sixth international congress of protozoology

p iiw

SIXTH INTERNATIONAL CONGRESS OF PROTOZOOLOGY 5-11 JULY 1981, WARSZAWA, POLAND

organized and sponsored by the Nencki Institute o f Experimental Biology in Warsaw, with the assistance of the Committee on Cell Biology, Polish Academy of Sciences (Member of ECBO) and the Protozoological Section o f the Polish Zoological Society on behalf of the International Commission o f Protozoology

OFFICERS OF THE ORGANIZING COMMITTEE OF THE CONGRESS

President S. DRYL, Warszawa Vicepresident and Chairman of Scientific Sessions L. KUZNICKI, Warszawa Vicepresidents A. CZAPIK, Krakow A. GREJBECKI, Warszawa M. JERKA-DZIADOSZ, Warszawa W. KASPRZAK, Poznan, Secretary General S. L. KAZUBSKI, Warszawa Executive Secretary E. WYROBA, Warszawa Assistant Secretary General H. REBANDEL, Warszawa Treasurers B. SKOCZYLAS, Warszawa I. WITA, Warszawa Editors of Proceedings S. DRYL S. L. KAZUBSKI J. PLOSZAJ

INTERNATIONALCOMMISSION OF PROTOZOOLOGY

S. DRYL, Warszawa, Poland P. C. C. GARNHAM, ASCOT, England K. G. GRELL, Tubingen, G. F. R. B. M. HONIGBERG, Amherst, U.S.A. S. INOKI, Osaka, Japan J. JADIN, Antwerpen, Belgium S. L. KAZUBSKI, Warszawa, Poland L. KUZNICKI, Warszawa, Poland J. LEE, New York, U.S.A. J. LOM, Prague, Czechoslovakia R. B. Me. GHEE, Athens, U.S.A. B. A. NEWTON, Cambridge, England J. R. NILSSON, Copenhagen, Denmark R. NOBILI, Pisa, Italy I. J. POLJANSKY, Leningrad, U.S.S.R. P. de PUYTORAC, Aubiere, France I. B. RAIKOV, Leningrad, U.S.S.R. B. R. SESHACHAR, BANGALORE, India D. T. SPIRA, Jerusalem, Israel J. H. TERAS, Tallin, U.S.S.R. W. TRÄGER, New York. U.S.A. E. VIVIER, Villeneuve d’Ascq., France

ACKNOWLEDGEMENTS It is a pleasure to acknowledge generous financial support o f the Congress by the Polish Academy of Sciences, the International Union o f Biological Sciences and the United Nations Environment Programme. On the cover: Copper-plate of 1771, Warsaw, by Canaletto

P R O G R E S S

I N

P R O T O Z O O L O G Y

abstracts

of p a p e r s

submitted

to

VI I n t e rnational Congress of Protozoology WARSZAWA,

W A R S

Poland,

Z A W A

July 5-11,

1981

19 8 1

1 Prevalence of T r i c h o m o n i a s i s V a g i n a l i s in Imo State, Nigeria A L E X A N D E R D. W O Z U Z O ACHOLONU, Unive r s i t y of Lagos,

Lagos,

Coll e g e of Medicine,

NIGERIA

Trichomoniasis vag i n a l i s in N i g e r i a was recently r e v iewed and its p rev alence at Lagos U n i v e r s i t y T e a c h i n g Hospital reported (Acholonu,

1980. Nig.

J. Parasit. Vol.

1, in Press).

It was

shown to have b e e n r e c orded in o nly two States of Nigeria and Lagos)

(Oyo

in the W e s t e r n par t out of the 19 States of Nigeria,

and the fact that m u c h of the w o r k on it so far has been in and around the U n i v e r s i t y C o l l e g e Hospital, versity T e a ching Hospital,

Ibadan and Lagos U n i ­

Lagos wa s brought-out.

A nee d for

survey or epid e m i o l o g i c a l studies in v a r i o u s p arts of the country in order to assess the m a g n i t u d e of t r i chomoniasis vaginalis as a public h e a l t h p r o b l e m s in the coun t r y along wit h other sexually tr a n s m i t t e d d i s e a s e s

(STD's) w a s stressed.

The

present report is an ef f o r t to assess the p r e v a l e n c e of Trichomonas v a g i n a l is i n fection in an East e r n State,

Imo,

compare it w i t h the s i t uation in the W e s t e r n part and c o n t r i ­ bute to the m e a g r e kno w l e d g e on its d i s t r i b u t i o n in the country. The study is based on 794 h i g h v a g i n a l swabs urethal swabs

(U.S.)

(H.V.S.),

178 male

a n d 5550 m a l e an d female samples obtained

from out-patients at the Ow e r r i General Hospital and ranging in age from 1 to 60 and above y e a r s as w e l l as 447 H.V.S.

from

adult out- p a t i e n t s from Our Lady's Hospital Emek u k u in Imo State, and covers a pariod from A u g u s t 1978 to M a r c h 1981. 119

(9.6%) of 1241 H . V . S . , 6

(3.4%)

of 178 U.S. an d 74

of 5530 urine samples w e r e p o s i t i v e w i t h T. vaginalis. prevalence of

(1.3%) The

vag i n a l i s in swabbed females is o n l y slightly

higher than that r e c orded at the Lagos U n i v e r s i t y Teaching Hospital

(8.2%) w h i l e w i t h U.S. an d u r i n e it is slightly lower

(4.1% and 1.83% respectively).

The o c c u r r e n c e of T. vaginalis

infection in Imo State is r e p o r t e d for the first time and supports the statement that tr i c h o m o n i a s i s va g i n a l i s is not uncommon in Nigeria,

(loc. cit.).

2 Fauna and Ecology of Free-Living Ciliates of the Northern Part of the Caspian Sea F .G. AGAMALIEV, Azerbaijan State University, Baku, USSR Various regions of the North Caspian (west and east ooasts, oentral part,avant-deltas of the Volga and the Ural) were pro­ spected and 204 species of free living ciliates found ( 166 in the microbenthos ,102 in the periphyton and 73 in the plancton) Many species were common for two and even three biotops. SpecieB of Holophrya, Prorodon. Lacrvtnarla, Tracheloraphis, Trachelonema, Litonotus, Frontonia, TintInnopsis, Aspidisoa, tforticella were among the commonest. Specific psammophilic species occurred mainly In the sands of the Mangyshlak bay, of the west coast and of the central region. Despite low salinity (3-5 °/oo) of the North Caspian, many species found here proved to be common with the more saline Middle Caspian (70.1% of the species found), and South Caspian (66-5 %)* However, some spe­ cies (Chilodonella rlglda, Ophryoedena macrostoma, O.atra, As­ pidisca costata) were found only in the North CaspTan. In ge­ neral , there is a rather high degree of community of the spe­ cies composition between various parts of the Caspian sea. Psammophilic species are the dominant ones in the North Caspi­ an microbenthos, the fine sands (Mo = 0 .1 - 0.4 mm) being the richest in both species number (137) and cumulative population density (up to 6 millions per square metre). The ciliates are most abundant in the top layers of the sand (0 -2 cm) and in coastal regions, and more diverse when the content of the or­ ganic matter in the sediment is moderate ( 1 - 1 .2 % of the dry weight). Among periphytonic ciliates, 73-8 % of the species found were common with the Middle Caspian, and 70.4 % with the South Caspian. However, their population density in the North Caspian reached only 0.7-1.5 million per square metre, as com­ pared with 4-6 millions in other parts of the sea. Most periphytonio North Caspian ciliates are eurythermal. The motile forms are capable of diurnal vertical migrations. The growth of the periphytonic ciliates on test slides is -most repid in shallow-water, wave-protected regions during the warm season (spring and summer).As to the planctonic ciliates of the North Caspian, they had 64*8 % and 58*5 % of species in common with the Middle and South Caspian»respectively. In the North Caspi­ an, their vertical distribution was poorly expressed due to small depths. Only in the avant-deltas »where the water is very turbid, the ciliates occurred only in the surface layers (0 -1 and 0-3 m ) . Two peaks of the abundance of the planctonic cili­ ates were revealed, one in spring and the other in autumn. A zoogeographical analysis of the fauna composition of the North Caspian, in con^parison with other geographical zones, revealed no significant endemism of the free-living ciliates of this region, the fauna consisting mainly of cosmopolitlo species.

3 Genetic, nutritional and physiological eradication of Plasmodium yoelli nlgeriensls malaria in the young and adult Albino mice and Ash wistar rats. PHILIP U. AGOMO, National Institute for Medical Research, Yaba, Lagos, Nigeria. It has been known that some species of malaria parasites are non-lethal to their hosts. However, the mechanism (innate or acquired) of this non-lethality as studied with several malarial parasite species and hosts, is not well understood. Using the blood-stage infection of Plasmodium yoelli nigériensis malaria in rodent hosts, several observations have been made in our labo­ ratory to indicate that this parasite can be killed by genetic, nutritional and physiological mechanisms. The materials and methods used consisted of the injection of 10^ P yoelii nlgeriensls parasitized red blood cells intravenously into groups of young and adult Swiss albino mice or Ash wistar rats (5-6 weeks old and 5-6 months old) before or after treatment/feeding with (a) a non­ rodent diet, chicken growers mash (b) oral glucose-electrolyte therapy and (c) chloroquine 7-chloro-4-(4-dietyhlamino-l-methyl butyl amino) quinone diphosphate. The mean percentage parasitaemia was obtained by making thin blood smears on slides, fixing with methanol, staining with phosphate-buffered giemsa stain and enumerating parasites with the use of oil immersion light microscope and xlOO objective. Results were ex­ pressed statistically after repeating experiments at least twice and performing each analysis with not less than 6 hosts. Results showed that Swiss albino mice were more susceptible to Plasmodium yoelii nigeriensis infection than Ash wistar rats. The young mice or rats were more susceptible to this infection than the adults. Chloroquine was more effective in parasite modulation in adults than in the young. Mice and rats fed on chicken growers mash were completely resistant to this dose of parasite infection. Increase in weights of groups of these rodents placed either on conventional diet or on chicken diet, showed no obvious differences. Terramycin/LA, one of the ingre­ dients present in chicken growers mash but not in rodent diet had no anti-parasitic effect. Oral rehydration fluid, a glucose-electrolyte therapy, modulated the infection in mice. It is suggested that several mechanisms are operating in the eradication of Plasmodium yoelii nigeriensis infection by innate or non­ specific means. These mechanisms include the genetic differences between , _.ia rats, age aifterences (ana reticulocyte numoersj Detween tne k b u or the old and the young, differences between RBC membrane receptors for drug binding, osmotic stress and the nutritional status of the hosts's RBC. These mechanisms should be well-studied, maximized and utilized for the benefit of mankind.

4

Immunoemplification Activity of Glucan in Experimental Amebic Infections SOHAIL AHMAD, M.U. SIDDIQUI and AFROZ-UL-HAQ, Parasitology Laboratory, Department of Microbiology, Oawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Indie Intestinal and extrainte3tinal infections of Entamoeba histolytica generally tend to be chronic because of the ease with wnich reinfections can take place. Since the indivi­ duals normally subjected to this predominantly tropical infection are not always able to change their situation, the chances to avoid or minimize the risk of reinfection are rather few. Chronicity of the infection is further ensured due to lack of a sterilizing immunity. A large number of workers around the world are of the opinion that absence of protective immunity following an amebic infection is due to a somewhat weaker antigenic stimulation provided by these parasites. Efforts were made in this laboratory to amplify the antigenic stimulation in animal models by making use of several non­ specific immunopotentiating agents. Immunoemplifying activity of a polysaccharide extract from Saccharomyces cerevisiae was evaluated by assaying the extent and the magnitude o7 humoral and cell-mediated immune responses in experimental amebic infections. A suspension containing 30mg/ml B 1-3 glucan in GJ.5M sodium chloride /supplied by courtesy of Prof. Pierre □. Jacaues, Biochemical Cytology, Centre Interuniversitaire ae Recherches Multidisciplinaries en * Affections Parasitaries et Mecanismes de Defense de L'Hote, Brussels, Belgium/ was used for simultaneous inoculation with Ej_ histolytica antigen samples. An immunizing dose consisting of i.5‘ml portion of a sonicated antigen extract /6 x 10° amebae/ along with 0.5 ml glucan suspension was inoculated intradermally. Each animal received a total of 4 injections over a period of 8 weeks. Humoral antibody titres were monitored during the course of immunization by means of indirect hemagglutination /IHA/ tests. The cell-mediated immune responses from the test and control animals were assessed on the basis of results obtained from skin hypersensitivity reactions and blast transformation experiments. Activity of B glucan as an immunopotentiating agent was also compared with a few more conventional immunoadjuvants like BCG and Tetanus Toxoid.

5

Experimental Transmission of Babesia bigemina and Anaplasma marginale into Splenectomised and intact local cattle breeds using infective larval tick of Boophilus decoloratus AKINBOADE O.A., Department of Veterinary Microbiology and Parasitology, University of Ibadan, Ibadan, Nigeria

In an experimental infection of Babesia bigamina and Anaplasma marginale into clean and healthy intact and splenectomised local breeds of cattle, the intact,animals showed some positive parasitaemia of B. bigemina from the 4th day of infection up to 2 weeks after which no B.. bigemina was found. Babesia parasites were found Initially at low levels from the 3rd day of infection in splenectomised cattle and this level of infection gradually increased until about 2 weeks when all the cells at any viewed field had been virtually parasitised. This subsided gradually and were overtaken by heavy A. marginale infection as from 20th day post-infection. After about a month, it became difficult to locate the parasites since most of the red cells had been destroyed and the blood had become watery. There was no Anaplasma organism found in the blood of intact animals during the experiment.

6 32

P-labelled compounds released during encystment by Acanthamoeba castellanil: Correlations with appearance of encystment enhancing "activity., ROBERT A. AKINS and THOMAS J. BYERS, Department of Microbiology, The Ohio State University, Columbus, Ohio, USA Encystment of A. castellanil (strain Neff; clone 0S4) Is proportional to the concentration of an encystment enhancing activity (EEA) that accumulates In culture medium during postlog growth, glucose starvation, or treatment with berenil or ethldium bromide (Akins and Byers, 1980, Devel. Biol. 78, 126). Ultrafiltration and gel filtration of EEA Indicate that it is a com­ pound, or compounds, of low molecular weight (nominally ~1,000-3,000). Other studies suggest that EEA may be associated with a modified nucleotide component; therefore, we have tried to label EEA with p. 32 Monolayer cultures were incubated in 0.05-0.10 uCl H_ PO. (Pl)/ml through­ out culture growth and/or during encystment Induced By berenil or glucose starvation. Culture medium was recoveggd at various times during labelling and then analyzed for the presence of P-labelled compounds by PEI anionexchange thin-layer chromatography (TLC). One dimensional chromatograms of labelled medium developed in an acidic high salt buffer (1.0 M NaCl, 1.5 M KH_PO^, pH 3.4) revealed TCA-precipitable activity that remained at the origin, plus Pi and five additional labelled spots. Another four spots were obtained if cell homogenates were analyzed. We have been unable to perform reliable biological assays for EEA with extracts from TLC chromatograms, however, several lines of evidence in{JJcate a correlation between the presence of EEA and one or more of the Plabelled TLC spots. First, several of-the spots became labelled In paral­ lel with increases in the bioassayable EEA titer during postlog growth and during berenil- or glucose starvation-induced encystment. Second,^bioactivity and several of the more slowly migrating TLC spots adsorbed to acidified charcoal and co-eluted in 2.8% NH^OH. Third, EEA bioactiVity was associated with peaks containing a subset of the slowly migrating H jC spots when labelled medium was fractionated by column chromatography using Blogel P4 or DEAE-Sephadex. To date, we have succeeded in identifying several candidate spots, but have not yet established with certainty that EEA becomes labelled in our system, or if it does, that it is only one compound. We have been able to rule out a number of nucleotides, including c-AMP, as candidates for EEA, but have not yet identified the labelled compounds most closely correlated with bioactivity. Further progress toward this objective is being aided by a more sensitive bioassay which utilizes new ameba variants having very high sensitivity to EEA and by other ameba variants (mutants?) that produce higher titers of EEA, thus, substantially increasing the supply of EEA for further purification and chromatographic studies. Supported by NSF grant # PCM-8011696.

7

Toxoplasma gondii; Transmission and Scanning Electron Microscopy of Host Cell Penetration by Parasites of Different Strains GALINA T. AKINSHINA, Department of Biology and Cultivation of Parasites, Martsinovsky Institute of Medical Parasitology and Tropical Medicine, Ministry of Public Health USSR, Moscow Possible mechanisms of penetration of host cell/chicken fibroblasts, L-cell, macrophages /by toxoplasmes and their exodus from infected cells were derived from transmission and scannning electron microscopy of cell cultures infected by three strains of T. gondii: RH, highly virulent and Expert/E-18/ and Thalllandier /T-9/ which were isolated from human placenta by G. Desmonts. Two-day-old cell cultures were inoculated by endozoites and then fixed every 10 minutes during the first hour of incubation at 37°C. Virulent toxoplasmes /RH/ seemed to be invading the cells by active penetration: the parasite usually attaches and enteres the cells with the extended anterior pole that is accompanied by an obvious damage of the membrane host cell with the opening formation. In contrast to the high virulent strain endozoites of E-18 and T-9 strains are often adsorbed on the cell with the posterior pole. Scanning electron microscopy on the initial stages of entry process showed: pseudopods of macrophages surround the parasite by type of phagocytosis while a small portion of L-cell protrudes toward the anterior pole of toxoplasma and seems to be absorbed by L-cell. The parasite however does not remain inert that was confirmed by sharp constriction of its body and different function of spiral subpellicular microtubules. Mode of exodus of toxoplasmes from parasitized cells was active with the perforation formation in host cell membrane. Based on our findings and on the data of Lycke and Norrby /1966/ on enzyme-like penetration-enhancing factor /PEF/ of different strains a hypothesis on the mechanism of Toxoplasma penetration will be presented.

8 RNA Depolymerase Activity in Extracts of Entamoeba histolytica RICHARD A. ALBACH and VIRAPONG PRACHAYASITTIKUL, Department of Microbiology, University of Health Sciences/The Chicago Medical School, North Chicago, Illinois, USA RNA depolymerase(s) activity was detected in a supernatant of a crude homogenate of £. histolytica and ribosome derived ribonucleoproteins (RNP's) recovered from sucrose gradients (Prachayasittikul and Albach, 1981, Bact. Proc.). Coincubation experiments of such extracts with rat liver RNA suggested that depolymerase activity was: 1) lost at 70-75 C for 5 min; 2) inhibited by DEP (37 C 5“20 min); 3) not apparent in a Tris HC1 buffer (37 C 20 min). In concert with the report of Azhar and Mohan-Rao (1978, Zbl. Bakt. Hyg. 230:270-8), who identified a soluble RNase, we found that activity was substantial in 0.05 M phosphate buffered saline (PBS) at pH 7-5, and Mg2+ inhibited activity. However, in contrast to the above report, Ca2+ did not inhibit activity. Subsequent RNase assays were done with several extracts of amebae to determine the relative distribution of activity, Amebae were homogenized and the following samples were recovered: 1) an 8,000 x g pellet; 2) a 23,000 x g pellet; 3) a supernatant which was further fractionated on sucrose gradients to yield three fractions, viz., a 1»S fraction, and the two amebal RNP's (2l*S and 36S). Prior to enzyme assay, the pellets were washed 5x in PBS and the three supernatant fractions were subjected to a preliminary isolation procedure by dialysis of samples in PBS buffered k M urea followed by distilled water to remove the urea. The non-dialyzable material from these three fractions were adjusted to pH 7*5 with PBS and used for assay of enzyme. RNase assays-were done on all fractions in PBS using dialyzed yeast RNA as substrate. Reaction mixtures (30 min at 37 C) contained 1 ml extract (enzyme), 1 ml of 1% purified yeast RNA and 0.5 ml PBS. PCA containing uranyl acetate was added, the samples were chilled, centrifuged, and the OD 260 of the supernatant determined. One unit enzyme is defined as' the amount of enzyme required to release 0.01 OD260 acid soluble material/30 min. Most activity was associated with the k S fraction. The relative activities (total enzyme units) of these fractions were: 4S * 5,000; 2l»S = 225; 36S * 140; 8,000 x g pellet = 2.5; 23,000 x g pellet = 1. This was consistent with the fact that most RNase detected by Azhar and MohanRao (1975) was in soluble form.

9

Pood Relationships of Freshwater Ci liâtes with Other Hydrobionts I.H. ALEKPEROV, Institute of Zoology, Academy of Sciences of the Azerbaijan SSR, Baku, USSR

Observations of both living and fixed material have shown the important role of diatoms in feeding of the ciliates, especi­ ally of Hypostomata and Cvrtophorida. Ten specimens of Trithigmostoma steini IBlochman) contained from 22 to 63 ingested and partially digested diatoms «species of Prorodon contained 7 to 45 diatoms per cell, depending on the season, and Clima­ cestomum emarginatum Stokes contained up to 24 diatoms per cell« Species of Hassula are the most prominent Cyanophyceaefeeders, their cytoplasm being usually full of ingested bluegreen algae. As to predatory ciliates, we repeatedly observed Monodinium balbianii Pabre-Dom. to attack not only Paramecium but also Tritnlgmostoma Cucullulus (Mull.), T. steini (Èlockman), Euplotes gracilis Kabl and others. Cases or ingestion of various ciliates and even rotifers by Coleps hirtus Nitsch were also observed« In 24 hours, 25 specimens of C. hirtus have completely ingested the body of Spirostomum minus killed by a short-term drying« Studies of feeding of other hydrobionts on ciliates are also of interest« We observed intense fe­ eding of chirbnomid larvae on Hassula sp«, each larva ingest­ ing at average 8-16 specimens per hour« Ingestion of ciliates by Cyclops sp« was also frequent, the intensity of utiliza tion of the ciliates by the copepods being however not very high. Cases of ectocommensalism of the perityichs on various hydrobionts included Yorticella nebulifera Mull, and Carcheslum lachmanni Kent epizoic on cladocerans and copepods. Vortlcella anabaena Still«, V, campanula Ehrb« and Thuricola folliculata From.-Kent on”algae. ïhe presence or absence of zoochlorellae in the cytoplasm of ciliates has long been con­ sidered a taxonomic character« However, recent data show this character to be a seasonaly cyclic one. In our conditions .Pa­ ramecium bursaria Focke occurred only with zoochlorellae, But the intensity or pigmentation of the latter showed considerab­ le variation with time«

10 d i l a t e s of the Water Basins of the North-East Azerbai­ jan, A.R.ALIEV, Institute of Zoology, Academy of Scien­ ces of the Azerbaijan SSR, Baku, USSR Investigation during 1979-1980 of the d i l a t e fauna of six rivers, two lakes, and a mineral spring in North-East Azer­ baijan revealed 142 species and forms of d i l a t e s belonging to three subclasses. Running waters contained 48 , stagnant ones, 121 species, and 24 species occured in both; 66 spe­ cies were new for Azerbaijan. The d i l a t e fauna of the Kala-Alty mineral spring (29 species) was not strongly different from that of other wa­ ter basins of the region. However, it contained 12 species (Bleoharisma steini. Monochilum frontatum. Lembadion lucens, Euplotes trisulcatus. and others) which so far were not re­ corded in other basins of the region. In stagnant waters, the fauna was more diverse in shallow regions than in deep ones, in running waters, in protected bays and temporary flood-land pools. Holotrichs predominated in stagnant, and spirotrichs in running waters. in the former, the mass spe­ cies were Lacrymaria olor. Lionotus lamella, Loxophvllum helus. Coleps hirtus. CTtesselatus. Paramecium caudatum. Halteria grandinella, and in the later, Coleps hirtus. Uronema sp., Cyclidium citrullus, Lacrymaria coronata. Spirostomum teres, ^tr'drnïïidium mirabile. Lohmaniella spiralis, and Euplotes gracilis. ~The fauna varied both in species number and population density with the seasons, though some species occurred the year round (Coleps hirtus; C.tesselatus. Chilodonella cucullulus. Euplotes patella) . In stagnant waters, the maximum number of species (38 ) and population density (8,2 million specimens per m 2 ) were recorded in spring, and the minimum (6 to 10 species, 1 . 3 million specimens per m 2) , in winter. In the Agzibyr lake, the ciliates occurred in the se­ diment down to 5 cm depth (in pure sand); deeper layers con­ tained much H 2 S. Lionotus lamella, Spirostomum teres, Aspidisca costata were rrequent in the 4.0-4.5 cm layer, though reaching a combined density of only 1.5 million per m2. The vertical distribution of the ciliates in the sediment depen­ ds on the season. Some stagnant water bodies (Agzibyr lake) had regions where toxic gases (H2S, CH 4) accumulated. Some polysaprobic species (Spirostomum teres, Metopus sp., Paramecium putrinum) showed mass development in these conditions (up to 4-5.5 millions per m 2). In the composition of ciliates of the reservoirs (in the main in the Agzibyr lake) the sea forms are alBo of iauportance (in all 7 species). The analysis of ciliate fauna of the North-East Azer­ baijan reservoirs shows that these species of ciliates in the main are euryxebiontic, most of them can stand the fluctation of oxygen from 0.5 to 13.0 mg/ 1 .

11

Symposium C

Membrane Recycling in Ciliates, RICHARD D. ALLEN, and AGNES K. FOK, Pacific Biomedical Research Center and Department of Microbiology, University of Hawaii, Honolulu, USA

Ciliates provide a unique model for the study of membrane flow and reutili­ zation within one cell. The alveolar system underlies the cell membrane limiting and outlining the sites of exocytosis and endocytosis at the cell surface. Electron-opaque markers administered in pulse-chase studies can be used to follow the endocytic vesicles and phagosomes and, in turn, the flow of membrane within a cell. Using these techniques a complex flow of membrane has been observed in the digestive vacuole system in Paramecium. The removal of "phagosome recycling vesicles" from DB C> BAP. Under the experimental conditions of this study, approximate threshold values for the three compounds and their effect on growth rate are DBT, 1 x 10"6M; DBC, 2.5 x 10"6M; and BAP, 2.5 x 10"5M. Media pH also affects the toxicity of the compounds; generally, more acidic conditions produce enhanced toxicity. These studies model the biological effects of these PAH compounds in the aquatic environment and justify the concern for regulating their release into the environment.

27 Saroocvstis ovIfelIs (Elmerildae, Sporozoa, Aplcomplexa): Modes of Asexual Reproduction In the Cyst Stages T.V. BEYER, J.S. GRIKENENE, N.V. SIDORENKO, Institute of Cytology Acad. Sol. USSR, Leningrad, USSR According

to the generally accepted viewpoint,

metrocytee of

Sarcocystls,are reproduced by endodyogeny to give rise direotly to merozoites.

However, this conception hardly fits

both

the cytological picture of endodyogeny and the ultrastructural organization

of the metrocyte. Large shapeless metrocytee are

characterized

by deep infoldings

in their 2-layered pellicle

and lack the apicomplexan organelles: ronemes, subpellicular microtubules.

conoid, rhoptrles, micMetrocytee are not polar

zoites,8inoe the cell polarity is commonly assigned by the p o ­ sition of the conoid,

rhqptrieB and micronemes.

The E M study

performed on the asexual reproduction of cyst stages of S.ovifelie has suggested

that

fers from endodyogeny.

the mode of metrocyte division dif­ The nucleus of the dividing metrocyte

bee(»aes hypertrophied, the nuclear envelope disappearing. The nucleus proper makes numerous out growings

taking up irre­

gular shape due, in part, to numerous vacuoles the cytoplasm. thus

differing

In metrocytes,

that appear in

The nucleoplasm retains its granular structure from the fine granulated adjacent the cytokinesis is accomplished

cytoplasm. by means

of

deep pellicular infoldings that become even more extended into the cytoplasm

to divide eventually the mother cell

into 4 or

more daughter cells ( rather than into two as by endodyogeny). The newly formed cells are not yet merozoitesI These still re­ semble metrocytes but differ from them in acquiring progressi­ vely a less

folded pellicle

and

anlagen of the apicomplexan

organelles. Such cells are referred to as "intermediate cells" (IC). Most advanced IC generations start to reproduce by endo­ dyogeny giving rise to both IC and merozoites. The latter seem to be the ultimate product

of the asexual reproduction in the

cyst thus corresponding to the gamont stage of other Sporozoa. Merozoites themselves

are unable

their transformation

into gametes

host only.

to divide

within the cyst,

taking place

in the fined

28 Kinematics of Spreading and M i g r a t i o n of Physarum p ol y c e p h a l u m Plasmodium, SOFIA I.BEYLINA, A N A T O L Y A.BUDNITSKY, D O N A T B.LAYRAND, N I N A B.MATVEEVA, V L A D I M I R A.TEPLOV, Institute of Biological Physics, A c a d . Sei. USSR, Pushchino, 142292, M o s c o w Region, USS R

The area changes on the p lane of s u bstratum during the spreading and m i g r a t i o n of p l a s m o d i u m were analyzed using the time-lapse m i c r o c i n e m a t o g r a p h i c data. Th e area m e a s u r e m e n t s w ere m a d e by weighing contoiir-cut figures and s t e r e o l o g i c a l l y . In some e xpe­ riments thickness changes in local regions of living plasmodium were registered optically. Th e study was m a d e on plasmodia form­ ed from isolated p r o t o p l a s m i c drops. In the m o s t of the cases spreading of plasm o d i u m from the drop after regeneration of c ontractile activity is isotropic: a thin pseudopodial sheet on the p e r i p h e r y of the dro p forms along the w hole p e r imeter or the m o s t part of it, the shape of plasmodium remains nearly round as the frontal zone and zone of channals appear and inner holes arise with formation of strands. After transformation into polyt a c t i c form the plasmodium often conti­ nues to spread r a d i a l l y from the center. A s to the rate of spreading, the process can be divided in several phases: the latent phase (L), w h e r e d eformation of the drops is not accompanied by a change of the area, the phase of slow spreading (S.) at w hich the area linearly increases nearly two-fold and the phase of rapid spreading (S«). Growth of the area of spreading in S_ is also essent i a l l y linear and can be approximated as a rule oy one or two straight lines. Whe n p r o p a ­ gation is isotropic the rate increases in each subsequent linear segment,when anisotropic - the sequence can be disturbed. The area remains constant for stable d u m b b e l l-shaped plasmodia. Visua l i z a t i o n of channels and arising of holes on the sheet d u r ­ ing strands formation are not accompaned by any change in the rate of spreading area increase. The frequency of contractile activity oscillations is shown to be m a x i m a l in phase S^, the transition into S_ phase is characterized by appearance'1'of a lower frequency; The transformation of plasm o d i u m into a triangular migrational form is considered. V a riations of the total area of migrating plasmodium and the areas in zones defined by secants normal to the dir e c t i o n of m i g r a t i o n are analyzed. The area changes are shown to have a linear character. The extent of flattening cyc­ lically changes during m i g r a t i o n due to v a r i a t i o n of relative rates of the increase in the area of the leading portion and the decrease of the trailing one. C o mparing the kinematics of moving behavior for plasmodium and fibroblasts (Smolyaninov, Bliokh, 1976) it is noted that they are essent i a l l y common.

29 In-vltro cultivation of Plasmodium falciparum from an Indian tribal population P. K. BHATTACHARYYA and T. M. GHOSH Department of Protozoology, School of Tropical Medicine, Chittaranjan Avenue, Calcutta-7000 73, India, P.O.B. 12380 Failure of malaria control by the existing chemotherapy and vector control measures have led us to look for ah immunological approach for malaria control. The in-vitro cultivation of malaria parasites therefore comes in. It is a pre-requisite for the source of malaria antigen, a primary requirement for any immunological studies of the disease. Since the successful cultivation of P« falciparum by Träger and Jensen (1976), we have been attempting the in-vltro cultivation of the parasite, being collected from an Indian tribal population at the Ajodhya Hills of Purulia district. West Bengal. This area is situated about 3 6 0 Kms north-west of Calcutta with an average altitude of 3 6 O meters. We are using the modified candle-jar technique of Träger and Jensen and observed the following: P. falciparum does not show the normal erythrocytic schizogony in in-vitro cultivation in the presence of a higher percentage of alkali resistant "haemoglobin (HbF) and/or sickle cell haemoglobin (HbS). Gametocytogenesis of P. falciparum in the presence of HbF/HbS in-vitro has been a prominent feature. A very low parasitemia occurs in individuals having higher HbF or HbS in endemic and non-endemic areas of P. falciparum. Possible interactions of P. falciparum with these abnormal haemoglobins will be discussed. Financial support of the Indian Council of Medical Research (ICMR) is gratefully acknowledged.

30

Symposium on Trichomoniasis

, Bialystok

Trichomonacidal Activity of New Water-soluble Derivatives of Polyenic Antibiotics DANUTA BIALASIEWICZ, ALICJA KURNATOWSKA, JOLANTA KWA!§NIEWSKA Department of Medical Biology and Parasitology BMI, Medical Academy, Lddi, and ZUZANNA KOWSZYK-GINDIFER, Tarchomin "Polfa", Warsaw, Poland To characterize biological properties of original Polish water-soluble polyenic antibiotics /1979,Acta Pol.Pharm.,36 ï 173/ were estimated the influence of N-methylglucamine salts of N-glucosyl amphotericin B, nystatin and polyfungin deriva­ tives on axenic strains of Trichomonas vaginalis /T.v./ on Roiron-Ratner medium. The mycostatic effect of these compounds was previously shown /1981,Acta Pol.Pharm.,38, in press/. The lethal concentrations of new salts for 50 % of T.v. popu­ lation /CL ç q /, after 30 min., were calculated from the linear regression equation on the basis of dose-response curve, accor­ ding to the method worked out by Kadlubowski /1961, Wiad. Parazytol., 7 : 515/* As the new derivative of polyfungin appeared to be the most trichomonacidally active it was used for further investiga­ tions in the course of which its action against 30 axenic strains of T.v. was estimated. Analysing the distribution of CL 50 values for 29 strains it was found that they are in large zones of variation from 8.79 to 2390 mg%; one T.v. strain was not sensitive to new salt of polyfungin. The difference betwe­ en the lowest and the highest concentration value CL c q of the compound obtained for different strains is statistically signi­ ficant / P < 0 ,001 /; arythmetical mean + standard error of the CL 50 value for 29 strains of T.v. is 357 ± 3.55 mg%. The influence of these compounds on the growth curves of T.v. population was investigated in continual experiments; the con­ ditions: axenic culture of T . v . on Roiron-Ratner medium at 37° C; adequate compound concentration dissolved in buffer solution of 0.85 % NaCl, pH 6 .8 ; 50 000 T. v . in 1 cnr of the medium in "0” time; measurement in Thom-Zeiss chamber for 72 hours at six hour intervals; for every T.v. strain 4-5 concen­ trations of compound- were used; control - T.v. strain on Roiron-Ratner medium without examined compound. The analysis of growth curves was made by calculating /Kadlu­ bowski method/ the growth coefficients /r/ and the maximal number of population /Nm „/ in relation to the control./1969* Wiad.Parazytol.,15 : 439/; the growth coefficients were cal­ culated for the logarythmic phase /+ r/ and the dying phase

/ - r/. It was shown that all investigated compounds inhibited the growth of T . v . population. New derivative of polyfungin showed particularly high antitrichomonad activity as compared to other investigated compounds.

31 Repetitive D N A Cont e n t in P l a s m o d i u m b e r g h e i :Its Relevance in Gametog enesis

BIRAGO cî,BUCCI-ORFEI A?,DORE

e Î,FRONTALI

et,ZENOS! P?,

+ L aboratorio di B i o logia Cellulare e Immunologia, ° L a b o ratorio di Parassito l o g i a , I s t i t u t o Superiore di Sanità, Roma,Italy

The repetitive fraction of P,berghei genome

( 16% of total DNA)

appears to be essential for successful conjugation,as revealed by o o c y s t formation,in the m o s q u i t o e s , b u t d i s p e nsable for v ege­ tative reproduction in the verte b r a t e h o s t .P .berghei NK65 s train,originally p r oducing viable g a m é t ocytes,has b e e n syringe passaged in mice for m a n y m o n t h s , a n d the following parameters periodically measured:

a)amount of r e p e t i t i v e DNA,determined by

kinetic ren a t u r a t i o n experiments op t i c a l l y monitored;

b)pre­

sence of m o r p h o l o g i c a l l y identifiable gametocytes; c)formation of oocysts in m o s q u i t o gut. The observations w e r e c o ntinued up to the p oint w h e n viability of the still m o r p h o l o g i c a l l y i d e n t ifiable gametocytes is spon­ taneously and irreversibly lost.The loss of capacity of active gametogenesis is accomp a n i e d b y a r e d uction of the repetitive fraction from 16% to 3-5% of the entire genome. Several pure clones w e r e deri v e d from the NK65 strainsall of them contain the d i f ferent sexuated and asexuated forms. The possible role of gene a m p l ification as the molecular basis of d i f ferentiation into sexuated forms is discussed.

32 Non protein nitrogen as a factor influencing

the popula­

tion density of rumen ciliates in vitro Jolanta Bochenek, P.Muszyhski, T.Michalowski Department of Animal Physiology, Institute of Zoology, University of Warszawa, 02-089 Warszawa, Zwirki i Wigury 93»* Poland The relation between the population development of the rumen protozoa and presence of N.P.N. /non protein nitro­ gen/ in the food is still not well known. In this experiment the rumen protozoa Entodinium caudatum and Eudiplodinium affine were cultivated in vitro in the solution of mineral salts. The urea was the sole source of nitrogen or the supplement of the plant protein. Barley starch, cellulose, pectins, soya protein, finely ground Leguminosae seeds and hay were the solid ingredients of the diets used. There was no ascertained possibility of long - term cultivation of the ciliates using urea as the sole source of nitrogen irrespective of the kind of carbohydrate added. The urea supplementation of food comprising the flour from vetch, field-pea, lupin, beans and pea stimulated the development of the ciliate protozoa population which was manifested by the protozoa number increase of ca 25 -9S& when,compared to the controls. The negative effect on supplementation of soya protein with urea was also observed.

33 Positionnement des cinétosomes oraux au cours de la régénération buc­ cale chez Stentor ooeruleua. Jacques BOHATIER, Département de Zoologie et Protistologie, B. P. 45, 63170 Aubière (France) La différenciation et la mise en place des organelles buccaux chez stentor ooeruleu8

ont été étudiées en microscopie électronique ê transmission pen­

dant la régénération orale. Une phase d'intense prolifération cinétosomienne conduit d'abord à la réali­ sation d'un primordium qui est un champ cinétosomlen inorganisé. A l'inté­ rieur du champ, on observe, de manière précoce, des associations de cinéto­ somes par deux, l'un pourvu de dérivés postciliaires, l'autre d'un dérivé transverse, les deux cinétosomes d'un même couple étant disposés obliquement l'un par rapport à l'autre. L'étude des étapes successives de la morphogénèse montre : a) que les diffé­ rents couples ont des orientations quelconques au sein du champ cinétosomlen, ce qui permet de le qualifier de "champ anarchique" ; b) que la différencia­ tion ultérieure de la ciliature orale gauche (adorale), puis droite (parora-

1e) peut être interprétée, au moins au début pour la ciliature adorale, com­ me une mise en place progressive de ces couples clnétosomiens. Dans la partie gauche du primordium, et ê tous les niveaux, les couples s'a­ lignent régulièrement selon un même axe transversal; les membranelles adora-

1es doivent donc, dans un premier temps, être considérées comme des succes­ sions transversales de paires cinëtosomiennes. La troisième rangée de ciné­ tosomes n'apparaît que secondairement. Dans la partie droite du primordium les couples réalisent un alignement lon­ gitudinal. Le positionnement précis des couples s'effectue progressivement et aboutit â une configuration de type "stlchodyade" au niveau du champ frontal du Cilié et de type "paires inversées" au niveau du cytopharynx régénéré. L'ultrastructure de la parorale chez le S t entor est, par conséquent, compo­ site. Ces observations montrent d'une part la possibilité d'élaboration d'une parorale du type "stichodyade" chez un Polyhymenophora et illustrent d'autre part le fait que la mise en place d'un couple cinétosomien dans la position d'une paire ou d'une dyade chez une même cellule est directement fonction du territoire cellulaire.

34 Ecological Characteristics of Unicellulars (Ciliata, Flagellata) in the Baltic Sea EIMIRA BOIKOVA, Institute of Biology of the Academy of Sciences of the Latvian SSR, Riga, USSR The study of ecosystems as complete functional units appears to be a necessity, however the information about unicellulars of the Baltic Sea is rather scarce. Investigations on the spatial distribution of unicellulars have been carried out in the coastal waters in 1978 during the complex expedition of the Institute of Oceanology of the Academy of Sciences of the USSR, but the investigations in the central Baltics were per­ formed in summer of 1979 during the III Soviet-Swedish expe­ dition. Vertically, under homothermal conditions, the amount of infusoria changes gradually. Changes in quantity as well as in specific composition of protozoa

have been observed in

stratification of temperature and salinity. In flagellates the amount decreases for 1-2 degrees vertically after being maximum on the surface layer. In the coastal area an increase in density of unicellulars horizontally has been observed in the zone of blending of marine and fresh-waters, but in the open Baltics it takes place in the area of the Gotland Sea. Facts on biomass clearly speak about the prevailing role of flagellates in summer plankton of unicellulars. High density of protozoa

can be explained by the fact that the Baltic Sea,

being a shelf sea, belongs to the area of the World ocean of high productivity. The first results obtained from complex investigations on two groups of unicellulars show high suscep­ tibility of protozoa

to the marine environment and their eoolo-

gical importance in plankton communities of the Baltic Sea.

•35 Ultrastruotural Changes of Micronucl eus of Paramecium bur sari a induced by Local UV-irradiation. OLGA N. BORCHSENIUS and SERGEJ I. FOKIN, Laboratory of Invertebrate Zoology, Biological Institute, Leningrad State University, Leningrad, USSR The ultrastructural changes of generative nucleus was studied at different times after local UV-irradiation* in 0.5-7 h, 2, 6, 20 days and 2 years. It was found that the irradiation (ex-

p

positional doze - 260 J/m ) doesn't entail to the loss of micronuclei (MI) in descendants. I n our early studies with light optics these UV-clones were identified as amicronuclear ones. I t shows that the question about the presence of MI in P. bursaria cells is very complicated and should be carefully reconsidered. B y electron-microscopical study the changes of micronuclear envelope were revealed. I n 2-6 days after UV-irra­ diation MI became multilobed and the breaks of micronuclear envelope were observed. During the month after irradiation the total amount of chromatin in MI diminished. Also the process of chromatin decompactization was observed. But some of such MI were on the anaphase stage of division. The change of morphofunctional state of MI causes the macronuclear fragmentation. This fact suggests the existence of nuclear interactions.

36

Enhacement of the Fluorescence Intensity of Dansyl Labelled Paramecium Evoked by Removal of Surface Components G. BOTTIROLI, E. WYROBA*, and P. GIORDANO Centro di Studio per 1 Istochlmica, Pavia, Italy and Department of Cell Biology, Nenckl Institute of Experimental Biology, Warsaw, Poland Cytofluorometric and microspectrographic analysis has been applied to examine cycloheptaamylose-dansyl chloride /CDC/ binding to trypsin pretreated cells. Such a supravital and mild treatment has been shown to remove the most external components of Paramecium surface coat and evoke faster response to homologous antiserum /E. Wyroba - Cell Tiss. Res. 182, 1977/. Ciliates /P. aurelia, strain 299 s axenic/ have been digested with trypsin /0.25 mg per ml of incubation medium containing about 104 cells/ for 30 min at 4°C and subsequently washed twice with Tris-HCl buffer, pH 7.6. Labelling with cyclo­ heptaamylose-dansyl chloride was performed at room temperature for 20 min. Intensity of fluorescence emitted by single cells has been measured with a Leitz MPV microphotometer using a variable circular diaphragm. It has been found that fluorescence intensity of treated cells was much higher than in the control: the ratio of intensities varied from 3 to 4. On the other hand no difference was observed in the emission spectra obtained by means of a Leitz microspectrograph: the labelled digested ciliates as well as the control ones showed maximum fluorescence at the same wavelength. This indicates the specific binding of CDC to trypsin-modified surface coat demonstrating that our method of supravital labelling of Paramecium with cycloheptaamylose-dansyl chloride /E.Wyroba, G.Bottiroli and P.Giordano, 1981/ enables to follow the alterations occurred on the cell surface*

37 POSITION TAXONOMIOUE DE DACTYLOSOMA D ’APRES L'ETUDE ULTRASTRUCTURALE Y. BOULARD, I. LANDAU, Zool., M.N.H.N., Paris et E. VIVIER, Biol., Univ. Sei. Lille FRANCE

Des hématies de sang de Grenouille, parasitées par VactutoAOma. examinées au microscope électronique, ont montré la présence de zoïtes et de différents stades de schizogonie. Les zoïtes, placés dans une vacuole parasitophore, présentent la structure typique des Apicomplexa : pellicule trimembranaire avec micropores, comple­ xe apical avec rhoptries, micronèmes, conoïde et anneau d'insertion des microtubules sous-pelliculaires. L'examen du cytoplasme révèle les ultra* structures classiques : vésicules multimembranaires, mitochondries, golgi réserves lipidiques ; mais aucun grain d'amylopectine n'a pu être identifié.

fUUlCUum,

Les images de schizogonie montrent des divisions nucléaires de type eimerien ; la génèse des mérozoîtes s'effectue par un processus d'exogénèse assez classique qui peut cependant débuter assez profondément et donc s'apparenter partiellement à de 1 'endogenèse. Ces résultats entraînent une modification complète de la posi­ tion taxonomique de VcutLtljZo&omOL. En effet celui-ci était placé au voisina­ ge des BabeA-ia, et Th&lCe/Ua et constituait le genre type des Dactylosomidés, au sein des Piroplasmes. Or il s'avère que Vadtijto&onCL s'apparente aux Eimeriidés par la présence d'un conoïde et par le mode de division nu­ cléaire ; il diffère des Piroplasmes par l'existence d'une vacuole parasitophore et par l'organisation cellulaire. En conséquence, le genre Dactylosoma ne peut être conservé dans le groupe des Piroplasmes, pas plus qu'il ne peut être inclus dans les Hemosporidies : il doit être rapproché des Eimeriidés o& sa position précise sera déterminée après connaissance de son cycle complet.

38

Phosphorus and nitrogen excretion rate by free living planktonic Ciliates LUCYNA BO'.VNIK-DYLlftSKA, Department of Hydrobiology, Institute of Ecology, Polish Academy of Sciences, Dziekandw Lesny near Warsaw, 05-150 {.omianki, Poland The excretion of two dissolved nutrients: inorganic phosphorus P-P04 and nitrogen N-NH4 by several species of free living planktonic Ciliates was measured in laboratory conditions. Excretion rate of phosphorus was measured by using blue-moli bdenian method with addition of ascorbic acid and following extraction to hexanol by Standards Methods /1968/. Nitrogen was determined after Prohazkova /1968/, bispirazolone method. It was found: phosphate and ammonia excretion rate is dependent on Ciliate biomass. Relationship between excretion rate - E/in/jg x specim. x hour / and individual weight - W/dry weight in mg/ in following equations of regretlon have been established: for phosphate - E = 0.00154 x w"0,515 for ammonia - E = 0.02047 x w“0,738 PsNratio in products of excretion was about 1:3 in smaller organisms /as Colpes hirtus, Epistylis rotans/ and increased to about 1:5 in higher i.e, Trachellus ovum and Dileptus anser

39 Evidence for a unique method of feeding: The early feeding stage of the ciliate Ascophrys Deroux,1974 P. C. BRADBURY, Department of Zoology, North Raleigh, USA, G. DEROUX, Station Biologique, and A. CAMPILLO, Institut des Pilches, 29211

fine structure of the rodor Campillo and Carolina State University, 29211 Roscoff, FRANCE Roscoff, FRANCE

Ascophrys rodor, a parasite of Palaemon serratus has been identified as an apostome ciliate, because its tomite has a rosette and an infraciliature resembling those of foettingeriid apostomes. The sole diet of the feeding stage is the exoskeleton of its host. The tomite settles on the surface of the exoskeleton and secretes a heavy cyst wall which surrounds it completely except at the mouth. The ciliate undergoes a striking metamorphosis to the feeding stage, the fine structure of which resembles neither the Ascophrys tomite, nor the feeding stage of any other apostome yet described. Unlike Terebrospira or Synophrya which tunnel through the exoskeleton, Ascophrys remains on the surface, digesting the exoskeleton, presumably by secreting enzymes that are released through the oral hatchway in the cyst and attack the exoskeleton directly beneath the cyst. The exoskeleton visibly suffers a step-wise degradation. The soluble products of degradation are taken up in food vacuoles by the ciliate. Gradually the ciliate, cyst wall, epicuticle, and all, sink into the cavity formed by the loss of exoskeleton as food. The feeding apparatus is unique. The cytostome is surrounded by short cilia. A cytopharynx bounded by complex microtubular and dense elements extends deep into the cytoplasm. The arrangement of these cytopharyngeal elements as a tube and.the changes in their configuration along the length of the tube suggests that the tube acts as a pump. The phagoplasm is full of the organelles that store membrane for food vacuoles. Numerous vacuoles containing materials of widely varying densities are scattered through the cytoplasm which is filled with lamellae of endoplasmic reticulum. Other conspicuous organelles in the cytoplasm are tracts of tubules,, bearing patterns of granules on their surfaces. Similar tracts' of tubules in another ciliate have been implicated in the function of the water expulsion vacuole. The appearance of the cytoplasm at this stage in the life cycle confirms the view that the early feeding stage is a period of intense physiological activity involving all the organelles.

40 Réexamen de 1 'ultrastructure de Trypanophie g r o b b e n i , Flagellé parasite de Siphonophores G. BRUGEROLLE et M. CHARNIER, Zoologie Clermont-Fd. et Faculté des Sciences de Nice (France)

L'étude ultrastructurale montre que l'organite sécrétoire antérieur de la forme flagellée de T. grobbeni est entouré par une seule membrane dépourvue de crête et ne peut être assimilé â un kinétoplaste qui est un dérivé mito­ chondrial. La recherche cytochlmique des polysaccharides révèle la présence de nombreux granules glucidiques dans le cytoplasme de la forme flagellée et de la forme grégarinienne. Il ne paraît pas exister de différence fondamentale entre la structure de la forme flagellée et celle du grégarinien (mitochondrie, périplaste, réserves). Plusieurs caractères tels que : l'insertion des 2 flagelles, le périplaste avec 3 membranes superposées, des micropores et des microtubules sous-ja­ cents, la forme des crêtes mitochondriales et les réserves rapprochent ce parasite de Spiromonae perforons, de certains Dinoflagellés et des Sporozoaires.

41

Symposium B

In vitro Cultivation of Trypanosoma (T.) brucel

RETO BRUN, Swiss Tropical Institute. CH-4051 Basel, Switzerland

Considerable advances in the continuous cultivation of the Trypanozoon subspecies (Trypanosoma brucel, T_. rhodeslense, _T. gambiense) have been achieved during the past 5 years, especially with regard to the blood­ stream stages. Since the beginning of the century several culture media have been developed for the procyclic trypomastigotes. However, the de­ velopment to the metacyclic forms could so far not be induced in a quan­ titative and synchronized way. The only system in which metacyclic forms occurred regularly in small numbers used head-salivary gland explants of tsetse flies (Cunningham S Honigberg, Science 197, 1279, 1977).

Bloodstream forms of 2 monomorphic T_. brucei stocks could for the first time be continuously grown over a feeder layer of bovine fibroblast-like cells (Hirumi et al.. Science 196, 992, 1977), but this system does not allow the propagation of pleomorphic 7_. brucel stocks. Recently a series of new in vitro systems for the cultivation of bloodstream forms has been developed in our laboratory. Several feeder layers isolated form labora­ tory animals were cross-checked with different sera to test for growth support of 3 Trypanozoon subspecies. The best system found for all 3 sub­ species consisted of fibroblast-like cells from embryos of Microtus montanus, a North American vole, with HEPES buffered HEM and 15% inactivated rabbit serum. For T_. rhodesiense and T_. gambiense 15% human serum gave even better results. The in vitro bloodstream forms retained their ihfectivity for mice, and a surface coat was always present. These forms could be cyclically transmitted through tsetse flies and the metacyclic forms from these flies could be brought back into culture.

The in vitro culture for bloodstream forms offers new possibilities to study the phenomenon of pleomorphism and antigenic variation. It can be used to test drugs, and as a source of antigen for immunization studies. Finally a new test could be developed for the characterization and isola­ tion of human serum resistent bloodstream populations.

42 Influence of chemotrypsln in the iji vitro growth of Leishmania mexicana ama zonensis in mouse peritoneal macrophages and in_ vivo in albino mice

ELIZABETH BRUZUAL, Escuela de Medicine versidad Central de LUCILA ARCAY & FELIX TEJERO, Instituto cias, Universidad Central

J.M. Vargas, Fac. de Medicina, Uni — Venezuela, Caracas, Venezuela. de Zoologfa Tropical, Fac. de Cien— de Venezuela, Caracas, Venezuela

Mouse peritoneal macrophages were activated with 0.5 ml GKN solution intraperitoneally administered one day before the were extracted by wa — shing the mouse peritoneal cavity with GKN. The harvested liquid was cen — trifuged at 800 rpm/ 8 min.; the sediment was resuspended in Medium 199 and the macrophages were placed in Leighton tubes, 125000 in each. They were iin cubated at 37°C for 2 hr. in 5% CCL. The tubes were separated In three gro­ ups: 1.- macrophages + chemotrypsin 1:1000; 2.- macrophages + chemotrypsin 1:5000; 3.- control, macrophages without treatment. Each tube was infected with 250000 Leishmania mexicana amazonensis amastigotes derived from a sus­ pension prepared with an homogenized cutaneous lesion passed through sterile gauze with penicillin and streptomycin added. Leighton's slips were extracted, fixed with methanol and stained with May-Grunwald-Giemsa 30 min., 2, 12, 16, and 24 hr after inoculation. Twenty fields (400X) of free and intracellular forms were counted on each slip. With the results obtained we can appreciate that chemotrypsin 1:1000 favours growth and multiplication of free forms, which reach maximum va^ lue 16 hr. after inoculation, later abruptly diminishing until 24 hr. The intracellular forms are slightly favoured after 16 hr. When chemotrypsin a m centration is 1:5000 phagocytosis is clearly enhanced; in this group the fewest free forms are observed in the whole experiment. The controls exhi­ bit a free form normal distribution (gauss) around 12 hr. The intracellular forms decrease from the second hour. In vivo chemotrypsin influence in albino mice was studied. Albino mice were inoculated in the tail base with a suspension containing 4X10° L_. m. amazonensis amastigotes in o.l ml saline solution. Chemotrypsin was used T:500 with different inoculation routes; one parenteral group and another intradermal. Lesions 1n the parenteral group were smaller than in the 1ntra_ dermal one or the controls, but survival was greater 1n the first one (8 months) 1n comparison with the other (4 months). In vivo chemotrypsin has a bimodal effect, as is usual in many inespecific agents. The histopathology of infected and treated mice was studied in relation to the controls.

43 Activity of ATPase in Mitochondria of Acanthamoeba oastellanii Neff MAiiGORZATA B U D Z I N S K A and JAN MICHEJDA, Biochemistry,

U n i v e r s i t y of Poznan,

D e p a r t m e n t of

Poznan,

Poland

Mitochondria were isolated from trofozoites from stationary phase of agitated cultures, grown on Neff's medium with genera­ tion time of 8 hours, and collected between 1000 g and 8600 g. The activity of Mg-ATPas6, measured in the presence of phosphoenolopyruvate /PEP/ and pyruvate kinase /PK/ was about 60 and -1 -1 500-600 nmoles x min x mg prot. in intact and sonicated /or tritonized/ mitochondria, respectively. K

ATP

1,3 mM was estimated regardless of the presence or absence m , ATP of 0.3$ Triton X-100; when measured without PEP+PK, Km 1.3 mM

and 7 was found in intact and tritonized mitochondria, respecti­ vely. Activity of AP-ase was negligible. Optimal ATP/Mg ratio was 2-4, depending on the presence or absence of PEP+PK and on the degree of integrity of mitochondria. Maximal activity was obtained by pH 7.4, regardless of the conditions of measurements. Dicyclohexylcarbodiimide /DCCD, 15 pM/ and azide /150 pM/ inhi­ bited ATPase activity by 90$, while oligomycin /25 pg x mg” ^ prot/ only by 75$. Half-maximal inhibition was obtained at 0.5 pg x mg

prot. oligomycin,

10 pM azide and 1 pM DOCD. ADP was a

strong competitive inhibitor of ATPase activity /K^=0.8 mM/. ATPase was'activated by some cations in the decreasing order Mg^>Mn>Co, while Fe and Ca were without effect. OTP was almost as effective substrate as ATP, while CTP was not. Presence of native inhibitor of ATPase was studied by digestion with tripsin and by the relase of inhibition by previous energization of mitochondria. Activity and kinetic parameters of ATPase were the same in mitochondria from exponential as well as from stationary phase in spite of somewhat less efficient oxidative phospborrflation and higher activity of alternative respiratory pathway in mitochondria from the exponential phase. Several differences !»• tween -the present results and those obtained by Seilhamer / r. ~n / due to d i ^’ferent technic of mi to­ chondria, isolation and acti vity mease

wents will be discussed.

44

Membrane-associated ATPase of Tetrahymena pyriformis Cilia S.A, BURNASHEVA, Dach Institute of Biochemistry, Acad. Sci. USSR, Moscow, U.S.S.R. ATPase activity protein has been solubilized from membrane of Tetrahymena pyriformis cilia by treatment of isolated cilia with Triton X-100 and the removal of the axonemes by high-speed centrifugation. Electron microscopy and electro­ phoretic analysis show that the ATPase is the result of the solubilization of the membrane proteins and it did not result in the solubilization of the axonemal proteins. Membrane-associated ATPase was purified by gel filtration on Sephadex G-200 and by chromatography on DEAE-Sephadex A-50. Analysis by SDS-acrylamide gel electrophoresis of cilia membrane ATPase revealed two closely spaced bands with apparent weights of 93400 and 85200 dalton. Some properties of ATPase were examined: activity of cilia membrane ATPase is stimulated only by Ca2+, is specific for ATP with optimum pH 7.5. The characteristic feature for this ATPase is inhibition of enzymatic activity by EGTA and by both NA+ and K+ . The role of membrane-associated ATPase in movements of cilia is discussed.

45

T e n t a c l e c o n t r a c t i o n in D i s c o p h r y a collini R.D. B U T L E R a n d C A R O L E M. HACKNEY,

Departments

o f Bo t a n y a n d Zoology, U n i v e r s i t y o f Manchester, M a n c h e s t e r M1 3 9PL,

England

Th e c o n t r a c t i l e t e n t a c l e s of D. c o l l i n i cont a i n a m i c r o ­ tubular axoneme c o v e r e d b y a f i b r o u s e p i p l a s m a n d s u rrounded at its base b y a fibr o u s collar. be indu c e d by u.v.,

Tentacle retraction may

e l e c t r i c a l s timulation o r b y h i g h

external levels of d i v a l e n t cations.

W i t h i n t r acellular

e l ectrodes the m e m b r a n e p o t e n t i a l ha s bee n m e a s u r e d as -30 m V and retraction oc c u r s in r e s p o n s e to c u r r e n t s of as little as 1.25 nA.

T e n t a c l e s retract in r e s p o n s e to the 44

applic a t i o n of c a t i o n s i n cluding Cu 4 .4 .

Zn Ca

44

, Ba an d M g

4.4.

, Sr

44.

, Mn

4.4.

, Ni

,

444

and La .

, but are p a r t i c u l a r l y s e nsitive to

All r e t r a c t i o n s are b l o c k e d in the p r e s e n c e

of the c h e l a t i n g agents e t h y l e n e d i a m i n e t e t r a a c e t a t e or e t h y l e n e g l y c o l t e t r a a c e t a t e

(EGTA).

(EDTA)

X-ray microanalysis

of g l u t a r a l d e h y d e - o n l y f i x e d c e l l s show Ca to b e p r e s e n t in the c y t o p l a s m i c g r o u n d sub s t a n c e an d e l o n g a t e d e n s e b o d i e s (EDB) b u t n o M g w a s detectable.

In C a - r e t r a c t e d c e l l s the

EDB s h o w a t h r e e - f o l d i n c r e a s e in c a l c i u m and it is sug g e s t e d that t hey act as l e v e l - c o n t r o l l i n g reservoirs. T e n t a c l e s o f g l y c e r i n a t e d c e l l s retr a c t on the addition of reaction m i x t u r e c o n t a i n i n g ATP,

C a ++ and M g + + .

A d d ition

of h e a v y m e r o m y o s i n ca u s e s the a p p e a r a n c e of decorated, fibres in t h e e p i p l a s m a n d e l e c t r o n - d e n s e m a t e r i a l in the fibrous c o l l a r region.

O n retr a c t i o n the axonemes pass into

the cell b o d y b u t no c o n s i s t e n t chan g e s occur in their m i c r o t u b u l e configuration.

It is suggested that the con­

tractile m e c h a n i s m p r o b a b l y involves an interaction with actin-like fibres localised in the fibrous collar and epi­ p l a s m w h i c h is m o d u l a t e d b y . c a l c i u m availability.

46 Cortical Morphogenesis d uring the Excystment of Gastrostyla steinii P U RIFICACION CALVO. NAVARRO. Biologia.

A N T O N I O T O R R E S a n d A N G E L M.

Departamento de Microbiologia. Sevilla.

GUTIERREZ-

F a c u l t a d de

Spain

Cortical morphogenesis during the excystment of Gastrostyla steinii has bëen studied using protargol stage of that m o r p h o g e n e s i s

staining.

The initial

is t he a p p e a r a n c e of a field of

d i s p e r s e k i n e t o s o m e s w h i c h u p o n p r o l i f e r a t i o n r e a r r a n g e in t w o discrete regions.

One of them gives both undulating membrane

and fronto-ventro transverse primordia,

T h e U M ' s p r i m o r d i u m is

formely o r iented perpendicular to the cirral one but soon moves to a p a r a l l e l p o s i t i o n . the oral primordium.

The other kinetosomal region originates

Apparition of right marginal cirral pri­

m o r d i u m also h a ppens e arly d u r i n g the morph o g e n e t i c a l events. The morphogenesis of dorsal ciliature begins w i t h the f o r m a t i o n o f t h r e e p r i m o r d i a l d o r s a l rows. right row breaks

T he p r i m o r d i u m of

into two fragments which converge to form a

single caudal cirrus three caudal cirri.

in s u c h a w a y t h a t f i n a l l y o n l y a p p e a r The two dorsal oblique rows are originated

f r o m t h e a n t e r i o r r e g i o n s o f m a r g i n a l cirr i .

47 The Parasite/Host Cell Interface in Microsporidian Infections ELIZABETH U. CANNING, Department of Pure and Applied Biology, Imperial College of Science and Technology, London, SW7 2AZ The mechanism by which microsporidia invade host cells, by inoculation into the cell cytoplasm through a polar tube which pierces the cell plasmalemma, ensures that the initial stages lie in direct contact with cell cytoplasm. Subsequently the parasite/host cell interface may be of 3 kinds: a) without additional membranes; b) modified by interpolation of a membrane of parasite origin; c) modified by interpolation of a membrane of host origin. (a) Direct contact between parasite plasmalemma and cell cytoplasm. In certain genera, e.g. Nosema, the only modification occurs during sporogony when amorphous, electron dense material is added as a coat, external to the plasmalemma. The coat divides with the sporont e.g. N. lepocreadii in Platyhelminthes (Canning & Olson, unpublished results). In Pleistophora typicalis, parasitic in the muscle fibres of fish (Canning & Nicholas,1980) the secreted coat on the plasmalemma is copious and is present around the early division phases. It is permeated by a system of channels which connect with a layer of vesicles at the parasite/host cell interface. It is not certain whether these membrane formations are derived from parasite plasmalemma or are a proliferation of the sarcoplasmic reti­ culum or T-system of the muscle fibres. In sporogony the coat separates from the plasmalemma, is modified by further secretions and persists as a sporophorous vesicle (pansporoblast wall) around a large group of sporoblasts and spores: thus it ultimately provides an effective barrier against exchange of materials between host cell and parasite. Vavraia culicis in mosquitoes has a similar but somewhat thinner coat of secretions at the interface (Canning & Hazard, unpublished results). (b) Development of an additional membrane of parasite origin. A distinctive group of microsporidia, many or perhaps all of which are dimorphic, produce haploid spores after meiotic nuclear divisions. The spores, varying in number according to the number of nuclei in the sporogonial plasmodium (Loubes, 1979), are enclosed in a sporophorous vesicle of enigmatic origin. In Pleistophora simulii the vesicle wall appears to materialise as a separate membrane external to the plasmalemma and becomes thickened by amorphous electron dense material on its outer border (Canning & Hazard, unpublished results). Since true membranes are thought not to originate de novo, the origin of the sporophorous vesicle as thus described must be questioned, although its parasitic source is not in doubt. (c) Development of a vacuolar membrane of host origin. Encephalitozoon cuniculi develops within a vacuolar membrane, for which there is good evidence of host origin (Weidner 1975). In view of the inoculative mode of entry of microsporidia into cells, this vacuole cannot be a phagosome. It is possible that meronts become encircled by host cell endoplasmic reticulum and that degeneration of the e.r. inner membrane follows, rather in the manner of autophagic vacuole formation. A sequence of this kind is most clearly demonstrated in Glugea anomala (Canning & Lom, unpublished results) where the early stages lie in direct contact with cell cytoplasm, the so-called "cylinders" are girded completely by e.r. and the stages of sporogony lie in a vacuole bounded by a single membrane. An interesting variation on this theme is exhibited by an Encephalitozoon sp. in lizards (Canning, 1981) where the vacuoles divide with the meronts. Division of autophagic-type vacuoles has not hitherto been reported. References: Canning, E.U. 1981. Parasitological Topics: presentation volume to P.C.C. Garnham. J. Protozool. special publication. Allen Press Inc., Kansas; Canning, E.U., Nicholas, J.P. 1980. J.Fish Pis., 3^,317-38; Loubes C. 1979. J.Protozool, 26,200-8; Weidner, E. 1975. Z. Parasitenk. 47,1-9.

Symposium D

48 S t r u c t u r e and F u n c t i o n of Hydrogenosoiaes j a Review A P O L E N A CERKASOVOVA,

Institute of Parasitology,

slovak A c a d e m y of Sciences,

166 32 Prague,

and J A R O S L A V KULDA, F a c u l t y of Science, 128 44 Prague,

Czecho­

JIftf C E R KASOV

Char l e s U niversity

Czechoslovakia

The cell of T r i c h o m o n a d i d a p o s s e s s e s n e i t h e r m i t o c h o n d r i a nor peroxisomes. les,

Instead it is a b u n d a n t in m i c r o b o d y - l i k e o r g a n e l ­

the hydrogenosomes.

S t r u c t u r e of the h y d r o g e n o s o m e r e s e m ­

ble s that of a peroxisome.

However,

the h y d r o g e n o s o m e is e n v e ­

loped by two c l o s e l y appo s e d u n i t m e mbranes. ability,

A limited p e r m e ­

specific tra n s p o r t m e c h a n i s m s and the absence of any

c o m m o n type of A T P a s e a r e so far r e c o g n i z e d b i o c h emical p r o p e r ­ ties of the h y d r o g e n o s o m a l membrane. ently a s e m i a u t o n o m i c organelle.

The h y d r o g e n o s o m e is appar­

T h i s s u g g e s t i o n is u n derlined

b y e l e ctron m i c r o s c o p i c o b s e r v a t i o n s of t r a n s v e r s a l binary f i s ­ sion of h y d r o g e n o s o m e s an d by p r e l i m i n a r y findings of the h y ­ d r o g e n o s o m a l DNA.

P r incipal f u n c t i o n of the hydro g e n o s o m e is

the ox i d a t i v e d e c a r b o x y l a t i o n of p y r u v a t e to a c e t y l - C o A cata l y z ­ ed by p y r u v a t e synthase and linked to the synthesis of ATP at the s ubstrate level.

The latter is m e d i a t e d by the h y d r o g e n o s o ­

mal enzymes a c e t y l - C o A succinate transferase, nase and n u c l e o t i d e p h o s p h a t e kinases.

succinate thioki-

Under ana e r o b i o s i s p y r ­

u vate synthase t r a nsfers e l e ctrons to hy d r o g e n a s e that couples the ele c t r o n s w i t h H+ ions to form ^

• U nder a e r o b i o s i s the elec­

trons are t r a n s f e r r e d to o x y g e n by a c y a n i d e res i s t a n t oxidase. H y d r o g e n o s o m e s r e d u c e 5 - n i t r o i m i d a z o l e drugs to a trichomonocidal intermediate.

Th e proc e s s is linked to the anaerobic func­

tion of p y r u v a t e synthase.

R e c e n t l y o b t a i n e d m u t a n t s resistant

to m e t r o n i d a z o l e d i s p l a y neither a c t i v i t y of this enzyme nor that of hydrogenase,

indicating that the hydroge n o s o m a l pyruvate

o x i d a t i o n need not be e s s e n t i a l for trichomonads. sistant m u t a n t s however, enzyme activities.

The drug r e ­

re t a i n h y d r o g e n o s o m e s w h i c h show some

T h e two of the enzy m e s identified,

(NADP)-malate d e h y d r o g e n a s e

(decarboxylating)

the NAD

located in the

m e m b r a n e and the N A D - m a l a t e d e h y d r o g e n a s e located in the matrix m a y have s ignificant r e g u l a t o r y functions in trichomonad m e t a b o ­ lism.

Probably,

the role per f o r m e d by hy d r o g e n o s o m e s within the

cell is m o r e complex than o r i g i n a l l y assumed.

49 Further studies on the life cycle of sarcosporidians in the intermediate host, fcOFIE SERNA, Faculty of Sciences, Charles University, Prague, Czechoslovakia We have studied the way how the mouse-owl parasite Sarcocystis disperse invades the muscles of the intermediate host* Mice were inoculated with 8 x 10

sporocysts. The

asexual multiplication by multiple endopolygeny in the liver of mice is completed within 6 days and the merozoites thus formed are very slender, 5 - 6 x 2pm in size. Seven and eight days p.i. the parasites can be seen in the periphery blood of the host in the form of thick merozoites, 8 - 9 x 5/im in size. As far as can.be judged from the light microscope observation, the merozoites found in the peri­ phery blood stream seem to multiply by endodyogeny. The formation of muscle cysts started in our species already between 10 - 11 days p.i. The course of infection in very young animals was also studied in colaboration with dr J.Gut from the Institute of Parasitology, Czechoslovak Academy of Sciences. One-dayold mice were inoculated perorally with 10^ sporocysts of S.dispersa. Infected mice examined

28 - 69 days later

were found to harbour Sarcocysti3-cy3ts in their muscles. The muscles of the control non-inoculatod mice had no pa­ rasites.

50

Differences in the growth requirements of virulent and nonvirulent strains of Naegleria fowleri LUBOR CERVA, Department of Protozoology, Institute of Parasitology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia Nonvirulent strains of Naegleria fowleri are inhibited by low concentrations of trimethoprim in certain types

of

culture media. The growth of virulent strains remains un­ affected even in high concentrations of this drug.

The

inhibitory effect of trimethoprim is prevented by tracers of folic acid or leukovorin or such media components which contain the folic acid. Differences between virulent and nonvirulent N. fowleri strains, are expressed in other growth requirements, too. Low concentrations of serum, aminoacids and vitamins promoting the growth of virulent strains are unsufficient for the nonvirulent ones. Differentiation of environmental isolates of N.fowleri based on their growth characteristics appears to be more efficient than the use of time-consuming immunological and ultrastructural studies.

51

The India Ink Immunoreaction for the Diagnosis of Toxoplasmosis JOSEF CHALUPSKf, Department of Parasitology» Charles University, Prague 128 44, Czechoslovakia An immunoreaction for the diagnosis of toxoplasmosis using India ink / H R / is proposed. Comparative examination of 255 human sera b y the IIR and by indirect immunofluorescent antibody technique /IFAT/ demonstrated that the IIR is less sensitive than the IFAT but similarly specific. As simple, rapid and inexpensive method the IIR can be usefull especially i n large scale screening.

52 Studies ofi Pro too p a .1in id r from Indian Anoraks, AS IT KUMAR CHAM DR A and AMA7J3SH CHOTTDHUPY Calcutta University,

Calcutta - 700019,

Dept, of ^oo 1oov Tndia

Right species of Pro too pal'in a. seemingly distinct and now to science based on their morphometric measurements and Vinety arrangements,

spread over three different anuran

hosts (Kaloul a pu 1chra taprobanica Parker,

Rana tiqrina

Daudin and R. lironocharis limnocharis Wiegmann) have been communicated in the present paper. So far twelve species of Protoopalina have been reported from India by D i e (1933), uttangi (1951, 1961),

Earl (1974) and Mandal (1975) . Metcalf

(1918) erected the genus Protoopal ina and described a good number of Protoopalines(1923, 1940) from different parts of the globe and subsecruentiy categorised them into eight subgeneric groups

(I-VTt i ) . Amero (1966) named and establi­

shed eight subgenera based on Metcalf's subgeneric groups assigning significant morphological characters,

specially

the number and pattern of kineties on the right and left side, originating from the falx area. All the eight Pro toopal ina. snn. communicated in this paper have been accommodated in four different subgenera. These are P. (Caudlcul ata) Amero, Amero and p.

P.

(longicorpus) Amero,

(Pel lb at id is) subgen. nov.,

proposed by the authors,

P. (Ovalis)

the 1 ast being

in add ition to the eight subgenera

or Amero(iat6), corresponding the subgeneric group TI as suggested by Metcal f (1940)

to accommodate four protoopal ines

not having any posterior formation structure-.

nor with anv tail

53 Peroxidase Activity in Large Freshwater Amoebae C1CILY CHAPMAN-ANDRfiSEN^"Institute of General Zoology, University of Copenhagen, Denmark The large micfco-aerobic Pelomyxa palustris always contains bact­ erial endosymbionts, but no mitochondria; the light type (growth phase) is the most oxygen tolerant stage in the life cycle, it can survive, but not feed, in media in equilibrium with atmos. air for up to 14 days. It is, however, very sensitive to dying after 2 min exposure to 0.03, 0.003%

^2 .^ 2

#2 ^2 '

30 min,

48 hr resp., while the fully aerobic Chaos carolinense survives at the higher concentration, but dies rapidly when exposed to partially anaerobic conditions. Both P.palustris and C.carolinense contain peroxidase activity, as demonstrated by 20 min spectrofotometric recordings at 475nm of homogenates containing 0.13% 3 » 3 f-diaminobenzidine (DAB) and 0.01% H 2 O 2 , buffered to pH 7.4; the reaction was inhibited by IQ-5 M Na azide. When volume-measured Pelomyxa were cut into 2 pieces, of-which one served as uncentrifuged control (C), the other being centrifuged in vivo and separated into a heavy, cen­ trifugal part (H) and a light centripetal part (L), the ratios of peroxidase activity / pi cell volume in the 3 parts of one amoeba were, C:H:L::1:1.6:0.5 (mean of 6 cells). Light microscop­ ic cytochemical localization by the DAB method showed scattered activity in uncentrifuged amoebae, and confirmed that most of the peroxidase was present in the H parts of centrifuged Pelo­ myxa . especially in the most centripetal zone, the "small gran­ ule zone", wnich also contains lysosomes. The majority of the endosymbionts sediment more centrifugally, indicating that these organelles do not contain significant enzyme levels. Preliminary tests (bubble formation by living amoebae in HgC^PVP media, unpubl. method, P.Prent0) indicate that light type Pelomyxa contain no, or very little, catalase, while high activ­ ity was observed in Chaos. These results present further eviden­ ce of physiological and biochemical differences between the gen­ era Pelomyxa and Chaos. + supported by The Carlsberg Foundation.

54 Life-cycle of Trypanosoma avium bakcri and its host-specificity D.K. CHATTERJEE, Research Centre, Hoechst Pharmaceuticals Limited, L.B.S. Harq, ilulund, Bombay 400 000, India T. avium bakeri was recorded from naturally infected red-whiskered bulbuls (Otocompsa jocosa Linn.)« This trypanosome was found to be very similar to T. avium but was larger in every respect. Division stages in the vertebrate and in the invertebrate host (Aedes albopictus) was described earlier. Metacyclic forms were seen only in the mid and hind guts of mosquitoes and were infective to fresh hosts during experimental transmission. The parasite showed very little host restrictions and was able to infect 15 species of birds belonging to 10 different orders. A discussion on the systematic status of avian trypanosome will be attempted to indicate that the avian trypanosome in question Is a "Polytypic species".

55 The lengths of cilia and flagella may be controlled by cytoskeletal links between the plasma membrane shell and the doublet microtubules FRANK M. CHILD, Department of Biology, Trinity College, Hartford, CT, 06106, USA The effective function of cilia and flagella depends, in part, on the length of each shaft. We know that the length depends on the species, on the cortical location, on the idiotype of the organelle, on the cell's genotype, on the cell-cycle stage, on differentiation, nutrition, and on a variety of other factors. Nevertheless, we usually find, when these factors have been controlled, the particular shafts elongate to a reproducible length, with reproducible kinetics. Studies by myself and others have begun to analyze and define the several types of control which regulate the length. In this paper I consider only a type of control which may operate by the gradual development of length-constraining, structural interactions in the shafts themselves during the assembly process. The general form of the model has been published (Child,F.M., 1978, In: Dirksen,E.R., et al.(eds),Cell Reproduction; ICN-UCLA Symp.Mol.Cell.Biol.12: 351-358). Here I present a morphologically concrete version. Three kinds of structures play roles in this model. The first is the set of doublet micro­ tubules (DM) of the axoneme. It is well established that each DM assembles by addition of tubulin dimers to the distal tip of the DM, the initial assembly having originated proximally at the basal body which anchors the set of DMs. The second structure is the membrane shell (MS), a morphlogical element consisting of the organized array of extrinsic and intrinsic membrane proteins lying at the cytoplasmic surface of the plasma membrane. (The term "shell" is used by analogy to the well-studied shell, containing spectrin and actin, lying at the inner side of the membrane of human red cells. This cytoskeletal shell is isolatable, rigid, but deformable.) The MS of cilia and flagella is assumed to be a substantially rigid component. The assembly of the MS is assumed to occur by addition of its precursors to its proximal region (near the transition zone) and by the sliding of assembled MS up the axoneme during elongation. Evidence that the membrane of flagella does assemble proximally and slide up the shaft has been described by G.B. Bouck from studies of the way mastigonemes become attached to the outside of the flagellar membrane. Thus, the DM and MS are two, rigid, axially parallel structures which, because of antiparallel growth, must slide past each other as the shaft elongates. The third structure is the set of radially oriented cytoskeletal switch-linkages (SL) (first described by Pitelka and Child,1964, as membrane-axoneme connectives) attaching the MS to the DMs. Insofar as the SLs firmly connect the MS and DMs, the SLs will constrain the length-wise sliding. Since sliding of MS relative to DM must occur during elongation, the model proposes that each SL can exist in either an open or a closed state, determined stochastically with independence for each SL. . The con­ dition permitting sliding of MS over DM is that enough SLs are open during that time interval. The model next proposes two processes during elongations (1) a freerunning process, unconstrained by SLs, and (2) a maturation process by which SLs get assembled and begin to constrain gradually the free-running process as they increase in number. Computer simulations of this model give elonga­ tion curves approximating those of protistan flagella. The model appears to explain why (a) the rate of flagellar elongation is deceleratory and depen­ dent on existing length, and (b) flagellar length is independent of large shifts in the size of the precursor protein pool. The model also provides a basis for designing genetic, biochemical, and ultrastructural approaches to this problem.

56 Life Cycle Study of a Schizocystis sp.

(Schizogregarine

from an Oligochaete Vorm Eutyphoeua maaoni

Bourne

AMALESH CHOUDHURT, Protozoology Unit, Department

of Zoology,

University of Calcutta, 35 B. C. Road, Calcutta 700 019, A S I N CHATTERJEE, Department Calcutta 700 009,

t Sporozoa)

India and

of Zoology, Bangabaai College,

India

A descriptive'account on the schizogony, gametogony and sporogony of a Schizogregarine protozoa Schizocyatia sp. has been communicated in the present paper.

Vorth mentioning is the fact that for the first

time a terrestrial oligochaete earthworm Eutyphoeua masoni Bourne and its seminal vesicles are recorded as the host and habitat respec­ tively for any schizogregarina, and it is for the first time that a schizogregarine protozoa are

reported from India.

Schizont of the large vermiform Schizocystis sp. is 400yum long, its 225 sq.^um area at the broader anterior end contains 50-60 nuclei with a diameter of 2-3.5 yum, the posterior and more mature narrower end of the schizont contains 15-20 nuclei with a diameterrange of 5-7.5 yum. Gametocysts and sporocysts measure 20-27.5 yum x 6 - 1 0 ^um and 25-30^um respectively. sporozoites measuring 13.8^um.

Spherical spores contains

The Schizogregarine under report

exhibits apparent resemblance with Schizocystis gregarinoides Leger (1909), the only described species of the genus from an arthropod host.

Their mensural differences and biology have been discussed.

To accommodate the present species under the sub family Schizocyst inae and the genus Schzocystis of the family Caulleryellidae, some emendations of character-range for the sub family and genus have been suggested.

57

Speciation in Fresh Water Amoebae I'iA»?ISA CIGADA LEONARD I, Instituto di Zoologia, Via Celoria 10, I 20133 Milano, Italy Fresh water amoebae can be used to study speciation. Amoeba /Mayorella/ spumosa G. i3 the most common species in Italy. Plain, mountain and cultured populations of this species were Studied. Also the Ugandan form was studied. Physiologic aspect as well as morphologic aspect were considered: the former turned out to be more determinate and more precise from the quantitative point of view than the latter. The changes of size and the fission rate of each clone in every month of the year, characters which were used to calculate the variations of the biomass, were studied in particular. It came out that amoebae cultures under standard conditions in laboratory show the same characters as the wild amoebae they originated from. Therefore these physiologic characters, that originally must have been related to the ecologic conditions, somehow set up in the offspring of the different populations. The characters, both morphologic and physiologic, of lombard amoebae show little differences in the various environments whereas the characters of Ugandan amoebae seem to characterize a species similar to Amoeba spumosa but different enough to be considered as a new

species /Amoeba lyncaea Cigada L., 1980/.

58 Control of cell growth and cell division: Analysis of the mutant conical of Tetrahymena thermophila GONTER CLEFFMANN and ECKHARD SCHAFER, Institut fUr Tierphysiologie der Justus-Liebig-Universitat, Wartweg 95, 6300 Giessen, FRG

In the mutant conical of Tetrahymena thermophila the division plane for cyto kinesis is dislocated towards the posterior end of the cell. The resulting daughter cells differ on the average by 50% in cell mass. The plane of macronuclear division is not related to the unequal cell division. Therefore, the cells differing in size obtain similar sized macronuclei. Since this mutant is stable and the average cell size of the population is constant the differences in size have to be compensated. Cell kinetic studies demon­ strate in accordance to the findings of Doerder et al. (J.Exp.Zool. 192, 237, 1975) that the small cells have long generation times and the large cells short ones. However, the compensation of cell size needs more than one cycle. Small and large cells furthermore have been analysed cytophotometrically with respect to rate of increase in protein and RNA. These rates are slower in small cells than in large cells. Two conclusions can be drawn: Firstly, the cellular growth rate depends on the mass of cytoplasm rather than on the amount of temples in a given cell. Secondly, it is evident that for the compensation of cell size the length of.cell cycles are re­ sponsible and not the growth rate which would even increase the differences in size.

59 On the fine structure of a microsporidian belonging to Thelohania.muscular parasite of a freshwater gammaridean RADU CODREANU and DOINA CODREANU-BALCESCU,Institute of Biological Sciences»Bucharest,Romania The Qammarus balcanicua Schlferna,1922 populating the Southern Carpathian streams may have their segmental muscles transformed in white-yellowish stripes by the presence of a microsporidian which we provisionally ascribe to Thelohania muelleri(Pfeiffer. 1894)»Its regularly octosporous moruloid pansporoblasts with a diameter of 8-9p in the fresh state,include under a thin persis tent envelope the typically 8 spores,ovoid-pyriform,refractile, 4-5x2,4p each,possessing a basal vacuole.In electron microscopy the early vesicular undulated sporonts,5,5x5-5x4p,show diploca?rya of 2,8x2,5p(the two nuclear calottes together) with a dark concentration on each side.The vacuolar cytoplasm containing ribosomes is bordered by a unit membrane.Attaining 8p in size, the sporonts form inside a fine pansporoblastic envelope 4 uninucleated daughter-cells,5x2,7p,linked one to another by an is­ thmus. It is not a usual rosette-like plasmotomy,the dividing products being uprolled in a transitory chain.All the intersti­ ces between the sporogonic cells and their pansporoblastic mem­ brane become densely filled with a secretion of sinuous rough microfilaments.A last bipartition of the first four sporogonic cells leads to the 5,5x2,lp octosporoblasts with a doubled bor­ der. This orthomitosis presents the spindle tubules attached to the depressed centriolar plaque lacking polar vesicles.Within the thickened double-walled shell,the mature spore contains a unique large lobate nucleus surrounded by many ribosomes rows with a paracrystalline texture.From its apical distinct ancho­ ring disc,the linear portion of the polar filament traverses the upper sporal half,through the entirely vesicular polaroplast,and forms after this exceptional length,only 4-5 basal turns.The compact filaments of the intersporal secretion incre­ ase in size and some larger tubules appear among them.The octospores of a muscular Thelohania parasitic in a Gammarus from a nearly situated stream,show however 8-11 turns of their polar filament and the intercellular secretion seems to be attenuated.

60 On the ultrastructure of a parenteral microsporidian cf. Noaema serbica Weiser,1963,parasitic in Lymantria diapar DOINA CODREANU-BALCESCU and RADU CODREANU,Institute of Biological Sciences»Bucharest,Romania The studied gypsy moth caterpillars came from forests of the south-western part of Romania and were considered as carrying the nuclear polyhedrosis virus,but their microscopic examinati­ on indicates the simultaneous presence of intracytoplasmic mi­ crosporidian stages»mainly in the fat body and silk gland.There the two pathogens occur generally in distinct cells closely si­ tuated, rarely in the same host-cell.The virus polyhedra are ex­ clusively located only in the hypodermal and tracheal tissues, whilst the microsporidian enter also into the segmental muscles which bear pouches of spores.In spite of similar tissular affi­ nities, this microsporidian is quite different from Nosema lyman triae Weiser,1957 by its ellipsoidal oblong spores of 6-7x23,5p with rounded ends.Therefore it corresponds probably to No­ sema serbica Weiser,1963 both in morphological as in geographi­ cal respect»representing the most northern position(45°N.L.) of the geonemic repartition of this meridional Lymantria pathogen. In ultrastructure,the young rounded,4x3}i sporonts show perma­ nent diplocarya,of 2-3,2x1,7p,double-walled with pores,embedded in a cytoplasm rich in ribosomes and vacuoles.They have few ergastoplasmic lamellae and a thin plasmalemma.The nuclei of the dividing sporonts exhibit their chromosomes attached on spindle tubules and also synaptonematic complexes.The telophasic sporoblasts preserve the diplocarya and become elongate after sepa­ ration, reaching 5,8x3,2p,sometimes with an electron opaque spot in each nucleus.The ripe spores being densely filled with ribosomal row8,the axial coupled nuclei present a polygonal outline with a 0,6-0,8^i diameter in each.The polar filament is very ob­ liquely coiled,forming 11-16,mostly 12-14 peripherical turns, the last distal 2-3 being more internal and narrowed.The apical ly inserted straight segment after penetrating the polaroplast between its upper lamellar part and the subjacent vesicular one is always directed towards the lower range of coils.Some homo­ genous spheroidal inclusions lay into the oval basal vacuole of the spore.

61

ARE THERE TWO SEPARATE PHOTOSENSORY TRANSDUCTION CHAINS IN EUGLENA ?

G.Colombetti.F.Lenci.V.Passarelli.M.Quaglia and G .Rinaldi-Fornaca Istituto Biofisica C.N.R. - via S.Lorenzo 24/2A, 56100 Pisa (Italy)

Many microorganisms exhibit motile responses to light stimuli,such as sudden alterations in swimming patterns known as photophobic (or avoiding) reactions. In Euglena gracilis these reactions are caused by

rapid var­

iations in flagellar position compared to that in normal swimming (flagel­ lar reorientation) and may be brought about by both an increase in light over a certain threshold (step-up photophobic responses) and a decrease in illuminance when the cells are aiapted to a certain light level under thresh­ old (step-down photophobic responses). Now the question arises : are there two different photoreceptor systems for

step-up and step-down responses,or are there only one receptor and two

different transduction chains ? Experiments performed on the wilde type and on a streptomycin-treated mutant of Euglena have shown that the mutant is only capable of exhibiting the step-up response,but the action spectra

in­

dicate that the same photoreceptor systems should be implied . On the other hand even in wilde type cells it is possible to find external conditions that almost cancel the step-down response but have no effect on the step-up reaction,such as for instance the effect of metals like Cadmium or the age of the culture. We tentatively conclude that in Euglena gracilis the two photophobic responses are very likely linked to the same receptor system,but that the transduction of the stimulus from the photoreceptor to the motor apparatus takes place through two different molecular pathways.

62

R. T. Discussion Tiie "PROTOZOA": An Unnatura l Grouping at Any Taxonomic Level JOHN 0. CORLISS, Department of Zoology, University of Maryland, College Park, 20742 USA

Although it has been convenient in the past to treat protozoan groups as comprising a single subkingdom, or even phylum, of microorganisms, sufficient evidence has now accumulated to consider such a conservative decision to be neither wise nor defensible. The "Levine Report" of 1980 lists seven phyla in a subkingdom Protozoa of the kingdom Protista. I suggest that the orga­ nisms called protozoa (with a small "p") should better be distributed throughout the first of the eukaryotic kingdoms, the Protista, in double or triple that number of phyla, with no subkingdoms indicated and with complete abandonment of the word "Protozoa" (with a capital "P") as a formal taxonomic name or unit. Most of the features that protozoa are said to possess in common are not exclusively protozoan characteristics: for example, "eukaryotic," "singlecelled," "microscopic" (in fact, not even all protozoa show the last two). At macromolecular and functional levels, protozoan cells often resemble the cells of organisms belonging to other kingdoms. Certain specialized struc­ tures, internally located or externally produced, may not be found elsewhere — but they are so exclusive as not to be found universally throughout pro­ tozoan groups, either. The protozoa in toto seem to manifest greater diver­ sity than unity. The very word "proto-zoa" is misleading, with its narrow connotation of "first animals." The fact that zoologists were mainly the first to study these organisms, while botanists claimed bacteria and algae, is today of historical interest only. Recognition of independent, specialized groups of protozoa as "phyla" within the vast kingdom Protista, itself defined essentially as the kingdom of basically unicellular-but-eukaryotic forms, should not, in general, pre­ sent difficulties. The great evolutionary diversity apparent has come about through many aeons of special adaptations, and even such disparate groups as the complex ciliates, with their kinetidal systems and nuclear dualism, and the all-parasitic sporozoa, with their complicated life cycles resulting from adjustments to the symbiotic life style, can be accepted as fitting nicely into the kingdom, as long as one does not attempt to "prove" their closeness phylogenetically. From some such separate lines of evolution have undoubtedly arisen the early ancestors of the other three eukaryotic king­ doms, exemplified by multicellularity, the Plantae, Fungi, and Animalia. The principal areas of difficulty in recognizing discrete phyla of pro­ tozoa are related to two major "protozoan" groups: these are the so-called "phytoflagellates" sensu lato and the highly enigmatic myxosporidians or "Myxozoa." The first, largely claimed by botanists and phycologists as be­ ing "algae" (another term best used in the vernacular sense only), involves not only nonphotosynthetic forms of obvious close affinity but also certain colorless "lower zooflagellate" groups not suspected of such phylogenetic kinship until studied by modern techniques (even the intriguing "gymnostome ciliate" Stephanopogon falls into this category). The second, the sometimes multicellular "Myxozoa," not only appears totally unrelated to any other protist group but is claimed, not without reason, to be a primitive (or secondarily degenerate?) group within the kingdom Animalia. I do not accept the latter hypothesis; but I do assign these forms to a highly isolated spot within the broad kingdom Protista. (Support of NSF grant DEB 79-23440 is gratefully acknowledged.)

63

The characterization of 71 strains of Acanthamoeba using starch gel electrophoresis of isoenzymes

MEHELAOS COSTAS and ALAN J GRIFFITHS Department of Microbiology, University College, Cardiff CF2 1TA, Hales

The technique of isoenzyme electrophoresis has been used successfully in the characterization of a number of genera of protozoa which are otherwise difficult to characterize due to a lack of morphological diversity. Following preliminary investigations of laboratory strains assigned to the genus Acanthamoeba of the acid phosphatase and esterase isoenzymes, it was decided that a wider more comprehensive survey using both monaxenic and axenic cultures was desirable. The extended survey included 71 strains representing 19 named species and 21 "unidentified" isolates. The strains fell into a number of groupings which did not Agree well with the species designations based on morphological criteria. The strains could be separated into a number of groupings, which were characterized by both esterase and acid phosphatase isoenzymes. The first grouping of 14 strains included strains of A castellanii, A terriola, A polyphaga, A rhysodes and one unidentified strain. The second grouping of 15 strains included strains of A castellanii, A polyphaga, A cuina and 7 unidentified strains. The remaining strains were placed in a large number of smaller groupings.

64

Relations

trophiques

chez

les P r o t o z o a i r e s

du sol

M a r i e - M a d e l e i n e C O U T E A U X , L a b o r a t o i r e d ’E c o l o g i e Générale, M u s é u m N a t i o n a l d ' H i s t o i r e N a t u r e l l e (C.N.R.S., E.R. 204), 4, a v e n u e du P e t i t C h â t e a u - 9 1 8 0 0 B r u n o y - F RA N C E

Il e st

généralement

sont e s s e n t i e l l e m e n t leurs

exigences

d'une

source

survivre

ne

admis qu e

bactériophages

sont pas

très

cependant,

strictes

de n o u r r i t u r e p r i n c i p a l e ,

grâce à des Dans

outre

est

chez

des

espèces

mycophages

lium ou i n g è re nt

les

spores.

La

dans

les

libère

sols

des

prennent

commence

p ar

numérique

Des

en é v i d e n c e

lots

de sol

il a été mis

champignons.

La relation

qui b r o u t e n t

le m y c é ­

indirecte

la m a t i è r e o r g a n i q u e fongique,

tant p o ur

cell e- ci

les b a c t é r i e s

c o n s i d é r a b l e q ue p o u r

expérimental

ont

été e n r i c h i s

en 5 e sp è c e s Humicola

de c ha mp i g n o n s

f u s c o - a t r a var fus-

a t ra et C l i t o c y b e n e b u l a r i s . Les

tés des

ont

Thécamoebiens

été m e s u r é e s

l'enrichissement

les e s p è c e s

qui

réagissent

Ces

résultats

Mr.

PUSSARD pour

sont mis

et

a permis

en p a r a l l è l e

les A m i b e s

amène â p r é c i s e r

l'écosystème

- sol

biodégradation

de

té du t u r n o v e r

des

et

les 9 mois qui

de m e t t r e

en é v i d e n c e du milieu.

avec ceux o bt e n u s

exigences

par

alimentaires

la f o n c t i o n des P r o t o z o a i r e s

la m a t i è r e

densi­

nues.

la p ar t

sols.

pendant

à c et te m o d i f i c a t i o n

La d é t e r m i n a t i o n des nantes

les

a été mis en p la c e p ou r

c o - a t r a , Stachybotrys

suivi

qui

la r e l a t i o n T h é c a m o e b i e n s - C h a mp ig no ns .

F u s a r i u m o x y s p o r u m , P y t h i u m sp.,

ont

de

s 'e n n o u r r i s s e n t .

Un p r o c e s s u s mettre

de

attaque

nutritives

une e x t e n s i o n qui

une

des

l'absence

de s ub st i t u t i o n .

r e l a t i o n p e ut être

la b i o d é g r a d a t i o n

substances

Protozoaires

trophique

que

capables

les b a c t é r i e s ,

l'importance

car on sait que

et q u ' e n

ressources, a l i m e n t a i r e s

les sols,

on sait,

ils sont

en é v i d e n c e directe

les P r o t o z o a i r e s

de

leur

dans

i n t e r v e n t i o n dans

o r g a n i q u e qui

assure

domi­

la

l'efficaci­

Clonal development and a kind of phenotypic change in Paramecium primaurelia TINA CRIPPA-FRANCESCHI, Istituto di Zoologia dell’UniversitK, via Balbi, 5 -

16126 Genova (Italia)

66 The Presence of Carotenoids in Free Living and Parasitic Flagellates BAZYLI CZECZUGA, D e partment of General Biology, Medical Academy,

Kiliriskiego 1, 15-230 Bialystok,

Poland

Column and thin-layer chromat o g r a p h y was applied by the author for analysis of carotenoids in Euglena rubida as free living and in Trichomanas vaginalis as parasitic representatives of flagellates. The results have shown that in Euglena rubida dominates

fi-carotene and several forms of its d erivatives ma i n l y astaxanthin, w hile in Trichomonas v a g i n a l i s , apart from its epoxide forms

p-carotene,

(^-carotene-epoxide and m u t a t o c h r o m e ) .

The author d i scusses the role of astaxanthin in the biology of free living flagellates species and the role of epoxide forms of

/3-carotene in the biology of parasitic species.

In

parasitic organisms an a t t ention should be paid to the import­ ance of carotenoids in the process of respiration, particular of carotenoids of the epoxide form.

and in

67 Cellular and Humoral Mediated Immunity in Cutaneous Leishmaniasis. Y.L. DANON, E. LIVNI, Z. HARARI, D. MICHAELI, Medical Corps, Israel Defence Forces and the Beilinson Medical Center, Petah Tiqva, Israel Cell Mediated Immunity (CMI) was studied in 33 patients with active cutane­ ous leishmaniasis, 12 immune donors with scars of leishmanial lesion (oriental sore) for more than 30 years, 40 healthy individuals after innoculation of Leishmania tropica and 20 control donors. CMI was assessed by study of Migratory Inhibiting Factor (MIF) production by lymphocytes when sensitized in vitro with Leishmania tropica - (LRC-L-137) extract as soon as two weeks after innoculation, repeating the test every second week to the sixth week. In all patients diagnosis was confirmed by skin biopsies, in most cases multiple skin lesions were observed. X

A

In all patients MIF production was assessed with an extract of 1(T and 1CT parasites and compared to MIF produced to common antigens as after PPD sen­ sitization, tests were performed 3, 6, and 12 months after diagnosis. In 70% of innoculated individuals, MIF production to Leishmania tropica appeared.two weeks after innoculation on the third week only 60% produced MIF and 50% on the sixth week. In no case we could detect leishmanial LRC-L-137 extract induced MIF activity before innoculation. In 12 patients with more than 30-year-old scars, 41% were LRC-L-137 MIF pos­ itives. Among 33 newly diagnosed patients, 58% were LRC-L-137 MIF positive three months after diagnosis, 45% six months after diagnosis and 34% 12 months after diagnosis. In 17 out of 26 of those patients, high IgE levels were detected. We conclude that MIF production due to Leishmania tropica LRC-L-137 sensiti­ zation test can be used as a measure of immunity and may in certain circum­ stances be of help in differential diagnosis.

Supported by ICRF

68

The Effect- of Histamine and its Antagonists on the Glucose Uptake and Utilization of Tet rahymena pyriforrois GL Strain 2s. DARVAS and G. CSABA Department of Biology Semmelweis University of Medicine Budapest, Hungary Previously the ability of Tet rahymena to react with hormones of vertebrates and to form receptorlike membrane pattern was demonstrated. In the present study the effect of histamine and its anta­ gonists, the H^ receptor antagonist phenindamine and the Hg receptor antagonist metiamide on the glucose uptake and utilization was examined. The starved animals were treated for 10 minutes/short term treatment/ with histamine and its antagonists in 10 M concentration solution. Other groups were cultured in medium containing histamine and its antagonists for 4 days, and after it they were starved and treated for 10 minutes /long term treatment/. After PAS reaction a cytophotometric analysis was done and the quantitative changes of polysaccharides /glycogen/ in the cells were demonstrated. By the short term administration of histamine the hexose content of cells increased significantly, while it was markedly decreased after the long term administration, probably as a result of speeding up utilization. As regards the action on the glucose uptake, H^ antagonist phenindamine affected only in a small degree while Hg anta­ gonist metiamide affected in the higher degree than histamine. The analysis of results suggested: - the cells response to histamine or its antagonists is not the same; - the histamine and its antagonists affect differently; - independently of mean values caused by histamine and its antagonists, the number of cells containing more glycogen in the population increased.

69 THE COMPARATIVE CHARACTERISTICS OF THE SIZES , THE GROWTH RATE AND POTENTIAL OF POLYXKNIC CULTURE OF ENTAMOEBA HISTOLYTICA IN SEVERAL MEDIA IT.KH. DAYANChair of Biology, Medical Institute, Yerevan, USSR The growth rate and potential of dysenteric amoeba in polyxenic culture were studied and the comparative biometry of diffe­ rent strains in different cultivation stages was made. The ex­ periments were carried out on 4 strains of EM histolytica iso­ lated from patients with amoebiasis and maintained in liquid nutrient media of Pavlova and Balamuth, in biphasal egg medium of Boeck and Drabohlav, modified by Sh.M. Matevossian. The observations of the growth and the amoeba count were made in chambers. During the determination of growth rate of EM his­ tolytica in the culture it was established that in different strains of dysenteric amoeba in Pavlova medium it is average 14-21 hours, and in Balamuth medium it is 27-33 hours. The growth potential of the tested strains in Pavlova medium was higher than that of Balamuth medium. The comparison of the biometrical measurement results of tro­ phozoite amoeba in culture has shown that the studied strains in Pavlova medium and in the medium of biphasal egg reached maximum sizes on the second day (20,87 +

0 ,3 2 ;

16,09 + 0,08;

17,97 + 0,41; 19,92 + 0,31 in Pavlova medium, and 16,25 +0,09; 14,36 + 0,05; 15,48 + 0,14; 15,29 + 0,11 in egg medium), and in Balamuth medium on the third day of cultivation (16,91 +0,1; 14,68 + 0,04; 17,5 + 0,3; 17,14 + 0,14); the amoeba reached the greatest maximum sizes in Pavlova medium, and the smallest maximum sizes in egg medium. Thus, the study of growth rate and potential of LM histolytica in culture and the biometrical results of trophozoites have shown the dependence of these indices on the composition of nutrient medium, as well as on the duration of cultivation.

70 Remote results of giardiasis treatment taking into account invasion of the small intestine HAZIMA

DEHKAN-HODJAEVA, .RYOYA

SHAKIROVA, Department of Fro

tozoal Diseases, The Isaev Uzbek Research Institute

of Medl

cal Parasitology, Tashkent, USSR Invasion of the small intestine

in humans and animals with

Giardia infection was confirmed by many workers. However the drugs in current

use (furasolidon, metronidazol, etc) are

absorbed essentially In the large intestine and exude with urine and feces. Therefore the efficiency of the treatment is not achieved. Acrichin and its analoges are Absorbed in duodenum and exude

with bile and kill the parasites in the

lumen and within tissue more efficiently. We compared the efficiency of metronidazol, furasolidon and aorichin in tre­ ating patients infected with Giardia with entero-hepato-biliary syndrome. The patients of the first group received three-cycled treatment ( furasolidon, metronidazol and fazigin). In the second group acrichin was added. Period of ob­ servation was 6 months - 4 years. Clinical and

laboratory

data show that the efficiency of the treatment in the first group was 35%, in the second - 92%. Thus the treatment of giardiasis

with acrichin proved the most successful. It

prevents the recurrences killing the tissue form of Giardia lamblia.

71 Voltage-clamp analysis of a mechanoreceptor response in the hypotriche ciliate Stylonychia mytilus JOACHIM W. DEITMER, Abteilung Biologie, Ruhr-Universität, D-4650 Bochum, Federal Republic of Germany

Mechanical stimuli to the cell posterior of ciliates elicit hyperpolarizing receptor potentials due to a conductance in­ crease of the membrane to K + ions (Naitoh & Eckert,1973, J.exp. Biol♦59,33-65)♦ These receptor responses have been investigated under voltage-clamp conditions in Stylonychia using two intra­ cellular microelectrodes. Mechanical stimuli were applied to cilia-free parts of the cell surface using a glass capillary (tip diameter of 2 to 5 pm) connected to a phonocartridge acti­ vated by DC-pulses (see de Peyer & Machemer,1978, J.comp. Physiol.127,295-266). Changes in the mode of mechanical stimu­ lation influenced the amplitude of the mechanoreceptor current, but had no effect on the time course of the receptor current. The reversal potential of the mechanoreceptor current was -88mV at [K]o of 1 mM, and varied with the [K]0 according to the Nernst equation (slope: 57-8 mV). Thus, the reversal potential was identical with the K +-equilibrium potential. The intracel­ lular K + concentration was calculated to be 55 mV, and did not appear to change during the exposures to [k ]0 of between 0.5 mM and 4.0 mM. The receptor current-voltage relationship was linear in the hyperpolarizing direction, but greatly non-linear in the depo­ larizing direction. The exponential decay of the receptor cur­ rent also cHanged with the membrane potential, its time con­ stant (7*5 ms at the resting membrane potential of -50 mV) increased e-fold with a hyperpolarization of 110 mV up to the reversal potential. Beyond the reversal potential the time con­ stant of decay appeared considerably reduced for small receptor inward currents before increasing again with further hyperpolarization. This suggests that the life time of the mechanically activated ionic channels (indicated by the time constant of current decay) is influenced by the membrane potential, simi­ larly as has been reported for chemically activated channels in the postsynaptic membrane (for references see Gage,1976, Physiol. Rev.56,177-247). Tetra^thylammonium (TEA) and 4-amino£yridine (4-AP) specifi­ cally blocked the K-mechanoreceptor current at relatively low concentration (TmM), while procaine, cesium and divalent cat­ ions had little or no effect on this receptor current. The re­ versal potential of the receptor current remained unchanged by these substances. TEA also prolonged the time constant of re­ ceptor current decay by up to 100%, while 4-AP and the other agents had .little or no effect on the current time course. It is suggested that these ciliates may serve as a model for stu­ dying the mechano-electrical transduction process at a cellu­ lar level. Supported by the D F G , SFB 114 'Bionach', TP A5

72 Variability of a quantitative character and macronuclear DNA content in exautogamous clones of Paramecium primaurelia M. UMBERTA DELMONTE-CORRADO, PAOLA RAMOINO, EDOARDO MARGALLO and TINA CRIPPA-FRANCESCHI, Istituto di Zoologia dell'University, Via Bal^ bi, 5

-

16126 Genova, Istituto Scientifico per la cura e lo studio

dei Tumori, Istituto di Oncologia dell'University, V.le Benedetto XV, Pad. B

-

16132 Genova (Italia)

Previous results (Crippa-Franceschi et al., 1977) showed that in Paramecium primaurelia stock 90 the temperature-resistance character varies during the development of exautogamous clones. Temperature-resistance has been measured by the death time at 42,5° C of single cells tested in the same stage of the cell cycle, at increasing age in fissions. A cytophotometric analysis has been carried out on Feulgen stained macronuclear DNA contents at fixed times during the course of the cell cycle and at succeeding levels of the clonal development, in the interval from to 34postautogamic fissions. Macronuclear DNA content has been observed to undergo variability. The correlation between such a variation and that of temperature-resistance will be discussed.

9

73 Intrageneric acid- phosphatases variability in Euplotes (Ciliata, Hypotrichida) COLETTE DEMAR, Laboratoire de Zoologie,Ecole Normale Supérieure, ^6,rue d'Ulm,F-75230 Paris Cedex et VERONIQUE MACHELON,Laboratoire de Zoologie 2, Université Paris-Sud, F-9I405 Orsay Cedex

lhe genus Euplotes includes marine and fresh-water stocks. Infrageneric classification is founded on dorsal argyromé patterns and numbers of fronto-ventral cirri. Many clones have been compared

on the basis of electrophoretic mobility

of acid-phosphatases. These clones belong to 3 classes »1- fresh-water, eurystomus-llke argyrome,9 fronto-ventral cirri; 2- marine.eurystomuslike argyrome,IO fronto-ventral cirri; 3 - marine,vannus-like argyrome, 10 fronto-ventral cirri « The zymograms of all marine clones are very similar,in spite of their morphological diversity,but are strongly dif­ ferent from those of fresh-water clones. A more detailed analysis of the variability has been performed among clo­ nes morphologically related to Euplotes vannus. They can be classified into 4 intrafertile and intersterile groups which may be considered as sibling species,since intraspecific variability for morphological cha­ racters (numbers of dorsal kineties and caudal cirri ) is not significantly lower than interspecific variability. They have been also classified on the basis of electrophoretic patterns. The two classifications are com­ pared and the relation between biochemical and morphological features is discussed. Some other enzyme systems are presently under study.

74

Symposium B

Axenic Cultivation of Entamoeba and Trichomonadidae; The State of the Art. Louis S. Diamond, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, 20205, USA. There are many parallels in the development of axenic culture systems for Entamoeba and Trichomonadidae. These will be featured in an abbreviated review of the history of these cultures. Media and techniques currently employed in the initiation and establishment of cultures will be reviewed. Refinement of the available media can be expected to result in improved yields, but acceptable levels of growth have been obtained for most research purposes. Cloning procedures will be discussed in a dual role, as a means of obtaining cloned colonies and their usefulness in investigations requiring critical assessment of viability. The challenges of unsolved problems will be highlighted. Relatively few Entamoeba or Trichomonadidae have been cultivated axenically. Availability of these cultures would further the study of the biochemistry, physiology, immunology, etc., of these parasites, which in turn would add materially to the general knowl­ edge of primitive eukaryotes. There are no completely defined media for either group of protozoa. Elucidation of their nutritional requirements will come about only through development of these media. Furthermore, such media would facilitate the investigation o f ■the physical and chemical nature of biomembranes, a subject presently of intense interest. With few exceptions, the interval between isolation of these protozoa and their axenization in vitro is inordinately long. Consequently, there is opportu­ nity for selection and/or mutation to occur during a period of high vulnerability. .Ways must be sought to reduce this interval in order to conserve those traits of the wild population present at the time of isolation from the host. Except for Entamoeba invadens. a reptilian parasite, the cystic stage of Entamoeba does not form spontaneously, nor can encystation be induced in axenic cultures. Thus, no means are availa­ ble for studying this important life-cycle stage of the other cultivable Entamoeba under axenic conditions. Because understanding of the biology of cysts is prerequisite to devising more rational programs for controlling transmission of these amebae, especially the human pathogen E. histolytica, priority should be given to developing methods for inducing encystation under the unique conditions prevailing in axenic cultures.

75 The Role of Lipids in the Adaptation of Tetrahymena to Low Temperature B. F. DICKENS, C. S. RAMESHA, G. A. THOMPSON, JR., Department of Botany, The University of Texas, Austin, TX 78712, USA Tetrahymena pyri formi s , strain NT-1, can survive rapid chilli no from 39° to 15° and adapt, over a period of 10-12 hours, to growth at the lower tempera­ ture. A key step in this acclimation process is the return of membrane fluidity to an optimal value following the initial rigidifying effect of chilling. We have dissected the molecular events accompanying the restora­ tion of normal membrane fluidity in various parts of the cell. Following a shift from 39° to 15° over a 5 min. period, fatty acid desaturases residing in the cell's endoplasmic reticulum quickly increase the degree of phospholipid fatty acid unsaturation in that membrane. Within a period of only 30 min. after reaching 15°, the physical properties of the endoplasmic reticulum, as measured by fluorescence depolarization of the added probe diphenylhexatriene, have already attained the state characteris­ tic of cells fully acclimated to 15°. However, the other cellular membranes lack fatty acid desaturases and attain the proper degree of fluidity by a much slower process of intermembrane phospholipid exchange. Thus regu­ lation of membrane acclimation throughout the cell is achieved via modu­ lation of fatty acid desaturase action in the endoplasmic reticulum. The molecular mechanisms governing fatty acid desaturase action are complex and incompletely understood. But the practical rewards (particularly in agricultural sciences) of understanding and being able to manipulate an organism's temperature acclimation system are so great that an intensive study of this model system is well warranted. Two separate but related responses have been discovered. The first involves an apparent induced synthesis of one or more fatty acid desaturase enzymes. The second appears to depend upon a selective stimulation of fatty acid desaturation as a direct result of the decreased membrane fluidity caused by low temperature. We have examined this latter response in some detail. The rapid changes in the physical state of two particular membranes - endo­ plasmic reticulum (microsomes) and ciliary membranes, have been studied by fluorescence depolarization techniques. In the case of endoplasmic reticulum or lipids extracted from these mem­ branes, curves of polarization vs. temperature (Dickens and Thompson, Bio­ chemistry '19^:5029, 1980) exhibited two break points (abrupt changes in slope) when plotted over the physiological temperature range. The tempera­ tures of the break points were characteristic of the cells' growth tempera­ ture and changed dramatically during acclimation to low temperature. The lipids of cilia also showed characteristic physical responses, but of a more subtle nature. An analysis of lipid molecular species indicated that small changes in fatty acid unsaturation can have a profound effect on the membrane's physical state. Supported in part by grants from the National Science Foundation, the National Cancer Institute, and the Robert A. Welch Foundation.

The ciliary junction. A unique membrane specialization in the ciliate, Glaucoma fevox

P. DIDIER, R. MONTESANO, L. ORCI, Zoologie, B. P. 45, Les Cézeaux, 63170 Aubière (France) ; Institut d'Histologie et d'Embryologie, Ecole de Médecine, 1211 Genève 4 (Suisse) In the oral region of the ciliate, Glaucoma f e v o x , the cilia of the second membraneile show an unusual morphological specialization. At the ciliary apex, the axoneme disappears, is replaced by a mass of electron-dense mate­ rial and the tips of the cilia have a hexagonal rather than a circular pro­ file. The hexagonal ciliary apices are closely apposed resulting in a geome­ trical pattern, with continuous membranes being separated by an extracellu­ lar space of uniform width (about 20 nm). This space is filled with a flocculent material which, in suitable sections, appears to be arranged into re­ gularly-spaced interciliary bridges joining adjacent membranes. In freezefracture electron microscopy, the sides of the hexagonal ciliary apices ap­ pear as flattened membrane facets containing (on the P fracture-face) many parallel rows of intramembrane particles which are oriented in a perpendicu­ lar or slightly oblique direction with respect to the longitudinal axis of the cilia. The close, regular spacing of these rows of particles results in the formation of a paracrystalline array which occupies the entire facet. The E-face contains rows of pits which appear to be in register with the rows of particles .on the P-face of adjacent ciliary membranes. This is the first report of a junctional differentiation occurring : (a) 1n a unicellu­ lar organism and (b) between contiguous cilia, and we propose to call it the "ciliary junction".

77

An Infectious Bacterium in the Cytoplasm of Paramecium caudaturn JOSEF DIECKMANN Drolshagenstr. 6, D-4-4Q1 Everswinkel, FRG In Paramecium caudaturn only few species of symbiotic bac­ teria have been observed, most of them in the. nuclei. Cytoplasmic bacteria in Paramecium caudatum have been only described by PETSCHENKO (1911)• However, they are differ­ ent from the here described bacteria. The here described bacteria are found in the cytoplasm of Paramecium caudatum only. They are gram negative, have a length of 4 - 10^um, and a width of 1.0 - 2.5^um. In some cases, especially in the paramecia of stationary phase cultures, the bacteria contain inclusion bodies of vari­ able size, which are refractile when observed with the dark phase-contrast microscope. The inclusion bodies look different from the R-bodies of kappa. Outside the host ciliates the bacteria are found to be motile and electron microscopic examinations have revealed the presence of flagella. The bacteria are highly infectious to different stocks of syngens 1, 5» 12, 15 of Paramecium caudatum, but are not infectious to several other ciliate species. Tt seems that only bacteria having inclusion bodies are infectious. The number of the bacteria per paramecium cell does not seem to be regulated by the host. In paramecia of statio­ nary phase cultures.the bacteria keep multiplying and the ciliates are killed. Fast growing (2 - J fissions per day) paramecia can grow out the bacteria.

78 Tolerance of Ciliates towards Tompcraturc Changes during Various Seasons of the Year G. UIETZ-HLBiiACHTtzr?, Private Hydrobiological Laboratory A-7142 Illnitz, Austria

During the year, the range of the tolerance of Ciliates towards temperature changes varies much in some biotopes, which are characterized by extremely high temperatures during summer and low temperatures in winter. For example, Plcuronema coronaturn and Cyclidium citrullus are not able to survive during winter time at temperatures higher than 32°C, a temperature range tolerable during summer. These features are characteristic for biotopes of shallow salty lakes of the pannonian area in Austria with an increased salinity of water during the summer season. The measurements have been undertaken with a heating-table of "Wild” on the microscope. Exact data will be given in “Wissenochaftliche Hefte aus dem Surgenland".

79 An e x a m p le o f r e l a t e d

s e m i s p e c i e s i n Euplotes Vannus, FERNANDO DINI, Istituto