboscid fiies and bit-ing midges were suggested as vectors of avian trypanosomes.We obtained five trypanosome strains from raptors (commonbuzzard, sparrowhawk, kestrel and lesser-spotted eagle), threefrom passerines (rook, blackbird and finch) and five fromblood-sucking insects (blackfiy, mosquito, hippoboscid fiy andbiting midge). Their kinetoplast disc was studied by electronmicroscopy and the minicircular component of their kinetoplastDNA (kDNA) was analyzed in agarose gels. Selected smallsubunit (SSU) rRNA gene s were PCR-amplified and sequencedusing internal primers. In an analysis of trypanosome strainsfrom birds and dipteran insects we have found a clear correla-tion between the minicircle size and the thickness of the kinet-oplast disc. Based on the analysis of the kDNA of fiagellatesisolated from blackfiies Eusimulium securiformeand E. latipe-swe conclude that they are very similar to the trypanosomes ofraptors, and that these dipterans are their vectors. Furthermore,in the phylogenetic trees based on the SSU rRNA sequences afthe trypanosome isolated from E. securiforme, this speciesbranched within the "avian trypanosomes" clade. We foundthree minicircle size categories in rour species of birds of prey,and all of them occur in buzzard. Dur data suggest that onetrypanosome species can develop in different hosts and that onebird species can be infected by several trypanosome species.

Groupement des Protistologues de LangueFranyaise (GPLF)May 23-26, 2000

38th Annual Meeting(Abstracts 54-78)

54Phospholipase A2-like Activity in Leishmania mexicana: Par-

ticipation in the Infection Process. H. ABDALA and P. LEPAPE, Unité de Parasitologie UPRES 1155, Faculté de Phar-macie, Université de Nantes, France.

Some obligate intracellular pathogens use phospholipases-de-pendent mechanisms for entry and survival inside host. We de-scribe in Leishmania mexicana a phospholipase-like activitywhere the highest rate of hydrolysis (25%) was found in themost infectious stage. The enzyme activity was inhibited by twospecific inhibitors of secretory PLAz [the p-BPB (10 f.1M)andthe phospholipid analog Decyl-octyl GPC (0.3 f.1M)]and by thecytoplasmic PLA2 inhibitor [AACOCF3 (15 f.1M)]with values

"of 30.1 ::'::2.2, 87.1 ::'::1.5 and 28.1 ::'::2 %, respectively. Theoptimal conditions for this globa! activity (pH 4.5 to 6.0, 37°)corroborates with the microenvironment found in the parasito-phorous vacuole of the host mononucleated cells. The PLA2-like activity detected in supernatants of promastigotes suspen-sions was Ca2+-dependent, resistant to heating and sensitive toreducing reagent. Further we studied the role of this PLA2-likeactivity inhibition in the invasion process. Parasite treatmentbefore challenge with p-BPB [10 f.1M]reduced the percentageof infected BALB/c macrophages by 56.7 ::'::7 % and the totalnumber of amastigotes per 100 infected and uninfected mac-

rophages by 51.8 ::'::7.4 %. Dur results suggest that this secre-tory PLA2-like could have an active role in the invasion pro-cess. This eventua!ly may be explained by an alteration of themembrane fiuidity caused by an arachidonic acid mobilizationfrom macrophage membrane phospholipids.

55Effects of Media on the Lipase Production by Tetrahymena

thermophila. C. B. PORTOIS,*,**,*** P. CAILLERET,** I.SADOVSKAYA,** C. DEWEER,* J. JEANFILS,** and J. DECONINCK, *Institut Supérieur d Agriculture, Lille, **Univer-sité du Littoral-Cóte dOpale, Boulogne sur met, and ***Fa-culté Libre des Sciences, Lille, France.

Different compounds were tested in fiasks on the whole li-pase production (intra- and extra-cellular) by Tetrahymena ther-mophila. Basis medium (MYE) was composed of skimmedmilk (1 % w/v) and yeast extract (1% w/v). Olive oil or othercommercial oils (0.2% v/v), tween 80, 20 (0.16% v/v) or arabicgum (0.2% w/v) were added. Media composed auly of yeastextract (w/v) were tested with or without glucose (1 % w/v).Fatty acids did not improve Tetrahymena's growth blit stimu-lated the lipase production. Added glucose had the same effect.Different tested oils did not strongly modify growth or lipaseproduction, blit with salmon oil, the lipase production was high-er and more constantly produced. Emulsifiers improved gener-ation time and finallipase production, blit not maximal biomass.

56Macronuclear Chromatin Structure during Resting Excyst-

ment of Colpoda inftata. M.G. CHESSA,* L. GALLUS,** L.TlANO,*** and M.U. DELMONTE CORRADO*, *Diparti-mento per 10 Studio del Territorio e delle sue Risorse, **Di-partimento di Biologia Sperimentale Ambientale ed Applicata,Universita di Genova, Genova, and ***Dipartimento di Biolo-gia Molecolare, Cellulare ed Animale, Universita di Camerino,Camerino, Italia.

The macronuclear chromatin structure of Colpoda inftata hasbeen studied in situ by image analysis, in order to investigatethe activity of regulating mechanisms during resting excyst-ment. Mean histogram on acquired optical microscopy imageswas ca1culated in 2- and 25-day-old cysts, coming from a stan-dard culture, as well as in l-year-old cysts coming from a se-nescent culture. Moreover, the region s corresponding to themost represented gray levels and ranging from 30% to 50% ofthe tota! area of each mean histogram, were identified. Regionsso defined were employed for a segmentation of the digitalimages, in order to identity macronuclear areas similar in theirchromatin condensation. This procedure allowed us to evidencethe dynamic structure of the macronuclear chromatin. In thehighly condensed macronuclear chromatin, the part unable todecondense is extruded as a pycnotic body; afterwards, the re-maining chromatin decondenses. The pycnotic feature of themacronuclear extrusion bodies appears similar to that of thedegenerating nuclei in animal cells undergoing apoptosis. Sup-ported by the 1999 grant of the University of Genoa.

57Flow Cytometric Comparison of Esterase and Radicals Ox-

ygen Intermediate Productions by Ostrea edulis HaemocytesUninfected and Infected by the Proti stan Parasite Bonamia os-treae. N. COCHENNEC and S. GARCIA IFREMER, Labora-