This paper describes a method which allows the amplification of Paramecium micronuclear DNA. Amacronucleate cells are first obtained by an appropriate treatment with nocodazole, a microtubule depolymerizing agent which blocks the elongation of the macronucleus and the distribution of the micronuclei at cell division between the two daughter cells; then, DNA from such cells is amplified by the polymerase chain reaction technique. We have applied this method to the problem of the central repeats of the G surface antigen of P. primaurelia (strain 156). The central repeats consist of a 74 amino acid sequence repeated in tandem. The sequence identity of these repeats is also found in the nucleotide sequence even at silent codon positions, suggesting the existence of a mechanism of identity maintenance acting at the nucleotide level. Mechanisms based on RNA secondary structure which are frequently proposed as an explanation of this phenomenon are unlikely to be valid in this case. One can, therefore, imagine that these repeats might originate from one micronuclear sequence through duplicative processes which could occur during the formation of the macronucleus. We have used the described technique to amplify the micronuclear version of the central repeats and showed that it is identical to the macronuclear version, thus ruling out the above hypothesis. Therefore, intragenic recombination appears to be the most likely explanation of the sequence identity of these central repeats.
We observed Plasmodium gallinaceum ookinetes in both intracellular and intercellular positions in the midgut epithelium of the mosquito Aedes aegypti. After epithelial cell invasion intracellular ookinetes lacked a parasitophorous vacuolar membrane and were surrounded solely by their own pellicle. Thus, the ookinete in the midgut epithelium of the mosquito differs from erythrocytic and hepatic stages in that the parasite in the vertebrate host is surrounded by a vacuole. The midgut epithelial cytoplasm around the apical end of invading ookinetes was replaced by fine granular material deprived of normal organelles. Membranous structure was observed within the fine granular area. Most ookinetes were seen intracellularly on the luminal side and intercellularly on the haemocoel side of the midgut epithelial cells. These observations suggest that the ookinete first enters into the midgut epithelial cell, then exists to the space between the epithelial cells and moves to the basal lamina where the ookinete develops to the oocyst.
Two strains of Acanthamoeba isolated from human brain tissue and a strain of Acanthamoeba isolated from a fish were compared with 10 species of Acanthamoeba belonging to groups 1, 2 and 3 based on their isoenzyme profiles and antigenic characteristics. A total of 12 enzymes were studied. The isoenzymes and antigens were electrophoretically separated on polyacrylamide gradient gels, and the patterns obtained were compared after appropriate staining for particular enzymes and reactivities with homologous and heterologous rabbit anti-Acanthamoeba antisera. One of the human strains (CDC: 1283:V013) was identified as A. healyi n. sp. because of its unique isoenzyme profiles for 11 of the 12 enzymes tested. The other human isolate was reidentified as A. culbertsoni because its isoenzyme profiles for 10 of 12 enzymes resembled those of A. culbertsoni, Lilly A-1 strain. Since the isoenzyme profiles and the antigenic patterns of the fish isolate as well were remarkably similar to those of A. royreba, it was considered as a strain of A. royreba. Polyacrylamide gradient gel electrophoresis appears to be a power-ful technique for the study of isoenzymes and antigens of Acanthamoeba.
The ciliated protozoan Paramecium contains hemoglobin in heterogeneous monomeric forms. In particular, Paramecium caudatum is characterized by the presence of a major component called Hb10 and a basic component named bHb. We found that in P. caudatum both of these hemoglobin components show some variation according to stock. The types and distributions of these hemoglobin components were examined on 16 stocks in five different syngens and one stock in an unidentified syngen using high performance liquid chromatography. The results indicate that in a variety of stocks the major component, Hb10, was divided into three types, A, B or A + B, and that the basic hemoglobin component was composed of a combination of two or three variants out of four possible, i.e. bHb 1, bHb 2, bHb 3 and bHb 4. Neither the Hb10 types nor the bHb variants, however, could be used to distinguish syngen in P. caudatum, since all of the Hb10 types and bHb variants were widely distributed over syngens and identical profiles appeared to some stocks in different syngens.
ABSTRACT. The cilia of Didinium nasutum are restricted to two girdles encircling the cell. Each row of cilia in both girdles is made up of two to three anterior pairs of kinetosomes followed by several single kinetosomes. Each single kinetosome has two sets of transverse microtubules, an overlapping postciliary microtubular ribbon, and a laterally directed kinetodesmal fiber. The pairs of kinetosomes are homologous to the oral dikinetids of other haptorians: the nonciliated kinetosome of the pair has a transverse microtubular ribbon that extends to line the membrane of the proboscis, a single short postciliary microtubule, and a nematodesma; the ciliated kinetosome has a ribbon of postciliary microtubules and two sets of transverse microtubules. The presence of these characters in Didinium invalidates Leipe & Hausmann's conclusion that the Didiniidae should be removed from the subclass that contains the other haptorians (Leipe, D. D. & Hausumann, K. 1989. Somatic infraciliature of the haptorid ciliate Homalozoon vermiculare (Kinetofragminophora, Gymnostomata) Ditransversalia n. subcl. and phylogenetic implications. J. Protozool., 36:280–289). In light of this, the justification for a subclass Ditransversalia is challenged and shown to be unnecessary.
Reinitiation of meiosis (maturation) of amphibian Bufo and Xenopus oocytes can be induced if Tetrahymena extract is injected into them. The activity differed from M-phase-promoting factor, because action of the former factor on the induction of maturation was inhibited by treatment of the oocytes with cycloheximide. Activity of M-phase-promoting factor was not detected in Tetrahymena extract regardless of the presence of cdc2 homologues in the extract. However, cycloheximide-resistant-maturation-inducing activity appeared in the recipients, when the maturation was induced by injection of Tetrahymena extract. Immunoblots using antibodies against cdc2 showed that injection of Tetrahymena extract induced fast mobility of the recipient cdc2 in the presence of the recipient protein synthesis. The same mobility shift of the cdc2 was also induced when M-phase-promoting factor containing Xenopus oocyte extract was injected into immature oocytes or when the immature oocyte extract was treated with alkaline phosphatase. These results indicate that meiosis-reinitiation-inducing factor of Tetrahymena functions upstream of M-phase-promoting factor to induce dephosphorylation of the recipient cdc2. Tetrahymena cdc2 homologues also showed fast mobility when the Tetrahymena extract was treated with alkaline phosphatase. Preliminary experiments showed that the meiosis-reinitiation-inducing factor of Tetrahymena was a soluble protein.
Trypanosoma congolense bloodstream forms preincubated with a high titer of anti-variant surface antigen (VSG)-specific antibody, a low amount of anti-VSG plus complement-active mouse serum (MS), MS alone, and trypsin were cocultivated with mouse peritoneal macrophages in vitro. Immunofluorescence as well as transmission and scanning electron microscopy revealed that upon attachment to the macrophages' surface, trypanosomes opsonized with anti-VSG/MS formed opsonized filopodia, which were rapidly internalized by the phagocytes. Although these cells attached as frequently as anti-VSG or trypsin-pretreated parasites, the rate of phagocytosis of anti-VSG/MS pretreated trypanosomes was reduced significantly. Trypanosomes pretreated with high antibody titers alone were lysed on the surface of the macrophages before phagocytosis was completed. Parasites opsonized with complement alone adhered only occasionally and were rarely phagocytosed. Trypsin-treated trypanosomes, which served as positive control cells, rapidly attached and remained intact until ingulfment by the macrophages was completed. Untreated control parasites did not attach to the macrophages and were not phagocytosed. Cocultivation of macrophages with anti-VSG/MS-opsonized trypanosomes caused internalization of the flagellum by membrane fusion. Filopodia formation by T. congolense is thus correlated with a marked reduction in phagocytosis even in the presence of only a sublytic antibody titer.
ABSTRACT. A method is described for obtaining large numbers of telotrochs from mass cultures of Vorticella convallaria. These free‐swimming cells contract slightly along the aboral‐oral plane when extracted with Triton X‐100, and thus appear more similar in shape to zooids than unextracted telotrochs. Cytoskeletal structures associated with feeding, such as the infudibulum and the cytopharynx, are visible in cytoskeletal preparations of these non‐feeding telotrochs and thus appear not to be disassembled during telotroch formation. The telotroch to stalked‐zooid transition proceeds rapidly through a set series of morphogenetic stages. After telotroch attachment, the aboral cilia cease beating and are resorbed. Within 7 min the cell is inverted bell‐shaped and the zooid begins feeding. Stalk elongation begins about 15–20 min after attachment, lengthening at the rate of 0.5 μ/min for the first hour and more slowly (0.1 μm/min) after that. Interestingly, these developmental stages are essentially the same as those described for the telotroch to zooid transition in the colonial peritrich Zoothamnium. This evolutionary conservation suggests that the precise sequence and timing of these events are critical for their successful completion. Furthermore, the facts that the telotroch to zooid transition occurs very rapidly, that the feeding structures are maintained throughout the transformation, and that basic cytoskeletal architecture is relatively unchanged is consistent with the hypothesis that the transformation occurs through controlled cytoskeletal rearrangements rather than by changes in gene expression.
ABSTRACT. Species belonging to the genus Diophrys Dujardin, 1841, are easily recognized due to possession of the usual complement of approximately seven frontoventral cirri, five transverse cirri, two left marginal cirri, and three large caudal cirri. Separation of these species has been based upon differences in cell length and width, the number and arrangement of cilia in dorsal kinetics, the configuration of the adoral zone of oral polykinetids, the number and distribution of cirri within cirral groups, and the number and arrangement of macronuclei. Jankowski used some of these characteristics to divide the genus into two genera, Diophrys and Paradiophrys, with several subgenera [Jankowski, A. W. 1978. Systematic revision of the class Polyhymenophora (Spirotricha), morphology, systematics and evolution. Tezisy Dokl. Zool. Inst. Akad. Nauk SSSR, 197839‐40. (in Russian); Jankowski, A. W. 1979. Systematics and phylogeny of the order Hypotrichida Stein, 1859 (Protozoa, Ciliophora). Trudy. Zool. Inst. Akad. Nauk SSSR, 86:48–85. (in Russian with English summary)]. Data obtained from light microscopic examination of stained (nigrosin‐butanol, Chatton‐Lwoff, and Protargol) cells in interphase or division supports and modifies the use of particular structural features of these ciliates for the purpose of taxonomic classification. The structural variability within and among populations of different species within the genera Diophrys (D. appendiculata, D. oligothrix, and D. scutum) and Paradiophrys (P. irmgard and P. multinucleata) is described. D. hystrix is redescribed as the type of the new genus Diophryopsis n. g. Comparative information on the cortical morphogenesis of division of selected species within each genus is reviewed. Two taxonomic classifications of these hypotrichs are discussed: 1) a listing of diagnoses and synonymies and 2) a binary key for identification of all species at the light microscope level. An alternative evolutionary explanation of variations among isolates is presented.
ABSTRACT The first known fossil slime mold with part of the plasmodium preserved, from Eocene‐Oligocene amber of the northern Dominican Republic, is described here. We assign it to the myxomycetes on the basis of its cytoplasmic structure. The paleoecological and evolutionary importance of this fossil is briefly discussed.
ABSTRACT. The nomenclature of protists is more complicated than that of plants or animals because more than one code of nomenclature applies (i.e. the taxa may be ambiregnal), because of the frequent absence of type material, and because of changing perspectives of the phylogenetic relationships of various groups of protists. These factors often lead to uncertainty over the meaning of names of taxa. If nomenclatural instability is to be avoided, some changes in current practices are required. The nature of the problems and some possible changes are discussed.
ABSTRACT. Blepharisma cells were attracted by a pellet of live bacteria (Enterobacter) which was separated from the Blepharisma suspension by a cellulose membrane (fractionation: M.W. 14,000). The cells, however, were not attracted by killed bacteria. Crude and heat‐treated supernatants obtained from bacterial suspension also induced chemoaccumulation of cells. These results suggest that the cells of Blepharisma detect certain small molecules, produced by live bacteria, that can pass through the cellulose membrane and are stable to heat. From the live bacteria supernatant, several ninhydrin‐positive substances were isolated by means of two‐dimensional thin‐layer chromatography. Several of the spots contained substances that attracted the cells, indicating that certain ninhydrin‐positive components, such as peptides or free amino acids (probably products of bacterial metabolism), may serve as a signal for food.
ABSTRACT. Gregarina coronata n. sp. (Apicomplexa: Eugregarinida) is described from the adults of the Southern Corn Root Worm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae). Measurements given are means, in micrometers, taken from mature gamonts in association. Primite: protomerite hemi‐ellipsoidal with basal tumidus, length 47.6, width 44.0, with cytoplasmic granule, apical crown apparent; deutomerite elongate ellipsoidal, length 227.9, width 81.3; epimerite absorbed into anterior in gamont, globular in trophozoite. Satellite: protomerite hemi‐ellipsoidal, truncated at association interface to appear trapezoidal, length 39.2, width 49.6, with cytoplasmic granule; deutomerite elongate ellipsoidal, length 240.6, width 80.2; epimerite absorbed into anterior in gamont. Association caudofrontal and often precocious, occurring during growth of trophozoites. Gametocysts spherical, 115.3 in diameter, 132.9 with hyaline coat; producing multiple oocyst chains under moist storage in 24–36 h. Oocysts very uniform in shape and size, dorsad: doliform, length 6.4, equatorial width 3.4, polar width 2.9; pleuron: dorso‐ventrally flattened, corpus concave with bicondylic termina; corpus height 0.98, width 4.44; terminus height 1.96, width 0.98.
ABSTRACT. The first ultrastructural study of the actinosporean genus Triactinomyxon was carried out on Triactinomyxon legeri from the intestinal epithelium of Tubifex tubifex. The developmental cycle starts with bi‐ and uninucleate cells. We propose that these cells may be an early proliferative phase of the cycle and may unite to give rise to the four‐cell stage, initiating pansporoblast formation. Valvogenic cells transform in the long stylus and anchor‐like projections of the spore. In the capsulogenic cells, the primordium of the polar capsules originates as a simple, dense, club‐shaped structure not observed in other actinosporeans. In all other respects, actinosporean ultrastructure follows more or less similar patterns. Comparison of actinosporean and myxosporean species gives evidence of considerable structural similarity, exemplified in both classes by the occurrence of cell junctions in their multicellular spores, identical polar capsules and their morphogenesis, cell‐in‐cell condition, pansporoblast formation, and presence of dense bodies (sporoplasmosomes) primarily in the sporoplasm. This unity of patterns speaks in favor of the postulated actinosporean‐myxosporean transformation, which warrants further study.
ABSTRACT Between May 1979 and August 1991, 48.7% (57/117) of the harvest mice (Reithrodontomys spp.) examined from 10 localities in Mexico, California and New Mexico had coccidian oocysts in their feces. A total of 46.7% (49/105) of the Reithrodontomys megalotis examined were positive for coccidian oocysts; this included samples from five states in Mexico (47.1%, 8/17), three counties in California (66.7%, 4/6) and two counties in New Mexico (45.1%, 37/82); 66.7% (8/12) of the Reithrodontomys montanus from one county in New Mexico also were infected. Only two coccidian species, Eimeria arizonensis and Eimeria langebarteli, were found in these hosts. Oocysts of E. langebarteli were found only in R. megalotis: in all three infected mice from Madera County, California, in the only mouse from San Bernardino County, California, and in 63% (5/8) of the infected mice from four states in Mexico. Oocysts of E. arizonensis were found in R. megalotis in Mexico, California, and New Mexico and in R. montanus from New Mexico. Sporulated oocysts of E. langebarteli differed slightly from those in previously published reports by having wider oocysts and larger sporocysts. Sporulated oocysts of E. arizonensis were variable in size, with those recovered from R. montanus significantly larger in length and width and sporocyst width than those from R. megalotis. The structure of the oocyst residuum was polymorphic, both within and between host species, and within the same mouse; it could appear as one large globule, two globules, several to many smaller globules, or as a compact mass of many small granules. Oocysts with a variable residuum were larger than those with one globule in all oocyst/sporocyst dimensions. Only 9% (5/57) of the infected mice were discharging oocysts of both eimerians when examined.
The ability of Giardia strains of the duodenalis type to grow in Keister's modified TYI-S-33 medium varies with serum lot. Recently, strains of Giardia including MR4, WB, and Human-1-Portland, have been cultivated in Keister's modified TYI-S-33 medium containing the serum substitute Ultroser G and have been cultured serially as least 40 times. An optimal concentration of 8% Ultroser G promotes maximal growth in Keister's modified TYI-S-33 medium for all three strains. This concentration of Ultroser G will produce a two-log increase in the number of trophozoites in approximately three days post-inoculation. Generation times for the trophozoites ranging from 6 to 11 h have been achieved in Keister's modified TYI-S-33 containing 10% adult bovine serum and from 8 to 13 h in Keister's modified TYI-S-33 with 8% Ultroser G. Despite the excellent growth of Giardia strains in medium containing Ultroser G, the maximum trophozoite density is only about half of that achieved in Keister's modified TYI-S-33 medium supplemented with 10% adult bovine serum. Comparisons of trophozoites grown with serum or the serum substitute reveal no discernable differences in morphology and motility. Additionally, these strains have been successfully cryopreserved and revived in Keister's modified TYI-S-33 medium supplemented with Ultroser G. Because Ultroser G is a characterized mixture of six main groups of ingredients (growth factors, adhesion factors, mineral trace elements, hormones, binding proteins, and vitamins), the variability in cell proliferation that may occur when changing serum lots should be minimized when using this product.
We investigated the effect of a cysteine proteinase inhibitor (E-64) and an aspartyl proteinase inhibitor (Pepstatin A) on asexual erythrocytic stages of Plasmodium falciparum in culture. These two protease inhibitors showed different patterns of activity. E-64 acted preferentially against trophozoite and schizont stages. After 48 h incubation at high concentrations of E-64 (28, 140, 280 microM), growth was totally abolished and the parasites presented characteristic enlarged food vacuoles. Morphological alterations were also seen after shorter incubation periods (6 h at 28 microM) or 12 h at the inhibitory concentration 50% (12 microM), but an additional culture period (24 h) in inhibitor-free medium allowed normal parasite development, demonstrating a parasitostatic effect. E-64 acts on parasite multiplication; the normal merozoite maturation was altered and the normal reinvasion process partially impaired. Pepstatin A used at the inhibitory concentration 50% (4 microM) killed the parasites before trophozoite development and had a major effect on schizonts maturation. No altered parasite development occurred during an additional culture period without Pepstatin A, demonstrating a parasiticidal effect. E-64 and Pepstatin A used in combination inhibit the parasite growth with a strong synergistic effect.
Macrophage-conditioned medium (M phi CM) prepared from mouse peritoneal macrophages activated in vivo with bacillus Calmette-Guérin (BCG) or Propionibacterium acnes and triggered with lipopolysaccharide in vitro contained tumoricidal and amoebicidal activity. The murine fibroblast cell line L929 was used as the indicator of tumoricidal activity and Naegleria fowleri amoeba was used to detect amoebicidal activity in M phi CM. The protease inhibitor, soybean trypsin inhibitor, decreased tumoricidal activity but had little effect on amoebicidal activity in M phi CM. Anti-TNF alpha antiserum inhibited tumoricidal activity in M phi CM. The antiserum reduced amoebicidal activity in BCG-activated M phi CM but had no effect on amoebicidal activity in P. acnes-activated M phi CM. Recombinant TNF alpha, rIL-1 alpha, or rIL-1 beta independently did not affect cytolysis of amoebae. Also, rTNF alpha had no effect on the growth of amoebae. Preparative flat-bed electrofocusing of BCG-activated M phi CM yielded fractions that exhibited different amoebicidal and tumoricidal activity profiles. Three domains of activity were analyzed (acidic, neutral, and basic). Anti-TNF alpha antiserum eliminated tumoricidal activity, but not amoebicidal activity, in fractions from the acidic domain. A combination of anti-TNF alpha and anti-IL-1 alpha antisera failed to eliminate amoebicidal activity in fractions from the basic domain. These results indicate that different factors are responsible for macrophage amoebicidal and tumoricidal activity. The amoebicidal factors in M phi CM affected cytolysis of several species of amoebae.
ABSTRACT The composition of the rumen ciliate fauna in 76 Kafue lechwe inhabiting a limited area in Zambia was surveyed and five genera containing 24 species with 16 formae belonging to the family Ophryoscolecidae were identified. Four new species belonging to Diplodiniinae were recognized and described as Diplodinium lochinvarense n. sp., Diplodinium leche n. sp., Diplodinium zambiense n. sp., and Metadinium ossiculi n. sp. In addition, Ostracodinium gracile form fissilaminatum Dogiel, 1932 was found for the second time and described as Metadinium fissilaminatum n. comb. The species composition was fairly unusual. Seven of the species have been found only in African wild antelopes and these species were found more frequently than cosmopolitan species. There was no evidence of isotrichid species. The average density of ciliates per 1 ml of the rumen fluid was 25.7 times 104, and the number of ciliate species per head of host was 10.8.
One hundred twenty non-autogamous wild-type strains of Euplotes crassus, collected over seven years, mainly from the Mediterranean coasts, were investigated for their mating interactions. The strains were mixed pair-wise and data from mating reactions were evaluated and organized by means of a specially constructed computer program. The program identified 38 strains with distinctive mating patterns which could be clustered in nine clumps, all of which were connected either directly or indirectly. Thus, all these strains appeared to be components of the same gene pool, even though direct genetic exchange between strains was not possible in every combination. Subsequently, the 38 strains were subjected to cytometric analysis and scored for zymic variations resulting from electrophoretic patterns of five enzyme systems (acid phosphatases, amylases, malic dehydrogenase, malic enzyme, and tetrazolium oxidases) of proved diagnostic value in the identification of Euplotes species. No significant discontinuities correlated with mating patterns was apparent from these analyses. It was concluded that the E. crassus strains analyzed are not properly divided in sibling species and it was consistently suggested to avoid a genetic partitioning of ciliate species endowed with high multiple mating type systems, in which the sets of wild strains brought under investigation with difficulty represent the natural dimensions of the species.
