Growth of Crithidia at High Temperature: Crithidia hutneri sp. n. and Crithidia luciliae thermophila s. sp. n.*

SYNOPSIS. Crithidia hutneri sp. n. and Crithidia luciliae thermophila s. sp. n. are described. Both flagellates can be grown in a defined medium over a temperature range of 15–37°C. The requirements for amino acids, vitamins, purine and hemin, and pH range were similar to those established for Crithidia fasciculata, although threonine was required as a growth factor for C. luciliae thermophila at high temperatures. Adenosine could be used by the 2 Crithidia as a purine source at 28 but not at 37 C.     

Entamoeba dispar: Cultivation with Sterilized Crithidia fasciculata1

Four isolates of Entamoeba dispar identified by their hexokinase and phosphoglucomutase isoenzyme profile and by their failure to react with Entamoeba histolytica-specific monoclonal antibody (4G6) could be grown in either Diamond's BI-S-33 medium, newly developed BCSI-S (Biosate cysteine starch iron-serum) medium, or casein-free YI-S medium in the presence of Crithidia fasciculata (ReF-1:PRR) sterilized by heating 56° C for 30 min and subsequent incubation with 1% hydrogen peroxide for 24 hours at 4° C. After the cultures were maintained for over 50 passages, the amebae were identified as E. dispar by isoenzyme analysis, polymerase chain reaction with E. histolytica- and E. dispar-specific primers, i.e. p11 plus p12 and p13 plus p14, respectively, and by negative reactivity with monoclonal antibody 4G6. The flagellates added to the culture were judged to be metabolically inactive based on the results of nuclear magnetic resonance spectroscopy, electron microscopy, and polarographic analysis. All of these findings suggest that E. dispar can grow in vitro with metabolically inactive C. fasciculata as a culture associate.     

Nutrition of the Trypanosomatids Crithidia acanthocephali and C. harmosa. Ribose and Adenosine: Substrates for C. acanthocephali1

Crithidia acanthocephali and C. harmosa share the same minimal nutritional requirements as the standard C. fasciculata. Crithidia acanthocephali is exceptional, however, in that it utilizes D-ribose, free or as nucleoside (e.g. adenosine), as carbon source.     

Labeling of Crithidia fasciculata DNA with [3H]Thymidine

Attempts at continuous labeling of Crithidia fasciculata DNA with [³H]thymidine led to a pulse-chase situation due to a cell-mediated conversion of thymidine to thymine in the medium. The uptake of thymine was slow compared to that of thymidine. Neither the addition of deoxyadenosine nor the sequential addition of several aliquots of [³H]thymidine had an effect on the pattern of labeling.     

The Sites of Action of Allopurinol in Crithidia fasciculata*

Reversal of the growth inhibition of Crithidia fasciculata by allopurinol requires both a purine and a pyrimidine. Hypoxanthine is the most effective purine in the reversal. Cell-free extracts were prepared which were capable of the decarboxylation of orotidine 5′-phosphate. Other enzyme preparations carried out the phosphoribosylation of allopurinol. By the use of [4-¹⁴C] orotidine 5′-phosphate (enzymatically prepared), it was shown that allopurinol ribotide (enzymatically prepared), but not the free base, inhibits orotidine 5′-phosphate decarboxylase.     

Effect of Ethidium Bromide on the Oxidative Metabolism and Enzyme Profiles of Crithidia fasciculata*

SYNOPSIS. Changes in the metabolism of Crithidia fasciculata ATCC 11745 when grown in the presence of ethidium bromide were studied. Ethidium bromide-grown cells had decreased respiratory activity as measured by oxygen consumption. More than 50% of the organisms cultivated in a defined medium containing 1.0 mg/liter of ethidium bromide became dyskine-toplastic and had decreased activities of particulate succinate and NADH-linked dehydrogenases as well as of soluble isocitrate dehydrogenase. These cells also had increased activities of particulate α-glycerophosphate dehydrogenase, soluble α-glycerophosphate dehydrogenase, malic enzyme, hexokinase, and malate dehydrogenase. Ethidium bromide-grown cells had a lower level of ATP and contained less DNA than cells grown in its absence.     

In vitro Transcription of Kinetoplast and Nuclear DNA in Kinetoplastida*†

SYNOPSIS. DNA-dependent RNA polymerases have been solubilized from homogenates of Crithidia fasciculata using gentle extraction procedures. RNA polymerase I and II are separated on DEAE cellulose at 0.07M (NH₄)₂SO₄ and 0.13M (NH₄)₂SO₄ respectively. RNA polymerase II is inhibited 80% by α-amanitin (25 μg/ml). Both RNA polymerases require DNA as a template, ribonucleoside triphosphates and Mn²⁺. The synthesis of RNA as a product is inhibited by DNase. RNase, pronase and actinomycin D. Purified kinetoplast and nuclear DNA can serve as templates for the RNA polymerases. Denatured DNA templates are preferred. The synthesis of RNA continues for at least an hour and is inhibited by trypanocidal drugs including suramin. antrycide, acriflavine, ethidium bromide and berenil. Complementary RNA synthesized in vitro from C. fasciculata kinetoplast DNA hybridizes with C. fasciculata kinetoplast DNA but not with C. fasciculata nuclear DNA or Blastocrithidia culicis kinetoplast DNA, Escherichia coli, T4 or calf thymus DNAs. The complementary RNA synthesized in vitro from C.fasciculata kinetoplast DNA sediments at 4–5S.     

Temperature-Enhanced Osmotic Growth Requirement of Crithidia*†

SYNOPSIS. An osmotic growth requirement for the trypanosomatid Crithidia fasciculata became conspicuous at 32 C. Osmo-supportive compounds were surveyed at 28 vs. 33 C, in “low-osmotic” defined medium. Effective osmotic support was provided by many compounds, e.g., glycerophosphate, sorbitol, mannitol, glycerol, Na isethionate, glycine, arginine, KCl, NaCl, NH₄Cl, and K₂SO₄. The nonspecificity of the requirement was thus evident, but inactivity of the presumably poorly adsorbable pentaerythritol indicated that osmotic pressure was a likely but insufficient condition for satisfying the temperature-enhanced growth requirement most clearly expressed as a need for osmotic support. Fortification of the medium with a combination of glycerophosphate, glycine, glycerol, and Tween 80 permitted good growth at 35 C. Possible relations between the temperature-enhanced osmotic requirement cell membrane damage, and morphological phases of Trypanosoma and Leishmania are discussed.     

Sequence elements essential for the rapid turnover of <Emphasis Type="Italic">Crithidia fasciculata</Emphasis> ornithine decarboxylase

Summary. Ornithine decarboxylase (ODC) has a very fast turnover in mammalian cells, but is a stable enzyme in T. brucei and other trypanosmatid parasites like Leishmania donovani. However, Crithidia fasciculata, which is a phylogenetically closely related trypanosomatid to L. donovani, has an ODC with a rapid turnover. Interestingly, C. fasciculata ODC, but not L. donovani ODC, is rapidly degraded also in mammalian systems. In order to obtain information on what sequences are important for the rapid degradation of C. fasciculata ODC, we produced a variety of C. fasciculata/L. donovani ODC hybrid proteins and characterized their turnover using two different mammalian expression systems. The results obtained indicate that C. fasciculata ODC contains several sequence elements essential for the rapid turnover of the protein and that these regions are mainly located in the central part of the enzyme. Present address: Department of Microbiology, Moyne Institute of Preventive Medicine, University of Dublin – Trinity College, Dublin, Ireland Authors’ address: Lo Persson, Department of Experimental Medical Science, Lund University, BMC D-12, S-221 84 Lund, Sweden     

The Ribonucleic Acids of Crithidia fasciculata*

SYNOPSIS. Crithidia fasciculata ribosomes were found to be 80S and to dissociate into 58 and 41S subunits; on 5 to 50% sucrose gradients, rRNA was separated into 25, 18, and 5S components. The molecular sizes of the heavier rRNA species, estimated by polyacrylamide gel electrophoresis were 1.24 and 0.84 M (×10⁶ daltons). The 25S RNA has a tendency to interact with the 18S RNA to give a complex that is difficult to separate by sucrose gradient centrifugation. The 25S RNA is also unstable and dissociates into 0.73 and 0.57 M components. The 18S RNA has molecular size (0.84 M) higher than the 0.7 M reported for most eukaryotes, but similar to that of Euglena and Amoeba. Ribosomal RNA hybridized 0.29% of the nuclear DNA. Mitochondrial RNA, extracted by a rapid procedure was resolved into 16 and 5S components in sucrose gradients.     

Effect of Colchicine on Cell Membrane and on Biopterin Transport in Crithidia fasciculata*

SYNOPSIS. Incorporation of ¹⁴C-labeled biopterin into Crithidia fasciculata was inhibited by 1 mM colchicine or lumicolchicine. These substances do not penetrate the cell membrane, hence they cannot interact with the subpellicular microtubules. In view of this, interference of colchicine with biopterin transport must occur on the outer surface of the cell membrane. Binding of colchicine to Crithidia was not temperature-dependent and did not exhibit saturation kinetics. These facts exclude a binding as in the case of tubulin, or similar proteins which may be present in the membrane. The results suggest an inhibition reflecting steric hindrance of the biopterin carrier system.     

Structure of the D-Mannan and D-Arabino-D-Galactan in Crithidia fasciculata: Changes in Proportion with Age of Culture*

SYNOPSIS. Cells of the insect flagellate Crithidia fasciculata contained mannan and arabinogalactan components, whose porportion varied with culture age, the former predominating during early stages, and the latter during the later stages of exponential growth and the deceleration phase. The mannan was a β-D-(1→2)-linked D-mannopyranan. The arabinogalactan had a complex structure containing, in part, a β-D-(1→-3)-linked galactopyranose main-chain substituted in the 2 positions by single-unit D-arabinopyranose side-chains and with some unsubstituted units.     

Comparison of Six Isoenzymes from 10 Species of Crithidia1

ABSTRACT. The electrophoretic mobility of six isoenzymes from 10 species of Crithidia from insects were compared. Five zymogram patterns emerged. Pattern I was presented by C. acanthocephali and Crithidia sp. from Euryophthalmus davisi; pattern II by C. hutneri and C. luciliae thermophila; pattern III by C. arili; pattern IV by C. luciliae luciliae, Crithidia sp. from Aedes solicitans, C. harmosa, and C. fasciculata; pattern V by C. oncopelti.     

Characteristics of Cytidine Aminohydrolase Activity in Trypanosoma cruzi and Crithidia fasciculata1

Cytidine deaminase (cytidine aminohydrolase, 3.5.4.5) is present in Crithidia fasciculata (a mosquito parasite) and in Trypanosoma cruzi (a human pathogen). The enzyme from C. fasciculata deaminated both cytidine and deoxycytidine, the affinity for the former being much lower than the latter. Affinities for both substrates are equal for the T. cruzi enzyme. The production of the enzyme in C. fasciculata was significantly stimulated by the addition of a number of pyrimidine nucleosides (cytidine, uridine, 5-bromouridine, thymidine, orotidine) to the culture media. Only cytidine stimulated enzyme production in T. cruzi. The enzyme from both organisms was unstable in air, even in the frozen state. Stabilization was achieved under anaerobic conditions.     

Effect of timing and exposure of sunflower pollen on a common gut pathogen of bumble bees

1. Several bee species are declining due to multiple factors, including pathogens. Ingestion of sunflower (Helianthus annuus) pollen can dramatically reduce the bumble bee gut pathogen Crithidia bombi, but little is known about how timing and exposure to sunflower pollen consumption affects pathogen load. 2. Two experiments were carried out to investigate how exposure to sunflower pollen relative to pathogen exposure affects Crithidia bombi in Bombus impatiens. Foraging trials with pollen‐producing and male‐sterile (pollen absent) sunflower lines were performed to investigate whether sunflower pollen affected pathogen transmission in a single foraging bout, and 7‐day laboratory trials were done to investigate whether timing and duration of exposure to sunflower pollen after infection affected C. bombi. 3. In foraging trials, pollen presence on inflorescences inoculated with C. bombi did not affect transmission (pathogen cell counts of foraging workers) 1 week later, suggesting that a brief experience with sunflower pollen concurrent with pathogen exposure is insufficient to reduce infection. In laboratory trials, consuming sunflower pollen for the first 3.5 days or all 7 days after infection reduced cell counts compared with a negative control pollen, but consuming sunflower pollen starting 3.5 days after infection did not. Consuming sunflower pollen for 7 days was significantly and substantially more effective than any other treatment. Thus, both duration and timing of exposure to sunflower pollen may affect pathogen load. 4. These results are important for understanding ecological disease dynamics in natural settings with free‐flying bumble bees, and may inform decisions about using medicinal diets to manage bumble bee health commercially.     

Isolation and Characterization of Kinetoplast DNA Networks and Minicircles from Crithidia fasciculata*

SYNOPSIS. Covalently closed kinetoplast DNA networks have been isolated from stationary phase Crithidia fasciculata cells by a technic involving selective pelleting of the networks at a low centrifugal field. Approximately 62% of the kinetoplast DNA of the cell was recovered free of nuclear DNA by simple differential centrifugation. Purified kinetoplast DNA networks were visualized both in the electron microscope and in the light microscope. Closed networks sedimented as a homogeneous band both in neutral and alkaline sucrose, with an s_20w in neutral sucrose of approximately 5 × 10³. Closed monomeric minicircles were isolated from purified networks by mild sonication and band sedimentation in alkaline sucrose. Several physical properties of closed monomeric minicircles were measured. These included molecular weight, buoyant density in CsCl, superhelix density and sedimentation coefficient.     

Pulse-Labeling of Kinetoplast DNA: Localization of 2 Sites of Synthesis Within the Networks and Kinetics of Labeling of Closed Minicircles*

SYNOPSIS. Short pulse-labeling of log phase Crithidia fasciculata cells with [³H]thymidine allowed the autoradiographic visualization of 2 sites of replication of kinetoplast DNA situated at the periphery of the networks and separated by 180°. Longer pulse-labeling led to the previously reported total peripheral labeling pattern. Pulse-labeled networks possess an intermediate density in ethidium bromide-CsCl equilibrium gradients between the densities characteristic of closed networks and open or linear DNA. Removal of ethidium bromide by several methods and treatment of intermediate band networks with RNase and pronase had no effect on the equilibrium rebanding pattern. Closed minicircles of Leishmania tarentolae are not labeled by a short pulse of intact cells with [³H]thymidine. A chase of ～ 3–4 hr is required for the appearance of radioactivity in closed minicircles, a time delay which implies the existence of intermediate events between replication and eventual covalent closure of the minicircles.     

Five Trypanosomatid Species of Insects Distinguished by Isoenzymes*

SYNOPSIS Blastocrithidia culicis, Crithidia deanei, Crithidia fasciculata, Herpetomonas samuelpessoai, Leptomonas seymouri and Leishmania tarentolae grown in cultures were compared by electrophoretic mobility for isoenzymes in 6 enzymes. All species were found distinct in these characteristics. Endosymbiotic C. deanei, which was identical to the aposymbiotic C. deanei in 5 enzymes, had an extra band in aspartate aminotransferase. No differences in isoenzymes were found between members of one species maintained in 2 different culture media.     

Metabolism of α-Glyceryl Ethers by Crithidia fasciculata. I. Study of the In Vivo Degradation of Exogenous Chimyl and Batyl Alcohols1

ABSTRACT. [¹⁴C]chimyl and [³H]batyl alcohols were added to Crithidia fasciculata cultures during the mid-log phase of cell growth, and the lipid extracts of the cells were analyzed for degradation products. C. fasciculata cells were able to take up exogenous glyceryl ethers, and in amounts as high as the endogenous lipid content. The glyceryl ether taken up by the cells was incorporated into lipids either prior to the ether bond cleavage or after degradation to fatty acid. The extent of degradation and the degree of incorporation of degradation products into cellular lipid were higher for chimyl than for batyl alcohol. Batyl alcohol was not metabolized efficiently, leading to the formation of large intracellular pools of free substrate. One product of glyceryl ether degradation was identified as alkyl-dihydroxy acetone, and was detected inside and outside of the cells. The data strongly suggest that this product is the first stable intermediate in the degradation process and indicate that the extracellular formation of alkyl-dihydroxy acetone is due to the action of exocnzyme; ecteted by the cells. The constant detection of alk I cnyl glycerol among the degradation products indicates the existence of a second mechantsm in C. fasciculata for converting the alkyl-to alkenyl-glycerol.     

Isolation and Properties of Flagella of Trypanosomatids*

SYNOPSIS. A procedure is described for the isolation of flagella of Crithidia fasciculata. Herpetomonas samuelpessoai and Leishmania tarentolae in a highly purified state and giving reasonably good yield. The 3 types of flagella give a similar electrophoretic pattern of proteins. It is shown that H. samuelpessoai and, to a lesser extent, C. fasciculata flagella confer protection against Trypanosoma cruzi infection.