ALBERT

Notes: One third of a collection of cloned Stylonychia pustulata micronuclear DNA PstI fragments were found to be of a similar size, consistent with their being members of a repetitious sequence family with a repeat size of about 160 base pairs. Cross-hybridization experiments confirmed that these small cloned fragments are related by sequence homology. Hybridization of the cloned repetitious sequences to PstI digested micronuclear DNA revealed a “ladder” of bands (step size = 160 base pairs), indicating that the repeats are found in tandem arrays. This is the first demonstration of highly repetitious, tandemly repeated sequences in a ciliated protozoan.

Notes: The erythrocytic developmental cycle of Plasmodium falciparum can be conveniently divided into the ring, trophozoite, and schizont stages based on morphology and metabolism. Using highly synchronous cultures of P. falciparum, considerable variation was demonstrated among these stages in sensitivity to chloroquine. The effects of timed, sequential exposure to several clinically relevant concentrations of chloroquine were monitored by three techniques: morphological analysis, changes in the rate of glucose consumption, and changes in the incorporation of 3H-hypoxanthine into parasite nucleic acids. All three techniques gave essentially identical results. The trophozoite and schizont stages were considerably more sensitive to the drug than ring-stage parasites. Chloroquine sensitivity decreased as nuclear division neared completion. The increase in chloroquine sensitivity was coincident with a marked rise in the rate of glucose consumption and nucleic acid synthesis. The rate of nucleic acid synthesis decreased as schizogony progressed while glucose consumption continued at high rates during this process. The degree of chloroquine sensitivity was not highly correlated with either metabolic activity.

Notes: Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRATat 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diaminobenzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A 〈 PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WGA, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M. The expression of lectin binding cell surface saccharides of T. rhodesiense WRATat 1 is related to the parasite stage. Sugars resembling α-D-mannose are on the surface of bloodstream trypomastigotes and culture procyclics; n-acetyl-D-galactosamine and D-galactose residues are on bloodstream forms; and n-acetyl-D-glucosamine-like sugars are on procyclic stages.

Notes: The cationic permeant fluorescent dye rhodamine 123 (R123) was used to stain Plasmodium yoelii-infected mouse erythrocytes. Fluorescence microscopic observations demonstrated that the parasite, but not the matrix of the infected erythrocyte, accumulated the dye. Differences in fluorescence intensity could not be found at the various developmental stages of the parasite; however, quantitation of the cell-associated dye revealed an increase in R123 uptake with parasite development. The retention of the parasite-associated dye, as measured by fluorescence microscopy and spectrophotometry after extraction of R123 with butanol, was markedly reduced by treatment of the infected erythrocytes with a proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and an inhibitor of proton ATPase, dicyclohexylcarbodiimide (DCCD). These results indicate that the accumulation and retention of R123 in P. yoelii reflect the parasite membrane potential and suggest that the parasite plasma membrane has a membrane potential-generating proton pump.

Notes: Distinctive organic-walled resting cysts of at least three different types with a highly conservative morphology appear to characterize specific orders or groups of genera within the Class Polyhymenophorea (Protozoa, Ciliophora), contrasting markedly with the great diversity of form seen in trophic stages. Polyhymenophorean ciliates have been considered in the past to form a cohesive class within the Phylum Ciliophora and, possibly, to represent the pinnacle of ciliate evolution. Evidence from cysts challenges the cohesive nature of the class, suggesting that the hypotrichs should be subdivided and that they have a different phylogenetic origin from the heterotrichs, tintinnids, and oligotrichs.

Notes: Yellow-brown, algal symbionts varying in diameter from approximately 5 μ m to 20 μ m, associated with solitary Radiolaria with spongiose skeletons (i.e. Spongodrymus sp.), exhibit fine structural features resembling the Prymnesiida (botanical class, Prymnesiophyceae). A large central vacuole is surrounded by a thin layer of cytoplasm containing plastids with lamellae composed of three thylakoids and granular pyrenoids with internal tubules immersed between the thylakoids. The pyrenoids lack internal thylakoid membranes. The nucleus is surrounded by a dilated cisterna of the nuclear envelope that also encloses the plastids and gives rise to saccules of the endoplasmic reticulum. The algal symbionts appear coccoid; hence no flagella nor surface scales were observed. The symbiont fine structure is compared to similar yellow-brown symbionts associated with Acantharia. Thus far, three kinds of algal symbionts have been observed to be associated with solitary Radiolaria: dinoflagellate, prasinomonad, and this apparent prymnesiomonad.

Notes: Ultrastructural observations of the cortically-located mitochondria of Tetrahymena thermophila revealed associations not only between the mitochondria and certain of the cortical microtubule bands, but also between the mitochondria and the epiplasm of the cortex. Most of the distal mitochondrial surface is close and parallel to the epiplasm; favorable views show bridge-like structures spanning the 20–10 nm gap between the mitochondrion and the epiplasm.Previous studies have shown that the placement of mitochondria in the cortex appears to be determined by certain of the cortical microtubule bands. This study, however, shows that mitochondrion-microtubule interactions account for only a small proportion of the total mitochondrial area associated with the cortex; the rest is accounted for by the epiplasm. A possible analogue of the spectrin layer of erythrocyte membranes, the epiplasm may be important in helping to arrange the intricately organized components of the ciliate cortex. Its involvement in apparently helping to “moor” mitochondria to their cortical sites is the first suggestion of any role in cell patterning played by the epiplasm.

Notes: Ce Tetradimorpha, rencontre en eau douce se présente soit sous forme sphérique pourvue de quatre flagelles et d'axopodes rayonnants, soit sous forme allongée avec a l'avant quatre flagelles associes a quatre axopodes et a l'arriére six a huit axopodes divergents. L'etude ultrastructurale révèle un cytosquelette axopodial de type centroplastidie comprenant un centroplaste lenticulaire homogéne, centre organisateur des quatre axopodes anterieurs et des six a huit axopodes posterieurs, auquel s'ajoutent les quatre cinetosomes des flagelles anterieurs. En outre, un deuxiéme éleément cytosquelettique incluant un microtubule associe chacun des quatre cinetosomes a l'axopode antérieur correspondant. Des cordons microfibrillaires réunissent axopodes et cinetosomes au niveau du centroplaste, puis a quelque distance du centroplaste les axopodes posterieurs. Les axonémes des axopodes comprenant de 5 a 30 microtubules sont constitues de triades, lorsqu'on peut détecter une organisation. Le noyau, a nucléole central est coince dans le cone axopodial posterieur, lui-méme entouré des dictyosomes. Par l'organisation du cytosquelette, par la structure des kinétocystes, par la structure des flagelles dépourvus de mastigonémes tubulaires, Tetradimorpha différe nettement de Ciliophrys marina. Comme le prévoyait Davidson (1975), il represönte bien un des chainons dans la série évolutive des Héliozoaires centrohélidiens. Mais il ne présente guère d'affinites avec les Chrysomonadines considerees comme la souche des Héliozoaires. L'intéret de ce Protiste dans l'étude de la differentiation et de l'evolution du cytosquelette est également présente.〈section xml:id="abs1-2"〉〈title type="main"〉ABSTRACTThis freshwater species of Tetradimorpha has a spherical body with four flagella and radiating axopods; it transforms into a pear-shaped cell that anteriorly has four flagella intercalated between four axopods and posteriorly has six to eight divergent axopods. Ultrastructural study reveals an axopodial cytoskeleton of the centrohelidan type comprising an homogeneous lenticular centroplast which acts as MTOC for axopodial microtubules. A second skeletal element is a microtubular linkage between the kinetosomes and the axonemes of anterior axopods. A microtubule embedded in dense material diverges from near the base of each kinetosomes and parallels the distal portion of the axoneme of each anterior axopod. A microfibrillar envelope around the centroplast links the axopodial bases to the kinetosomes situated just above. Close to the centroplast, microfibrillar strands link the axopodial axonemes to the kinetosomes. Axopodial axonemes are composed of 5 to 30 microtubules irregularly arranged except for some that form equilateral triangles. The nucleus containing a central nucleolus is constrained within a cone formed by the axonemes of the posterior axopods and surrounded by dictyosomes. By the cytoskeletal organization, the structure of kinetocysts, and flagella wthout tubular mastigonemes, Tetradimorpha differs obviously from Ciliophrys marina. As Davidson (1975) predicted, Tetradimorpha is an intermediate link in the centrohelidan lineage: however, it lacks the characteristics of chrysomonads, the supposed ancestors of Heliozoa. The contribution of this genus to the study of the differentiation and the evolution of the cytoskeleton is also presented and discussed.

Notes: Two-dimensional gel electrophoresis was used to identify the patterns of protein synthesis during initiation, and the patterns of membrane protein expression following initiation, in all of the mating types of the Tetrahymena thermophila B family. In addition, one-dimensional analysis was used to survey 125I-Concanavalin A-binding proteins. Although a large number of proteins was identified by each technique, no variation among the mating types was observed.

Notes: Cells of Tetrahymena pyriformis, T. thermophila, and Euglena gracilis were saturated with nitrogen gas at pressures up to 300 atm and rapidly decompressed. Damage was assessed by measuring post-decompression cell fragmentation or viability. Occurrence of intracellular bubbles was determined by cinephotomicrography performed during the decompression or by direct observations afterwards. The extreme gas supersaturations induced led to intracellular bubble formation and rupture in cells of Tetrahymena that contained food vacuoles, but only with supersaturations of 175 atm or higher; 225 atm left few cells intact. Bubbles were never observed in cells of Euglena or in Tetrahymena cells freed of food vacuoles, even when they were decompressed from substantially higher nitrogen supersaturations. Cells of Euglena were most resistant and were unaffected by supersaturations up to 250 atm.

Notes: The rapid, synchronous differentiation of N. gruberi from amoebae to flagellates is a useful paradigm to study aspects of cell differentiation, including regulation of the synthesis of proteins that are related to the changes in cell shape and motility, which occur during differentiation. The differentiation requires synthesis of new RNA and protein molecules to accomplish defined morphogenetic events. Specific new proteins, including the tubulins that form the flagellar microtubules, are synthesized at various times during differentiation, and particular mRNA species appear and disappear. The time course of the synthesis of the α and β subunits of flagellar tubulin is paralleled by the programmed appearance and disappearance of flagellar tubulin mRNAs. The evidence supports the hypothesis that the synthesis of flagellar tubulin is regulated by the transcription, and subsequent disappearance, of flagellar tubulin mRNA. Translatable mRNAs for two calmodulin-like calcium-binding proteins appear and disappear contemporaneously with those for flagellar tubulin. During differentiation the synthesis of actin, the major protein of amoebae, is selectively shut down, and translatable actin mRNA rapidly disappears. This description of the orderly appearance, utilization, and disappearance of the mRNAs for actin, calcium-binding proteins, and flagellar tubulin during differentiation provides means and motivation to investigate the mechanisms that regulate these events.

Notes: Earlier experimental work involving macronuclear implants in Stentor coeruleus has shown that the cytoplasmic cortex of the nuclear site 1) attracts the macronucleus and 2) holds it in place during interphase. Now experiments indicate macronuclei transferred with overlying cortex elongate in the direction of the transferred cortical pigment stripes, whether or not the transferred stripes realign in the direction of the host stentor's stripes. Therefore the third function of the cortex is to determine the direction of elongation and thus assure that both daughter cells at division receive part of the macronucleus.

Notes: During an electron microscopic study of Glugea stephani, three morphologically distinct tubular appendages that are continuous with the sporoblast plasmalemma were observed. The tubules were designated as: type I, 45–50 nm in diameter and 600–900 nm in length; type II, 25–35 nm in diameter, averaging 1300 nm in length; type III, 50–70 nm in diameter and with an indeterminate length, which often exceeds 3000 nm. Type III tubules contain regularly spaced, electron-dense particles that are approximately 30 nm in diameter. Since many genera of microsporida have some type of appendage, which may eventually be utilized for taxonomic purposes, we propose the formation of a system of serially numbered detailed descriptions of these structures to promote uniformity and clarity in future publications.

Notes: Cells of Paramecium tetraurelia, stock hrd, cultured in a micro-capillary containing 1 μl fresh culture medium, expressed mating activity through the whole cell cycle. Mating-reactive G2 phase cells can conjugate with cells of other phases. The G2 phase cells, which have double (4C) the normal micronuclear DNA content, undergo pre-meiotic DNA synthesis when conjugated with G1 phase cells. The micronucleus of the progeny from the cross between a G1 and a G2 cell becomes triploid.

Notes: Buffered solutions of KCl and NaCl were tested for their stimulatory effect on the germination of variously-aged spores of Vavraia culicis. Germination was optimal in 0.2 M KCl, pH 6.5 for one isolate, and, for another isolate, peaks of germination occurred at pH 7.0 and 9.5. Spores incubated for several hours in suboptimal solutions became unable to germinate under optimal conditions. After being returned to water, they regained their ability to germinate. Calcium chloride, magnesium chloride, and ammonium chloride inhibited germination. After ingestion by mosquito larvae, spores germinated near the posterior end of the midgut.

Notes: . Leishmania tropica promastigotes transport α-aminoisobutyric acid (AIB), the nonmetabolizable analog of neutral amino acids, against a substantial concentration gradient. AIB is not incorporated into cellular material but accumulates within the cells in an unaltered form. Intracellular AIB exchanges with external AIB. Various energy inhibitors (amytal, HOQNO, KCN, DNP, CCCP, and arsenate) and sulfhydryl reagents (NEM, pCMB, and iodoacetate) severely inhibit uptake. The uptake system is saturable with reference to AIB-and the Lineweaver-Burk plots show biphasic kinetics suggesting the involvement of two transport systems. AIB shares a common transport system with alanine, cysteine, glycine, methionine, serine, and proline. Uptake is regulated by feedback inhibition and transinhibition.

Notes: . Oxytricha strains used in biochemical studies have traditionally been grown in unaerated, unagitated culture tubes or Fernbach flasks. These cultures are limited in volume to about one liter and have a very nonuniform distribution of cells, with the majority of the cells at the very top or bottom of the medium. We have found conditions in which Oxytricha can be grown in 50-liter fermentation vats. The cultures grow to a uniform density of about 6000 cells/ml.

Notes: . The fine structure of the tomite of Foettingeria actiniarum (Claparède) was examined and compared with that of other apostome tomites. This stage in the life cycle has a unique configuration of kineties that form a spiral through the cytoplasm in the interior of the body. The structure and behavior of this internal spiral were evaluated as a mechanism for the storage of kinetosomes, an adaptation to the ciliate's two-host life cycle. The spiral is composed of nine ribbons of laterally compressed kinetosomes that are in contact with a thin electron-dense fibril. Paralleling the kineties of the spiral are conspicuous, swollen lamellae of the rough endoplasmic reticulum; these lamellae contain moderately electron-dense material. The spiral is associated with the large contractile vacuole and winds about the macronucleus. The tomite of Foettingeria possesses a single, robust, caudal cilium located in a pit, along with the nozzle-like pore of the contractile vacuole. The walls of the pit contain several trichocysts arranged radially about the caudal cilium and aimed into the pit.

Notes: . Fine structural studies of a specialized vesicle system associated with the endoplasmic reticulum (ER) of exo-erythrocytic Plasmodium berghei suggest that this system may be the equivalent of a Golgi apparatus. Patches of ER, randomly distributed in the cytoplasm of developing parasites, are formed of smooth and ribosome-studded cisternae intermingled with each other. The vesicle systems are located between as well as at the edges of ER aggregates and appear to be in different stages of budding from the cisternae. Prolonged osmication reveals distinct staining of the nuclear envelope and ER of the parasites as well as part of the Golgi apparatus of the hepatocytes. However, the small vesicles associated with the parasite's ER are unstained, as are the coated vesicles in the Golgi region of the liver cell. These sites in the parasite cytoplasm seem comparable to the concave surface of the Golgi apparatus in liver cells. The pinched-off vesicles fuse with others to form the prominent peripheral vacuolization characteristic of the nearly mature exo-erythrocytic form. The formation of these peripheral vacuoles and their subsequent fusion with the parasite membrane may be an exocytosis mechanism supplying the rapidly expanding parasite with new plasma membrane material.

Notes: . Ultrastructural cytochemical techniques were used to analyze the nucleus and the kinetoplast of epimastigotes of Trypanosoma cruzi. With the use of ethanolic phosphotungstic acid, which detects basic proteins, reaction product was seen in the chromatin and at the periphery of the kinetoplast. Thallium alcoholate, which interacts with DNA, stained strongly the whole kinetoplast and the chromatin. With the use of a silver impregnation method that detects acidic nucleolar proteins, silver granules were seen preferentially located in the central region of the nucleolus. With the EDTA method, which reveals the presence of ribonucleoproteins, staining was observed in the nuclear pores. Also 6–8 nm fibrils, 25 nm and 40 nm granules, which correspond to the perichromatin fibers, interchromatin granules and the perichromatin granules, respectively, were identified in the nucleus. The EDTA method also revealed the presence of 40 nm granules in the kinetoplast. These granules were seen mainly at the two extremities of the kinetoplast. Freeze-fracture images indicate that the nuclear membrane contains ca. 9 pores/μm2 of nuclear surface area. The mean diameter of the pores was 80 nm. All these results suggest that epimastigotes of T. cruzi have a very active nucleus and a high rate of nucleocytoplasmic interchange.

Notes: . When a streptomycin-bleached mutant of Euglena gracilis strain Z was cultured in the dark at 33, 26, or 15°C, the content of paramylon was higher at lower growing temperature while that of wax esters was higher at higher temperature. Transfer of the cells grown at 33°C–15°C decreased the wax ester content while increasing the paramylon content; transfer in the reverse direction caused reverse changes. On incubation with labeled acetate, the cells grown at 33°C showed more distribution of radioactivity in wax esters than the cells grown at lower temperatures. Apparently the two energy-reserve substances have different physiological functions.

Notes: The sessiline peritrich Ellobiophrya conviva n. sp. is described from marine ectoprocts of the genus Bugula, the first report of an ellobiophryid on bryozoan hosts. The new species is distinguished from others of its genus by its different body proportions, size, host, and structure of the clasping holdfast (for which the new name cinctum is chosen). Ellobiophrya conviva has been found only on B. neritina and B. turrita and shows a marked seasonal cycle of abundance. The family Ellobiophryidae Chatton & Lwoff is revised on the basis of new information provided by E. conviva, with the single species of the genus Clausophrya removed to Ellobiophrya as E. oblida Naidenova & Zaika n. comb. The genus Caliperia Laird remains unchanged. The two genera of the revised family are distinguished from one another by differences in the structure of the cinctum. Hypotheses are advanced to explain the morphogenesis of the cinctum and the evolution of ellobiophryids from other peritrichs.

Notes: The spatial and seasonal distribution of Paramecium bursaria in two small Indiana ponds was studied using a sampling grid. Very small (5.0 ml) samples were taken so that the individual microhabitats could be studied. The results were evaluated in comparison to the data collected for the P. aurelia complex collected in the same manner and at the same sites. It was found that P. bursaria exist in a clumped distribution, but that the distribution was not very different from random. Paramecium bursaria also exist at the surface and at the mud-water interface. Temperature does not seem to play a statistically significant role in determining population size. The breeding system of P. bursaria is optimized for an outbreeding population of low density. In comparison, the species of the P. aurelia complex exist in a very clumped distribution, are found only at the mud-water interface, and are inbreeders. The evolutionary strategies of the two types of paramecia are discussed.

Notes: L'étude du caractère planctonique de différentes spores d'Actinomyxidies montre une complexité croissante dans leur adaptation au milieu aquatique. Au contact de l'eau, les trois cellules épéisporales de chaque spore se transforment en flotteurs de forme différente suivant les espèces. Ces flotteurs peuvent s'unir entre eux en un style équivelent à un quatrième flotteur ou associer diversement les huit spores issues d'un měme pansporocyste. C'est le cas dans le genre Synactinomyxon dont la diagnose est modifiée pour inclure une deuxième espèce S. Iongicauda n. sp. Un type nouveau est décrit chez lequel la preéence d'ancres à l'extrémité des cellules épisporales permet de maintenir efficacement réunies plusieurs dizaines de spores émises simultanément. Nous avons observé dans les genres Aurantiactinomyxon, Synactinomyxon, Echinactinomyxon l'emission du sporoplasme. II est libére en entier et capable de se déplacer dans l'eau pendant plus d'une heure grǎce à des mouvements amoeboïdes. Chez Aurantiactinomyxon eiseniellae les études ultrastructurales montrent que l'enveloppe du pansporocyste, d'une part, les épispores et les capsules polaires d'autre part sont réalisées à partir de cellules distinctes et profondément modifiées. Quant au sporoplasme, autrefois décrit comme un plasmode avec de nombreuses paires de noyaux, il contient, en fait, des ensembles identiques dont chacun est constitué de l'union d'un noyau satellite et d'une cellule uninucléée.〈section xml:id="abs1-2"〉〈title type="main"〉ABSTRACTThe study of the planktonic character of different Actinomyxidia spores reveals increasingly complex adaptations to an aquatic environment. On contact with water, the three episporal cells of each spore transform into floats, the forms of which differ according to species. These floats can join together so that a fourth type of float is formed, or they can unite in various ways the eight spores originating from the same pansporocyst. This is the case in the genus Synactinomyxon whose diagnosis is modified to include a second species S. Iongicauda n. sp. A new type is described in which the presence of anchors at the extremities of the episporal cells permits several dozen spores that have been emitted simultaneously to be kept together. We have observed the emission of the sporoplasm in the genera Aurantiactinomyxon, Synactinomyxon, and Echinactinomyxon. It is freed completely and for more than an hour is capable of changing its position in the water by amoeboid movements. In the case of Aurantiactinomyxon eiseniellae, ultrastructural studies show that the pansporocyst envelope on the one hand, and the epispores and polar capsules on the other hand, are formed from separate but profoundly modified cells. The sporoplasm, however, sometimes described as a plasmodium with numerous pairs of nuclei, contains, in fact, identical complexes, each consisting of a uninucleate cell united with a satellite nucleus.

Notes: Paramecia detect and accumulate in or disperse from some chemicals. Cells do this by changing frequency of turning and speed of swimming. There are at least two mechanisms by which cells respond: one dependent on ability to turn, one dependent on speed modulation. There are also two classes of chemicals: those that require the cells' ability to turn in order to cause accumulation and dispersal (type I), and those that apparently require only speed modulation (type II). Attractants of type I cause qualitatively similar changes in behavior to repellents of type II and the converse; therefore, assays are needed to distinguish between these two classes of chemicals, despite qualitatively similar behavior of some attractants and repellents. We examined two assays of paramecium chemoresponse, T-maze assay and well test, to understand how the T-maze distinguishes between attractants of type I and repellents of type II and why the well test does not.

Notes: The morphology of spore germination in Didymium nigripes was studied using scanning electron microscopy and Nomarski phase optics. First, the outer spore wall splits, revealing a fibrillar inner wall. Remnants of the inner wall continue to cover the newly emerged amoeba. A single nucleus and a prominent vacuole are visible throughout germination. Germination is more rapid in glucose-peptone-yeast extract than in phosphate buffer. Germination is completely inhibited at 4°C, and is very slow at 18°C. Germination is most rapid at 26°C; at 21°C or 32°C it is slightly slower. Germination is reversibly inhibited by 20 μ/ml cycloheximide, but not by 200 μ/ml 5-fluoro uracil or 200 μ/ml proflavin. It is completely inhibited by 10-3 M Na azide.

Notes: Crithidia fasciculata (Anopheles, Culex, and Nöller strains), C. hutneri, C. luciliae thermophila, and Herpetomonas samuelpessoai were grown in a defined medium with different values of osmolarity at different temperatures. C. fasciculata (all strains) grew best between 300 to 500 mOsm; H. samuelpessoai, 400–500 mOsm; and C. hutneri and C. luciliae thermophila, 500–800 mOsm. At higher temperatures better growth was obtained at the upper osmolarities.

Notes: Crypthecodinium cohnii, a small marine heterotrophic homothallic dinoflagellate, has diversified into a complex of morphologically very similar breeding groups (biological species or sibling species), some of which have become widely dispersed. Membership of two clones in the same sibling species is shown by their sexual compatibility as determined by genetic complementation in zygotes formed from motility mutants derived from the two stocks. Membership in different sibling species may be inferrec when motility mutants of one strain do not complement those of another. Fifty-six clones representing seaweed enrichments from *** geographic sites have been found to belong to 28 sibling species; 35 clones are members of seven wide-ranging biological species, and 21 are single representatives of 21 other breeding groups within the ranges of the others. Of 174 clonal isolates in our possession, 168 conform in size and shape to C. cohnii. Six others which have smaller cells and only one-fifth the standard DNA and chromosome number belong, we believe, to another species. The C. cohnii complex provides a unique opportunity for the study of evolutionary divergence and geographical dispersion of a dinoflagellate.

Notes: Until recently, pansporoblastic microsporidia that produce a variable and large number of sporoblasts from a sporont have been included in a single genus, namely Pleistophora Gurley, 1893. Ultrastructural studies have been used to determine whether the resemblance of these species is fundamental or superficial. The results indicated that the multisporous pansporoblastic forms belong to at least three genera and, thus, that Pleistophora is a “composite genus.” The term pansporoblast was originally used for stages in myxosporidian development. The term sporophorous vesicle adopted from Gurley is suggested for the spore-containing vesicle in the Microspora. Three species were studied: Pleistophora typicalis, the type-species; Pleistophora culicis, for which a new genus Vavraia has already been proposed; and Pleistophora simulii. P. typicalis and V. culicis have isolated nuclei throughout their development, and the sporophorous vesicle wall enveloping the sporoblasts is derived from amorphous secretions laid down during merogony external to the plasmalemma. Pleistophora and Vavraia are differentiated principally in terms of the structure of the sporophorous vesicle wall and mode of division of the sporogonial plasmodium. The nuclei of young sporonts of P. simulii are in diplokaryon arrangement and undergo meiosis to give haploid nuclei in the sporoblasts. The sporophorous vesicle wall is membranoid and is laid down external to the plasmalemma at the onset of sporogony. A new genus, Polydispyrenia n. g., is suggested for this species, the affinities of which are closer to the dimorphic species of microsporidia than to Pleistophora or Vavraia. The terms “merontogenetic sporophorous vesicle” and “sporontogenetic sporophorous vesicle” are used to distinguish between the two groups.

Notes: Plasmodium berghei infection was more severe in pregnant than in nonpregnant mice. Infection initiated on gestation day 7 resulted in rapidly increasing parasitemia and deaths of all pregnant mice within 12 days, while some nonpregnant mice survived until day 21 postinfection. When mice were infected on gestation day 12 or 14, a proportion of mice died before parturition; but some animals survived to deliver living pups. Reduced birthweights and increased spleen weight to body weight ratios were seen in pups from infected mice as compared with pups from uninfected animals. Histopathological abnormalities of placentae from infected animals included degeneration of the normal labyrinthine architecture and thickening of the trophobast separating maternal and fetal blood vessels.

Notes: Tetrahymena pyriformis strain WH-14 secreted large quantities of intracellular proteases into its culture medium during growth. Extracellular enzymes were purified to homogeneity from cell-free medium by ammonium sulfate precipitation, CM-Sephadex column chromatography, gel filtration, and DEAE-cellulose column chromatography. The DEAE-cellulose eluates were separated into four peaks (P-I, P-II, P-III, and P-IV), each of which exhibited a different specific activity toward azocasein and α-N-benzoyl-DL-arginine-ρ-nitroanilide (Bz-Arg-Nan). These four forms of the protease showed similarity in amino acid composition, molecular weight (21,000–24,000), and antigenic reactivity. They had pH optima at neutral range. P-I showed the highest specificity to azocasein whereas P-IV was most effective toward the synthetic substrates. The Km values for hydrolysis of Bz-Arg-Nan were 2.4, 1.6, 1.3, and 1.4 mM for P-I, P-II. P-III, and P-IV, respectively, and the corresponding Kcat/Km values were 5.0, 9.4, 28.5, and 114.3 S-1.M-1. These properties of secreted proteases were compared with those of intracellular proteases purified by the same procedure except for the initial Triton X-100 extraction. There were similarities in specific activity toward two substrates, molecular weight, Km, pH optima, and antigenic reactivity between the proteases from two sources, providing evidence that the intracellular proteases may be secreted into the extracellular medium without modification.

Notes: The separation of extracellular protozoan parasites from host cells based on a difference in surface charge has been described. However, with Trypanosoma cruzi no method exists for the isolation of pure parasite stages from heterogeneous mixtures. Studies on the electrophoresis of mixed stage populations confirm significant surface charge density differences exist among epimastigotes, trypomastigotes, and amastigotes. In ascending order of electronegativity, amastigotes have the lowest charge density, try-pomastigotes next, followed by epimastigotes. A technique has been developed for the separation of purified populations of parasites based on these charge differences using a continuous free-flow electrophoresis apparatus. The separated populations are morphologically intact and maintain their infectivity to mice. This separation method is applicable for preparative and analytical isolation of pure stages of T. cruzi for biochemical and immunological studies.

Notes: Cellular levels of protein and two acid hydrolases, acid phosphatase (EC 3.1.3.2) and acid proteinase, were followed during cyst differentiation, arbitrarily divided into five stages, in the ciliate Histriculus muscorum Kahl. Extracellular enzyme activities were also measured. Protein content decreased gradually during cyst differentiation. In mature cysts the protein content was ca. 60% that of stationary phase organisms. The activities of both acid hydrolases remained unchanged during stage 1 and then decreased gradually; acid proteinase decreased more rapidly. Both enzymes remained slightly active in the mature cysts. The acid proteinase activity of stage 1 was reduced by cycloheximide treatment at time zero, whereas the enzyme was no longer sensitive to the inhibitor when treated at 1.5 h (late stage 1) after the first wash with encysting medium. Acid phosphatase activity was insensitive to the inhibitor. Extracellular release of acid phosphatase increased linearly at least until stage 5, although the extracellular release of acid proteinase was not detected. Cycloheximide blocked the extracellular release of acid phosphatase after stage 1. These results suggest that de novo synthesis of acid proteinase occurs during stage 1 and that lysosomes may play an important role during early stages of cyst differentiation.

Notes: SYNOPSIS. Thymidylate synthetase (E.C.2.1.1.45) has been demonstrated in unsporulated oocysts of Eimeria tenella. The properties of this enzyme have also been investigated in Tetrahymena pyriformis, as a protozoan model, and 7-day-old chick embryo, as a host model. The enzymes from E. tenella and chick embryo were inhibited by all concentrations of MnCl2 and MgCl2 tested. Tetrahymena pyriformis thymidylate synthetase was stimulated by low concentrations of both these cations but was inhibited by high concentrations. Subsequent data refer to chick embryo, E. tenella and T. pyriformis respectively: the apparent Km was 5.89 μM, 5.94 μM, and 0.53 M for the substrate dUMP: and 5.13 μM, 1.10 μM and 4.65 μM, respectively for the cofactor N5N10-methylenetetrahydrofolate. The pH optimum for the enzyme from both chick embryo and T. pyriformis was 8.0, with Tris-HCl buffer; activity of E. tenella thymidylate synthetase was still increasing at pH 8.2. The E. tenella enzyme was found to have a molecular weight of 4.6–4.9 × 105 daltons. The effects of nucleotides, inhibitors, and the omission of assay components on each enzyme are presented. Thymidylate synthetase from E. tenella is not greatly different from that of chick embryo, but does not resemble the enzyme from T. pyriformis. A case for using thymidylate synthetase as a chemotherapeutic target in the treatment of Eimeria infections remains. Indeed Eimeria may be considered as a model for infections caused by other protozoan parasites, such as Toxoplasma and Plasmodium, provided that suitable inhibitors can be found that are not toxic to the host.

Notes: SYNOPSIS. A protocol based on density differences between starved and fed cells and employing density gradient centrifugation has been devised to facilitate the isolation of auxotrophic mutants of cell lines derived from Tetrahymena thermophila strain B1868. First, a mass phenotype screening procedure was established whereby true auxotrophic mutants and slow-growing wild-type cells such as strain C* could readily be distinguished. Second, simulation experiments were performed in which wild-type cells starved first in non-nutritive buffer, then suspended in a defined medium lacking a single essential amino acid became significantly denser than the same cells when starved, then suspended in a complete defined medium. Finally, using the same protocol, a reconstruction experiment was carried out which resulted in effective separation of wild-type cells from cells of a tyrosine auxotroph. The overall procedure resulted in a 9-fold increase in the relative frequency of auxotrophic cells, while the density gradient centrifugation alone provided a 400-fold enrichment.

Notes: SYNOPSIS. Infectivity of Plasmodium gallinaceum (Brumpt) sporozoites isolated from midguts and salivary glands of experimentally infected Aedes fluviatilis (Lutz) was studied. The 2 populations were compared at 7, 8, and 9 days postisolation from mosquitoes, which were maintained at 27 C ± 1C and ∼75% relative humidity. Infectivity of the parasites was evaluated by the length of the prepatent period of the infection in 2-week-old chicks inoculated intramuscularly. Infection was caused by 7-day-old sporozoites from salivary glands, but not from midguts. Older sporozoites induced infection in all the inoculated chicks. The results suggested a somewhat higher infectivity of the 8- and 9-day salivary-gland parasites than of the oocyst sporozoites. However, unlike sporozoites from mammalian malaria, oocyst sporozoites from avian malaria were highly infective at this age.

Notes: SYNOPSIS. Sorogena stoianovitchae Bradbury & Olive, an epiphytic ciliate found in various parts of the world, has a trophic stage that feeds on members of the ciliate genus Colpoda. When grown in the presence of the food ciliate, it multiplies rapidly. When the cells become abundant they aggregate at the water surface on inserted plant fragments or floating pollen grains, the sides of culture dishes, or on floating films such as those deposited by bacteria or pollen grains. an aggregate mounds up and becomes ensheathed above the water level, after which the mass of cells called a sorogen rises aerially at the apex of a stalk deposited at its base. the tapering, noncellular stalk consists of a conspicuously furrowed sheath that encloses a mucilaginous matrix. At completion of stalk development the cells of the sorogen become encysted. the sorocysts are commonly discharged by fracturing of the drying sorus. Alternating light and dark conditions are required for sorocarp development.

Notes: SYNOPSIS. the antigenic types in populations of metacyclic trypanosomes of Trypanosoma brucei isolated from Glossina morsitans head-salivary gland trypanosome cultures and bloodstream forms in the early parasitemias produced from whole culture supernatant fluids containing metacyclic forms, were analyzed by the indirect fluorescent antibody test using clone-specific antisera. Metacyclic trypanosomes in cultures initiated with cloned bloodstream forms were heterogeneous with respect to their variable antigenic type (VAT). Trypanosomes comprising early parasitemias in immunosuppressed mice infected with metacyclics produced in cultures also had a range of VATs. Three of the VATs detected in the early parasitemias in mice have also been identified by other investigators in tsetse fly-transmitted populations of the same stock.

Notes: SYNOPSIS. A simple method is described for plating and cloning ciliates and other protozoa, based on a principle differing from that traditionally used for plating and cloning bacteria and other microorganisms. This procedure, referred to as the silicone-oil-plating-procedure (SOPP), involves vortexing small volumes of culture medium containing protozoa with larger volumes of a non-toxic silicone oil and plating the resulting unstable emulsion in small plastic petri plates. Discrete microdroplets of culture medium form containing protozoa entrapped and immobilized between the hydrophobic surfaces of the plastic petri dish and the oil. Protozoa, isolated by this method grow, divide, and multiply to form clones. the procedure may be used for plating and cloning protozoa in bacterized and axenic culture. Variations of the basic method may be applied to isolating protozoa from the wild, washing protozoa to remove microorganisms, screening for potential mutants, and for replica plating.

Notes: SYNOPSIS. Cyclic nucleotide phosphodiesterase [EC 3.1.4.17] was examined in Tetrahymena pyriformis strain NT-1. Enzymic activity was associated with the soluble and the particulate fractions, whereas most of the cyclic GMP phosphodiesterase activity was localized in the soluble fraction: the activities were optimal at pH 8.0–9.0. Although very low activities were detected in the absence of divalent cations, they were significantly increased by the addition of either Mg2+ or Mn2-. A kinetic analysis of the properties of the enzymes yielded 2 apparent KIII values ranging in concentration from 0.5 to 50 μM and from 0.1 to 62 μ M for cyclic AMP and GMP. respectively. A Ca2+-dependent activating factor for cyclic nucleotide phosphodiesterase was extracted from Tetrahymena cells, but this factor did not stimulate guanylate cyclase [EC 4.6.1.2] activity in this organism. On the other hand, Tetrahymena also contained a protein activator which stimulated guanylate cyclase in the presence of Ca2+, although this activator did not stimulate the phosphodiesterase. the results suggested that Tetrahymena might contain 2 types of Ca2+-dependent activators, one specific for phosphodiesterase and the other for guanylate cyclase.

Notes: SYNOPSIS. the cell size of Didinium nasutum was found to be dependent on the size of the Paramecium species available as prey. Didinium feeding on P. tetraurelia averaged 5.6 × 105μm3. the cell volume of Didinium increased with increasing prey size for the 5 prey species tested, to 9.1 × 105μm3 for Didinium feeding on P. caudatum. Didinium nearing a cell division ranged in size from 8.6 × 105μm3 on P. tetraurelia to 12.9 × 105μm3 on P. caudatum. the range in cell volume is such that Didinium feeding on P. caudatum are larger than the size at which Didinium divide when feeding on P. tetraurelia. This morphologic plasticity in cell volume allows Didinium to exploit a wide size range of Paramecium species as prey. It is proposed that the size of a Didinium may have profound effects on its ability to encounter and capture prey of different sizes.

Notes: RESUME. Chacun des 45–80 organelles adoraux de Bursaria truncatella O. F. Müller est constitué de 3 rangées de cinétosomes et l'aire buccale droite est couverte de nombreuses doubles rangées de cinétosomes. La stomatogenèse débute par la désorganisation et la résorption des organelles buccaux postérieurs. Puis, il y a désorganisation des rangées parorales de cinétosomes et multiplication des cinétosomes sur l'aire orale droite, en měme temps que sont rompues, selon une ligne oblique, un certain nombre de cinéties somatiques. La prolifération des cinétosomes aux extrémités des cinéties. de part et d'autre de la ligne de rupture, aboutit, d'une part, à la formation d'un champ anarchique qui est le primordium oral droit de l'opisthe, d'autre part, à la formation de nombreux doublets qui constituent chacun le primordium de chaque organelle adoral. Après la séparation des tomites, les cinétosomes de l'aire droite s'ordonnent en doubles rangées et les organelles adoraux se complètent par addition d'une 3ème rangée de cinétosomes. Les cinétosomes somatiques sont jumelés, reliés par 2 desmoses. Les fibres transverses postérieures et les fibres postciliaires forment de longs rubans de microtubules dirigés vers l'arrière et juxtaposés dans les crětes intercinétiennes. Les doubles rangées droites de cinétosomes buccaux sont assimilables à des stichodyades. Les organelles des cinétosomes adoraux portent des rideaux de fibres postciliaires convergents ou divergents. La rangée postérieure de chaque organelle est non ciliée. Par son type de stomatogenèse, par sa structure corticale, par l'ultrastructure des organelles adoraux, Bursaria appartient aux Colpodidea, ce qui suggère des remarques de plusieurs types.SYNOPSIS. In Bursaria truncatella O. F. Müller, each of the 45–80 adoral organelles is composed of 3 rows of kinetosomes, and the right buccal area is covered by many double rows of kinetosomes. Stomatogenesis begins by disorganization and disappearance of the posterior buccal organelles. Next, there is disorganization of the paroral rows of kinetosomes and multiplication of kinetosomes in the right oral area; at the same time, some somatic kineties are disrupted along an oblique line. Multiplication of kinetosomes at the extremities of the kineties, on both sides of the disruption, leads to the formation of an anarchic field which is the right oral primordium of the opisthe and the formation of doublets each of which constitutes an adoral organelle. After the separation of the tomites. the kinetosomes in the right buccal area position themselves, and the adoral organelles are completed by the addition of a 3rd row of kinetosomes. Somatic kineties are formed by successive pairs of ciliated kinetosomes united by 2 desmoses. the long posterior transverse ribbons and the postciliary ribbons extend posteriad, overlapping in the pellicular ridges. Oral rows of kinetosomes on the right can be compared with stichodyads. the adoral kinetosomes have convergent or divergent postciliary ribbons. the posterior row of kinetosomes in each organelle is not ciliated. By the type of stomatogenesis, the cortical ultrastructure, the ultrastructure adoral of its organelles, Bursaria belongs to the Colpodidea.

Notes: SYNOPSIS Free-living marine ciliates occur in the interstitial spaces of a wide vareity of filamentous and particulate substrata, on the surfaces of planar substrata, and in the plankton. In addition, they are found in association with a wide variety of plant and animal hosts. In this paper I review the progress during the past decade in understanding the distribution of marine ciliates, with particular emphasis on the relationship between ciliate biogeography and the species problem. It is concluded that as a general rule among marine ciliates, genera and species complexes are cosmopolitan. Specific locales may support a confusing array of sibling species or subspecific morphologic variants. Because the distributional processes and breeding biology of marine ciliates are only beginning to be understood, conventional ideas that marine ciliate species are cosmopolitan may require modification.

Notes: SYNOPSIS The subkingdom Protozoa now includes over 65,000 named species, of which over half are fossil and ∼ 10,000 are parasitic. Among living species, this includes ∼ 250 parasitic and 11,300 free-living sarcodines (of which ∼ 4,600 are foraminiferids); 1.800 parasitic and 5,100 free-living flagellates: ∼ 5,600 parasitic “Sporozoa” (including Apicomplexa, Microspora, Myxospora, and Aseetospora); and ∼ 2,500 parasitic and 4,700 free-living ciliates. There are undoubtedly thousands more still unmamed. Seven phyla of PROTOZOA are accepted in this classification—SARCOMASTIGOPHORA. LABYRINTHOMORPHA, APICOMPLEXA, MICROSPORA, ASCETOSPORA, MYXOSPORA, and CILIOPHORA. Diagnoses are given for these and for all higher taxa through suborders, and representative genera of each are named. the present scheme is a considerable revision of the Society's 1964 classification, which was prepared at a time when perhaps 48,000 species had been named. It has been necessitated by the acquisition of a great deal of new taxonomic information, much of it through electron microscopy. It is hoped that the present classification incorporates most of the major changes that will be made for some time. and that it will be used for many years by both protozoologists and non-protozoologists.

Notes: SYNOPSIS Leishmania donovani amastigote-to-promastigote transformation is inhibited by homogenates of infected hamster liver and spleen. This inhibitory activity is localized in the 100,000 g pellet fraction. Tests with lysates of adherent (macrophyages) and nonadherent (lymphocytes) spleen cells indicated that the inhibitory activity resided in the lymphocytes, specifically in the 100,000 g pellet fraction.

Notes: SYNOPSIS Antibodies induced in rabbits against Paramecium multimicronucleatum syngen 2 prevent sexually reactive cells from clumping, pairing, and forming cytoplasmic fusions. A biologic assay for the detection of these antibodies (designated blocking antibodies) is described. the blocking antibodies, unlike the immobilization antibodies, are produced against breis of sexually reactive cells and nonreactive cells of 2 types, nonstarved and immature. Isolated cilia from reactive cells of either mating type are weak immunogens for blocking antibodies. No correlation between the mating type specificity (III or IV) and these antibodies has been detected. Blocking antibodies can be absorbed with living cells, of which sexually reactive ones are the most effective absorbers, while immature ones are the least effective.

Notes: SYNOPSIS A method is described for the axenic mass cultivation of Paramecium tetraurelia strains 51s and 299s. the ciliate is grown in an enriched axenic medium developed by Soldo, Godoy & van Wagtendonk. Under continuous shaking on a rotary shaker, cultures were grown in one-liter Erlenmeyer flasks with 330 ml medium yield cell densities of 32,000 cell/ml and 20,000 cells/ml for strains 299s and 51s respectively. Doubling time is considerably shorter under these conditions than in the conventional static cultures. A 20-liter airlift bioreactor is described in detail which can be used successfully to otain up to 100 g wet weight of Paramecium in a single run; in this reactor the cell density reaches 38,000 cells/ml for strain 299s. and 23,000 cells/ml for 51s. This technic should facilitate the study of minor protein components of the ciliate.

Notes: SYNOPSIS The cadmium ion (Cd2+) was accumulated by Amoeba proteus in all cellular fractions, the highest level being associated with the cytosol fraction. On gel separation of the cytosol fraction, Cd-binding protein appeared in 2 peaks: one 〉45,000 MW (peak I) and the other 12,000 MW (peak II). Added cysteine increased the total Cd2+ taken up by the cells and resulted in disproportionate increase of Cd incorporated into the Cd-binding protein of peak II. the Cd-binding protein of peak II is analogous to the low-MW, Cdbinding proteins in Anacystis nidulans, Mytilus edulis, and to the metalloprotein of some vertebrates.

Notes: Sorogena stoianovitchae is an unusual ciliated protozoan with a life cycle characterized by the aggregation of individual trophic cells to form a multicellular sorogen that rises from the liquid culture medium surface by the secretion of a stalk. The noncellular stalk is a tapered, longitudinally furrowed structure composed of a fibrillar matrix that is initially hydrated, but with time dehydrates, the stalk becoming thin and brittle. This dehydration is of importance from the earliest stages of stalk formation since it results in the formation of the outer sheath-like region of the stalk that appears to provide much of the support of the stalk. Cytochemical tests of the stalk for polysaccharides (including acidic mucopolysaccharides) and proteins are positive. Proteolytic enzymes degrade the stalk. Lectins specific for glucose and N-acetyl-D-glucosamine bind to the stalk. Gas chromatography analysis detected the presence of fucose, glucose, glucosamine, and arabinose, as well as a variety of amino acids, predominantly glycine. The cytochemical and biochemical tests, the ultrastructural data, and the behavior of the stalk material suggest that the staik is composed of a matrix of complex protein-polysaccharide molecules.

Notes: Nosema disstriae, a parasite of the forest tent caterpillar, Malacosoma disstria, was cultured with cell lines UMN-MDH-1 (Malacosoma disstria), IPLB-1075 (Heliothis zea), and BTC-32 (Triatoma infestans). Infected cultured cells were used to infect the healthy cell lines. Electron micrographs of thin sections of 6-day-old cultures revealed infected cells that exocytosed vesicles containing vegetative and immature sporulating forms of the parasite. Some of these forms were believed to be responsible for intercellular transmission of the parasite. The spread of infection was augmented by culturing the cells at high densities; if the density was too low, there was little or no cross infection. Cross infection was inhibited, but not blocked completely, by high osmolality of the culture medium. The yield of spores from a confluent cell monolayer at the end of growth was generally 1–4 × 107 per ml of culture medium.

Notes: The fine structure of the trophozoite of Acanthamoeba palestinensis with a special emphasis on the Golgi complex, microbodies, and mitochondria has been examined. Golgi complexes are distributed throughout the cytoplasm but are most abundant in the perinuclear region. Usually two Goigi complexes are found in the same plane on opposite sides of the nucleus. One of them appears to be in an intimate association with the nuclear membrane. The region of contact contains compact cisternae, vesicles of various sizes, as well as granular and amorphous electron-dense material. Structural changes in the nuclear envelope are also observed in this area. A structure consisting of a Golgi complex and electron-dense microtubule organizing center, comparable to the centrosphere of other Acanthamoeba species, has been observed. Microbodies, surrounded by a single unit membrane and containing a granular matrix and tubular inclusions, are scattered throughout the cytoplasm. These organelles, circular (∼1 μm in diameter) or ovoidal (∼1 μm in length and ∼0.5 μm in width) in section, have often an irregular outline. These microbodies are probably the morphological equivalent of peroxisomes and glyoxysomes. Most mitochondria show a typical structure including tubular cristae and intracristal inclusions. Occasionally mitochondria with two apposed double membranes running through the midline are found. Such atypical cristae have never been reported in small amoebae before.

Notes: Heavy infections with enigmatic mobile organisms have recently been found in the blood of carp (Cyprinus carpio) in Central Europe. The organisms measure up to 15 μm, are variable in shape, and exhibit an unceasing twitching or dancing movement. Their developmental cycle starts with a primary cell enclosing a secondary cell. The former grows while the latter produces inside itself by a series of binary fissions and internal cleavages up to eight secondary cells, each of which encloses an inner (tertiary) cell of its own. In addition, up to four tiny cells with compact nuclei (“residual bodies”) also result from divisions of the secondary cells. Primary cells containing the products of the division of secondary cells finally disintegrate, releasing the secondary cells, which in their turn become new primary cells and repeat the cycle all over again. The structure and behavior of these organisms were so incompatible with existing ideas on myxosporean development that their myxosporean affinity was at first unrecognized. The final proof of their identity–appearance of myxosporean spores in sterile, experimentally infected hosts–is still to be presented. The interpretation of the myxosporean features of their life cycle (i.e., [1] the pericyte nature of the primary cell, [2] proliferation by disintegration of the pseudoplasmodial primary cell, [3] no rigidly fixed pattern in vegetative development), their ultrastructure (i.e., [1] characteristic bundles of microtubules and numerous free ribosomes in secondary cells, [2] lack of centrioles, [3] membranes enclosing the secondary cells within the primary cells), and facts on their epizootiology (i.e., [1] no success at transmission via leeches, [2] the occurrence of these organisms along with Sphaerospora renicola Dykova and Lom) suggest that they are stages of S. renicola from the kidney of carp. Similar mobile organisms were found in the blood of fry of two other fishes (Gobio gobi and Tinca tinca) which are also hosts for a Sphaerospora that infects the kidney. This suggests that these organisms represent an early phase in the developmental cycle in the genus Sphaerospora. The existence of cells enveloped one within the other (secondary and tertiary cells) in the developmental cycle, a characteristic myxosporean feature itself, is an intriguing parallel to similarly enclosed cells in sporogenesis of Paramyxea (Ascetospora).

Notes: The morphology and morphogenesis of the kinetofragminophoran soil ciliates, Fuscheria terricola n. sp. and Spathidium muscorum Dragesco & Dragesco-Kerneis, 1979, are described. Stained specimens (protargol) are characterized biometrically. The new species differs from the other species of the genus in its body size, body shape, number of kineties, length of extrusomes, and habitat. Both species have telokinetal stomatogenesis, which commences with a proliferation of kinetosomes at those kineties which bear the brosse. Fuscheria terricola does not have a complex perioral ciliature; indeed, it might be that this species has only monokinetids. Thus only a proliferation of kinetosomes and the separation of the kineties takes place in the prospective division furrow. In contrast, S. muscorum differentiates short dikinetid kinetofragments in the region of the division furrow, which are arranged to form the perioral kinety of the opisthe in the intermediate and late stages of the stomatogenesis. The right part of the perioral kinety develops first. This and other studies show that telokinetal stomatogenesis proceeds very differently depending on the differentiation of the oral ciliature; however, detailed studies on the morphogenesis of kinetofragminophoran ciliates are still too few in number for subtypes to be defined.

Notes: The apicomplexan family Barrouxiidae Léger, 1911 is reviewed and revised on the basis of present information. It includes the genera Barrouxia Schneider, 1885 with ten named species, Defretinella Henneré, 1966 with one named species, and Goussia Labbé, 1896 with 25 named species. The family is characterized by having bivalved sporocysts with a longitudinal suture line. Available information, admittedly spotty, is given for each species on oocyst, sporocyst and sporozoite structure, and on locus of sporulation. The following seven new combinations are made: Goussia flaviviridis (Setna & Bana, 1935) n. comb. in the gecko Hemidactylus flaviviridis; G. hyalina (Léger, 1898) n. comb. in an unidentified aquatic beetle; G. lacazei (Labbé, 1895) n. comb. in the centipedes Lithobius forficatus and L. martini; G. metchnikovi (Laveran, 1897) n. comb. in the gobies Gobio gobio and G. albipinnatus; G. schaudinniana (Pinto, 1928) n. comb. in the centipede Lithobius forficatus; G. stankovitchi (Pinto, 1928) n. comb. in the small bleak Alburnus alburnus, the bream Abramis brama, and the red roach Scardinius erythrophthalmus; Goussia sp. (Dogel' Akhmerov, 1959) nov. comb. in the freshwater fish Gnathopogon chankaensis.

Notes: Comparison of the electrophoretic migration patterns of proteins of active 40S and 60S ribosomal subunits isolated from nine amicronucleate strains of Tetrahymena of known phenoset revealed strain dependent differences which correlated with the phenoset classification of these strains as determined by Borden, Whitt & Nanney, who compared isoenzyme patterns.

Notes: Chromatin from a uninucleate dinoflagellate, Crypthecodinium cohnii, a binucleate dinoflagellate, Peridinium balticum, and a chromophyte, Olisthodiscus luteus, was examined by nuclease digestion and the results were compared to those from vertebrates. Gel analysis of the products of staphylococcal (micrococcal) nuclease digestion revealed a DNA repeat unit of 220(±5) base pairs for O. luteus and 215(±5) for P. balticum. Limit digestion gave a core particle of 140 base pairs, revealing that these longer repeat sizes are due to longer linker regions. No repeating subunit structure was found upon electrophoresis of digests of C. cohnii nuclei. Examination of the DNA fragments produced by DNAse I digestion of nuclei isolated from P. balticum and O. luteus showed the same ladder of ten base multiples as seen in chromatin from other eukaryotes. Examination of the kinetics of digestion by DNAse II of Peridinium chromatin revealed less susceptibility when compared to DNAse I digestions while 70% of Olisthodiscus chromatin and 35% of C. cohnii chromatin was sensitive to DNAse II. These data, taken together with previous results from Euglena, indicate that while algal chromatin is similar to that of higher eukaryotes in regard to DNAse I and II action, it differs in that the linker DNA is longer. In addition, the Hl-like histone from O. luteus and P. balticum is located in the linker DNA as in higher eukaryotes.

Notes: Changes in mean cell size, DNA and cell density were monitored at 6-h intervals for 72 h in populations of six species (eight clones) of marine dinoflagellates to determine the temporal relationships between the cell cycle events of DNA replication and cytokinesis. Batch cultures were maintained at 15 or 20°C on a 12-h light: 12-h dark photoperiod. Cell densities and size frequency distributions were determined conductimetrically and the amount of DNA within populations was measured fluorometrically. A variety of intra- and interspecific relationships were observed, ranging from parallel phasing of cell cycle processes to variations which involved the temporal uncoupling of DNA synthesis from the phased pattern of cell division which is characteristic of dinoflagellate cell cycles. Daily growth rates of individual populations varied from 0.05 (Gymnodinium nelsoni) to 2.08 (Amphidinium carteri) cell divisions day-1 and DNA doubling rates ranged from 0 to 1.14 day-1. Mean doubling rates for DNA were usually 30–40% lower than those for cells. The degree of difference in these rates and the amount of variability evident in cell cycle sequences may be major factors in determining the rate and extent of development of dinoflagellate populations in nature.

Notes: In Aedes cantator, Amblyospora sp. is transovarially transmitted and has two developmental sequences. The life cycle is initiated in the adult female with the release of sporoplasms from binucleated spores not bounded by membranes, lying free within host oenocytes. Sporoplasms infect the developing oocytes and are transmitted to the filial generation when the eggs are laid. In some of the female progeny that hatch from infected eggs, diplokaryotic cells infect host oenocytes and divide by binary fission during merogony. Sporulation and spore formation do not occur until a blood meal is taken by the host and they coincide with the development and maturation of the oocytes to complete the cycle. In other female and all male progeny, pathogen development occurs within fat body tissue of the host where diplokaryotic cells divide by multiple fission during merogony to spread the infection. Sporulation in this developmental sequence is characterized by the secretion of an accessory membrane and the meiotic division of diplokaryotic sporonts, which result in the formation of octonucleated plasmodia that undergo cytokinesis to form eight haploid spores which are not perorally infectious to other mosquito larvae. There is no increase in the prevalence of either type of infection in field populations during juvenile development, indicating that there is no direct horizontal transmission of the pathogen within any one generation. Data obtained from laboratory rearings of infected progeny, however, show that infections cannot persist relying solely upon maternal-mediated transmission and that some other mode of transmission must be operative for continued maintenance of this microsporidium in A. cantator.

Notes: Cells of Amoeba proteus and Chaos carolinensis that were in the process of phagocytosing large prey organisms were studied to find a structural basis for the generation of mechanical forces exerted by newly forming food cups. It was found that the food-cup walls facing prey organisms have a more prominent network of thin filaments inside the plasmalemma and that the glycocalyx covering the area is more condensed than usual.

Notes: Monoclonal IgG antibodies against sporozoites of Eimeria tenella were obtained from the ascites fluid of BALB/c mice. Oocysts, sporocysts, and sporozoites were exposed to medium 199, normal ascites fluid, or monoclonal antibodies 1A, 9D, 3D3II, or 2G8f. Specimens were then incubated with ferritin-conjugated goat anti-mouse IgG antibody. Ferritin was uniformly distributed over the surface of sporozoites exposed to 1A, 9D, or 3D3II; ferritin was localized in patches on sporozoites exposed to 2G8f. A uniform layer of ferritin was present on the inner layer of oocyst walls and on the Stieda body and outer surface of sporocysts exposed to 1A, 9D or 3D3II. In specimens treated with 2G8f, ferritin was present on the inner layer of the oocyst wall and the Stieda body, but not on the sporocyst wall. No ferritin was found on specimens exposed to medium 199 or normal ascites fluid. Monoclonal antibodies 1A, 9D, and 3D3II, but not 2G8f, caused complement-mediated lysis of sporozoites. These findings indicate that oocysts, sporocysts, and sporozoites of E. tenella contain common antigens specific for each monoclonal antibody tested.

Notes: Several divalent cation-dependent ATP phosphohydrolases associated with cilia, ciliary axonemes, ciliary membranes, pellicles, trichocysts, nuclei, mitochondria, microsomes, and soluble peripheral cell surface fractions of Paramecium tetraurelia were resolved in this study. Fifteen different activity bands were detected in whole cell sonicates or subcellular fractions by Triton polyacrylamide gel electrophoresis and ATPase activity staining. The ciliary surface membrane contained two major ATPase activities that were distinct from the enzymes associated with all other cell fractions.

Notes: During feeding a peritrophic membrane (PM) is formed in the gut of the tick Ixodes dammini, dividing the lumen of the gut into an ecto- and endoperitrophic space. Babesia and all food particles ingested with the blood meal by the tick are retained in the endoperitrophic space, the lumen proper. Only Babesia equipped with a highly specialized organelle, the arrowhead, are able to pass the PM and enter the ectoperitrophic compartment. During the crossing of the PM the arrowhead loses its density, suggesting that enzymes released from it dissolve the polymers in the PM, making passage of the parasite through this barrier possible. In the ectoperitrophic space the arrowhead of Babesia touches the epithelial cell. At the point of contact the membrane of the host cell starts to invaginate, and simultaneously the arrowhead's fine structure loses its highly organized pattern. The growing host membrane encircles the parasite and the arrowhead diminishes progressively in size. When the piroplasm is inside the host cell, the arrowhead can no longer be found. During invasion the host membrane often touches the parasite's plasma membrane at the site of a coiled structure, and the host membrane becomes ruptured and the nearby host cytoplasm appears to be lysed. Babesia inside the host cell is covered solely by its own plasma membrane; the invaginated host membrane is missing. It is postulated that the latter disintegrates during invasion by the parasite through the action of enzymes from the coiled structure. The parasite is surrounded by a halo of homogeneous material deriving most probably from the lysed host cytoplasm.

Notes: A technique for the separation of schizonts of Plasmodium falciparum is described. The different stages of the asexual cell cycle of the parasite were positioned according to their density in a continuous gradient of Percoll. Young trophozoites coincided with erythrocytes in a broad band corresponding to densities from 1.075 to 1.100 g/ml, whereas schizonts were concentrated at a density approximating 1.062 g/ml. The viability of the parasites was unimpaired by this procedure. Young trophozoites and schizonts continued their normal life cycle when cultured after the separation procedure. The percentage of recovery was high, reaching 80% of the initial quantity. Possible applications of the technique are discussed.

Notes: Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCCr̀30100, ATCCr̀30863, and ATCCr̀30896) and two strains of N. lovaniensis (ATCCr̀30467 and ATCCr̀30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCCr̀30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysine for ATCCr̀30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCCr̀30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCCr̀30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B12, nicotinamide, and Ca pantothenate) was required.

Notes: From several surveys of environmental sites, the virulent human pathogen, Naegleria fowleri, was isolated from a pond in Georgia, a sewage treatment plant in Missouri, and from the Potomac and Anacostia rivers near and in Washington, D.C. Widely scattered, sparse populations seemed only a potential threat to human health at the time of sampling. The data support an estimate that the sites sampled contain 10,000 typical, low temperature, bactivorous amoebae for each heat tolerant amoeba able to grow at 45° C. Heat tolerant competitors were much more common than N. fowleri. Naegleria lovaniensis, which is heat tolerant but nonpathogenic, was isolated from and downstream from an open air thermal pollution temperature gradient. Hot piles of composting sewage sludge yielded no amoeboflagellates, many heat tolerant (45–49° C) amoebae, and one thermophilic (52° C) Acanthamoeba. Features of the methods used include two-stage incubation to increase isolation of sparse organisms and distinction of N. fowleri from almost all other amoebae on agar plates. The flagellate-empty habitat hypothesis postulates a general model in which human intervention and/ or natural events remove usual competitors and the ability to transform to a motile flagellate confers an advantage in recolonizing.

Notes: The ultrastructure of the bloodstream form of Cryptobia salmositica in rainbow trout was examined during the acute phase of experimental infection. The arrangement of the major groupings of cytoplasmic microtubules originating near the basal bodies is similar to that in other bodonids. The cytostome is reinforced both by pellicular microtubules and an electron-dense plaque. Certain microtubules associated with the flagellar pocket serve as nucleating sites for pellicular microtubules. A flagellar rootlet, consisting of two parallel fibers which are bound together intermittently by electron-dense plaques, curves posteriorly from the basal body of the recurrent flagellum towards the kinetoplast. The basal body associated plaque on the kinetoplast membranes is duplicated at the same time as the basal bodies. Cytoplasmic microtubules are found in association with the plaque and the outer kinetoplastic membrane. A pulsatile vacuole, described for the first time in a hemoparasitic cryptobiid, lies adjacent to the post-flagellar pit. Smaller, interconnected vesicles of the spongiome are continuous with the pulsatile vacuole. Since a pulsatile vacuole occurs not only in free-living and ectoparasitic cryptobiids but in the hemoparasitic (=trypanoplasm) forms as well, this is no longer a character by which the genus Trypanoplasma may be separated from the genus Cryptobia. Possession of this osmoregulatory complex may allow the organism to survive outside of a host and fulfill a monoxenous life cycle, in addition to the usual heteroxenous cycle involving a leech as vector.

Notes: The life-cycle of the amoeboflagellate Tetramitus rostratus includes amoeboid, cyst, and flagellate stages. The ultrastructure of these three stages is illustrated, with particular emphasis on flagellate morphology. Amoeba morphology is typical of that of limax amoebas. Cysts, forming from trophic amoebas, are enclosed by a wall made up of two layers: ectocyst (ca. 70 nm), and endocyst (200 nm). The wall apparently forms from precursor material present in vesicles in the pre-cyst stage cytoplasm. Flagellate morphology is characterized by a well-defined top-shaped profile, maintained by microtubules under the plasma membrane. The flagellar apparatus or mastigont consists of four flagella, their basal bodies, sheaves of microtubules associated with two of the basal bodies, and several rhizoplasts (periodicity 20 nm). A deep, microtubule-supported, ventral invagination appears to function as a gullet. A small number of mitotic stages observed in amoeboid and flagellate individuals suggests similarity in the division process in both stages: intranuclear mitotic apparatus, nucleolus persisting through mitosis, no centrioles or basal bodies functioning as centrioles, difficulty in resolving chromosomes. The text compares ultrastructures of several amoeboflagellate organisms and evaluates the phylogenetic significance of those features common to different species. On the basis of this study, Tetramitus most closely resembles Naegleria spp.

Notes: The hypothesis is advanced that all freshwater Euplotes species with a 9 type 1 fronto-ventral cirrus pattern (E. patella type) depend upon bacteria-like endosymbionts. Aposymbiotic cells of these species are unable to divide. The hypothesis is based on the investigation of 40 different freshwater Euplotes stocks collected in Germany, France, the USA, and Japan. No symbionts were found in E. crenosus and E. palustris, freshwater species with 10 fronto-ventral cirri, nor in E. muscicola, a representative of the freshwater Euplotes group with a 9 type 2 fronto-ventral cirrus pattern (E. affinis type). Characteristic for the essential endosymbionts are multiple nucleoids, a feature described earlier for omikron, an indispensable symbiont of E. aediculatus. Although the symbionts differ from omikron and among each other in size, shape, and their average number per host, they are believed to be related to omikron. In two stocks a different type of bacterium was found in which no defined nucleoids can be detected. Transfer of this symbiont into aposymbiotic cells, originally carrying omikron, revealed that it can restore the ability to multiply. Similarly, omikron was also able to restore the ability to divide in cells freed of this symbiont. It is assumed that this different type of symbiont is a secondary invader of Euplotes which displaced the original omikron-like endosymbiont. Some of the stocks were found to carry, in addition to omikron-like symbionts, other symbiotic bacteria; E. daidaleos carries in addition an alga. The findings suggest that the freshwater Euplotes species with a 9 type 1 cirrus pattern are closely related to each other and evolved from an ancestor (probably of cirrotype 10) which already was dependent upon endosymbionts of the omikron type. It supports the view that the two subgroups of freshwater Euplotes forms with a cirrotype of 9 have evolved independently from each other from species with 10 fronto-ventral cirri by losing a cirrus at different positions.

Notes: Promastigotes of Leishmania mexicana mexicana attach to mouse macrophages in vitro in the absence of serum by a wheat germ agglutinin-like ligand on the surface of the promastigote that binds to the N-acetyl glucosamine moiety of a receptor on the surface of the macrophage. The binding is temperature dependent, and the macrophage receptor is trypsin, cytochalasin B, and glutaraldehyde sensitive. The promastigote ligand is proteolytic enzyme and glutaraldehyde insensitive. Uptake follows attachment and is assisted or inhibited as for attachment. Treatment of promastigotes with proteolytic enzymes uncovers a receptor for a serum component that binds strongly to a mouse macrophage receptor in vitro. The strain of mice donating the macrophages had little effect upon attachment and uptake except that A strain mouse macrophages attached fewer promastigotes in 10 min than those of outbred mice, but took up as many promastigotes over 90 min as those of outbred mice. Low responder Biozzi mouse macrophages took up more promastigotes than high responder Biozzi mouse macrophages. Normal unheated human, rabbit, and guinea pig sera lysed promastigotes and so inhibited their attachment to macrophages in vitro. Unheated immune serum showed an enhanced inhibition of attachment. Heated normal serum allowed attachment and uptake, while promastigotes treated with heated immune serum showed enhanced attachment to and uptake by macrophages. Treatment of macrophages in vitro with immune serum enhanced their ability to attach promastigotes and to engulf them. Repeated 90-min exposures of a population of promastigotes to uptake by mouse macrophages in vitro did not deplete the population of any sub-population more likely to be taken by macrophages. The first sub-population to be taken up survived better in macrophages over 24 h than subsequently engulfed sub-populations.

Notes: BALB/c mice were hyperimmunized with non-infectious extracts of either Leishmania braziliensis promastigotes or Trypanosoma cruzi epimastigotes. When spleen cells from these mice were fused with P3X63Ag8 plasmacytoma cells, the resultant hybridomas synthesized monoclonal antibodies which displayed specific reactivity by indirect immunofluorescence with distinct subcellular components of the parasites. These studies revealed that antigens associated with the flagellum and with a nongranular component of the cytoplasm would account for much of the serologic cross-reactivity observed between the two species. Conversely, antigens associated with surface and/or cytoplasmic granules and with an intracellular organelle believed to be the kinetoplast appeared to be species-specific.

Notes: The invasion of liver parenchymal cells by sporozoites of Plasmodium berghei Vincke & Lips, 1948, was studied in vivo using transmission electron microscopy. Livers of Brown Norway rats were examined 30 and 60 min after intraportal injection of 15 million sporozoites each. Sporozoites found after incorporation into vacuoles in hepatocytes were often located near a bile canaliculus at the lateral cell surface, surrounded by hepatocyte lysosomal structures; however, degradation of sporozoites caused by lysosomal digestion inside hepatocytes was never observed. Due to the crescent shape of sporozoites, serial sections were necessary to demonstrate the actual process of invasion of the hepatocyte. The hepatocyte's plasmalemma appeared to invaginate due to the sporozoite's action, thereby creating a parasitophorous vacuole. It was suggested that the sporozoite actively penetrated the hepatocyte; however, no visible depletion of rhoptries and micronemes was observed.

Notes: The human pathogenic amoeboflagellate Naegleria fowleri and the nonpathogenic species N. gruberi can be cultivated axenically but usually in different media. Naegleria fowleri 6088 has been adapted to grow in Balamuth H-4 medium, usually used to propagate N. gruberi nB81. and nB81 has been adapted to grow in supplemented Nelson's medium, usually used to propagate N. fowleri. N. gruberi nB81. grown in either medium, enflagellated 135 to 150 min after subculture to non-nutrient amoeba saline, whereas 6088 required 225 min. Naegleria gruberi nB81 grown in either medium was agglutinated by 100 ug concanavalin A/ml, whereas N. fowleri 6088 was not. Naegleria fowleri and N. gruberi grown in Nelson's medium became rounded to a greater extent upon chilling at 5° C and remained rounded longer than Naegleria grown in Balamuth medium. The specificity of the surface antigens was an inherent characteristic of each species and not dependent upon the propagating medium. but Naegleria grown in Nelson's medium was agglutinated more reproducibly and more effectively by antiserum. N. gruberi was somewhat more resistant to acriflavine, actinomycin D, cycloheximide, or tetracycline than N. fowleri, regardless of the culture medium. Naegleria fowleri 6088 grown in Nelson's medium, however, was more resistant to actinomycin D, daunomycin. mithramycin. sulfamethoxazole, or tyrocidine than 6088 grown in Balamuth medium. There are limitations on the validity of comparisons of N. fowleri and N. gruberi based upon cultures grown in different media.

Notes: Fine structural studies of the hydrogenosomes of Tritrichomonas foetus using an improved fixative reveal that they are enclosed by two closely apposed 6 nm membranes, which separate at some regions forming a large intramembranous vacuole where Ca++-binding sites are located. Fixation of the cells in a glutaraldehyde solution containing 5 mM CaCl2 and postfixation in an osmium tetroxide-potassium ferrocyanide solution led to the appearance of a reaction product associated with certain regions of the membrane of the hydrogenosomes and in the cisternae of the endoplasmic reticulum, in the recurrent flagellum, and in the plasma membrane. Treatment of ultrathin sections with EGTA removed the reaction product. These results, in association with others previously described, indicate the existence of several similarities between the hydrogenosomes and the mitochondria.

Notes: . One hundred eighty-eight stocks of Paramecium primaurelia. P. biaurelia, P. tetraurelia. and P. octaurelia were grown axenically and screened for variation in four different esterases and acid phosphatase using starch gel electrophoresis. Major observations: frequency of intraspecies variation for these enzymes is much lower in these four species than in other organisms; hypervariability for two esterases occurs in P. biaurelia both in isolates from worldwide locales and in a restricted locale; clustering of variations occurs in a high proportion of variant stocks in all four species; frequency of intraspecies variation is highest in Central and South America for all four species; and geographical differentiation is lacking between stocks in the same species both for common as well as variant phenotypes despite the cosmopolitan distribution of these species. These results are not correlated with adaptations that favor inbreeding over outbreeding. nor is the possession of bacterial endosymbionts strongly correlated with enzyme variation. When the riequency of intraspecies variation was examined for the aurelia complex of species as a whole for 13 enzymes, mitochondrial DNA, and ribosomal DNA, differences between enzymes in frequency of variation could be seen, ranging from less than 2% for seven enzymes to 12.4% for glucosephosphate isomerase, a value similar to that observed for malic dehydrogenase, mitochondrial DNA, and ribosomal DNA in P. tetraurelia. The percentage of polymorphic enzyme loci in the complex as a whole was found to be much lower than that observed for other organisms. For the species more intensely studied in this paper the level of genetic polymorphism was also much lower, although P. biaurelia showed greater variability for two of the enzymes.

Notes: Precursors of 2-aminobutanoic acid (2-ABA), found in the incubation medium of mixed rumen ciliate protozoa, were examined with washed or starved bacteria-free ciliates. Threonine and methionine strongly stimulated the formation of 2-ABA. Formation of 2-ABA by direct conversion of threonine and dethiomethylation of methionine was confirmed by radiotracer experiments with [U-14C]L-threonine and [carboxyl-14C] and [methyl-14C]L-methionine.

Notes: The effects of irreversible inhibition of protein synthesis by pactamycin in either infective forms of Trypanosoma cruzi or mammalian host cells on cellular invasion by this human pathogen were investigated. Treatment of bloodstream forms of T. cruzi with pactamycin markedly reduced their ability to bind either fibroblast-like cells of monkey origin or myoblasts of rat origin. The number of amastigote forms that could be established intracellularly was also significantly decreased with respect to control values obtained when mock-treated (medium alone) trypomastigotes were incubated with the cells. Pactamycin treatment also reduced the infectivity of T. cruzi trypomastigotes for mice as evidenced by both significantly reduced parasitemia levels and mortality rates when compared with those of control mice infected with mock-treated parasites. Inhibition of protein synthesis in the host cells neither prevented cell infection by untreated trypomastigotes nor altered the percentages of infected cells or the magnitude of the infection in vitro. These results indicate that protein synthesis is a requirement for cell invasion by T. cruzi and that the parasite can establish itself and replicate within cells relying on its own protein synthesis ability.

Notes: One hundred eighty-eight stocks of Paramecium primaurelia, P. biaurelia, P. tetraurelia, and P. octaurelia were grown axenically and tested for five esterases, visualized by starch gel electrophoresis, in a search for variant stocks. The five esterases can be distinguished on the bases of their substrate specificity, sensitivity to an inhibitor, and response to different growth conditions. This paper addresses the nature of the electrophoretic change in mobility of the variant stocks in order that species relationships can be more accurately assessed. Crosses carried out in all four species show that single genes determine the differences in mobility between variant and common subtypes. Extracts of variant stocks that gave similar patterns were run against each other, tested for their sensitivity to the inhibitor, and the pattern was compared to that found in extracts of stocks with variant and common subtypes in other species. The majority of the variants in P. primaurelia, P. tetraurelia, and P. octaurelia show an electrophoretic mobility characteristic of a common subtype, or a variant, in another species. The same proportion of variant subtypes as common subtypes have mobilities similar to esterase subtypes found in other species. Of the four species examined in this paper, P. tetraurelia and P. octaurelia appear to be most closely related on the basis of shared esterase subtypes. In P. biaurelia the mobilities of most of the variants are unique, as are the common esterase subtypes in this species. P. biaurelia stands out as having the greatest number of esterase subtypes, with very few of them homologous to subtypes found in other species. This observation supports the idea of greater diversification of stocks within P. biaurelia than for the other three species.

Notes: Glycogen phosphorylase and synthase activities were detected in the sonic lysate of rumen ciliates of the genus Entodinium. The ciliate phosphorylase had the following properties. The pH optimum was narrow and centered at pH 5.9. The activity was maximum at 30°C; above 40°C a rapid inactivation occurred. The Km value for glucose-1-phosphate (G-1-P) and for glycogen was 15 mM and 0.069% (w/v), respectively. NaF and ethylenediamine tetraacetic acid had no stimulative effect on the enzyme activity, though adenosine 3′,5′-monophosphate and theophylline activated it. NaHSO3 inhibited the enzyme activity at a concentration of 1 mM. The inhibition of glucose was noncompetitive for G-1-P. Glycolytic intermediates and nucleotides had a minor effect on phosphorylase activity. Glycogen synthase existed in two forms, glucose-6-phosphate dependent and independent forms: the proportion of the latter form increased with the decrease of reserve polysaccharide levels in the ciliates. Correlations between glycolytic enzyme activities included phosphorylase and synthase activities and reserve polysaccharide contents in the ciliates were determined, and a possible regulatory mechanism of polysaccharide synthesis and degradation was discussed.

Notes: The structure of the major protein of the pellicular membrane of Leishmania tropica was investigated. This protein is composed of two polypeptides, of ca. 50,000 d molecular weight, that were found to cross-react immunologically with the α and β subunits of pig brain tubulin. The polypeptides and pig brain tubulin subunits were partially digested with S. aureus V8 protease, and the peptides obtained analysed by SDS-polyacrylamide gel electrophoresis. A comparison of the patterns showed that the β subunits of Leishmania and pig tubulin have very similar primary structures, while the α subunits have evolved divergently. These experiments demonstrate that the major polypeptides found in the pellicular membrane of L. tropica are α and β subunits of tubulin. Immuno-electron microscopy indicates that the tubulin is located in the microtubules associated with the pellicular membrane of Leishmania. Arrays of microtubules were prepared by nonionic detergent treatment of the cells and observed by electron microscopy after negative staining. Optical diffraction reveals a 5 nm spacing between protofilaments in the microtubule and a 4 nm axial periodicity corresponding to the tubulin subunits. The pitch of the shallow left-hand three-start helix is 12°. A distance of 47 nm separates each microtubule from the next. These data show that the dimensions and supramolecular organization of the tubulin subunits in the microtubules are identical in the pellicular membrane of L. tropica and in mammalian brain.

Notes: Haemogregarina bigemina is redescribed from the blood of the marine fish Blennius pholis, and stages presumed to be those of the haemogregarine are recorded from the hematophagous praniza larva of the isopod Gnathia maxillaris. At College Rocks, Aberystwyth, Wales, the main study area, a high incidence of infection occurred in B. pholis. No exoerythrocytic stages were observed in these fish, nor was sexual dimorphism of the gametocyte evident. As in an earlier study, ecological evidence favored transmission by G. maxillaris rather than by leeches. Gametocytes, syzygy, oocysts, sporoblasts, and sporozoites were identified in the anterior hindgut of the isopod. The stages observed in G. maxillaris are compared with those of other haemogregarines described from the digestive tract of leeches. Mention is made of an intraleucocytic haemogregarine of another fish, Crenilabrus melops.

Notes: The nomenclature of three genera in the family Haemogregarinidae (Haemogregarina, Karyolysus, and Hepatozoon) has been reviewed and the following new names are introduced to replace homonyms or for previously unnamed species: Haemogregarina carlosi n. nom., in the erythrocytes of the lizard Lacerta ocellata; Haemogregarina tincae n. nom., in the stomach and intestine of the tench Tinca tinca; Hepatozoon insectivorae n. sp., in the leucocytes of the shrews Sorex araneus and Crocidura leucodon; Hepatozoon krampitzi n. sp., in the leucocytes of the vole Microtus oeconomus; Hepatozoon peromysci n. sp., in the leucocytes of the deermice Peromyscus boylii and P. truei gilberti; and Hepatozoon pallida (Pessoa et al., 1971) n. comb., in the erythrocytes of the snake Thamnodynastes pallidus nattereri.

Notes: Transmission and scanning electron microscopy of specimens of Paramecium multimicronucleatum treated with the Rio-Hortega silver-impregnation method as modified by Fernández-Galiano demonstrate that considerable deposition of silver occurs around the kinetosomes, especially at the level of the basal plate and also at the proximal end of the kinetosome. In addition, silver is heavily deposited within the kinetodesmal fibers, in the fibrous matrix that surrounds the postciliary and transverse microtubules, in the connective structures observed between the two kinetosomes of a pair and between the kinetodesmal fiber and the anterior kinetosome, and in the trichocysts. Differences and similarities in sites of deposit when other methods of silver impregnation are employed are discussed and the particular value of the present technique in studies of ciliate systematics and phytogeny is stressed.