Experimental Infection of Triatoma infestans and Rhodnius prolixus with Trypanosomatidae of the Genera Crithidia and Blastocrithidia*

SYNOPSIS. Dissections of Triatoma infestans and Rhodnius prolixus at various intervals after experimental infection with insect Trypanosomatidae of the genera Blastocrithidia and Crithidia (B. euschisti, C. luciliae, C. acanthocephali and C. mellificae) disclosed the protozoa to be surviving and in most instances undergoing reproduction in the experimental bugs for as long as 2 months following infection. Epimastigotes and amastigotes were seen in bugs infected with B. euschisti and choanomastigotes and amastigotes were observed in insects experimentally infected with the Crithidia. Since C. acanthocephali and C. luciliae were from clones, this is the 1st finding of these 2 structural types in this genus using cultures originating from a single choanomastigote. In view of the loose host specificity of the insect Trypanosomatidae as further emphasized by this work, it would appear that the necessity is increased for field workers in trypanosomiasis and leishmaniasis areas of the world to become well acquainted with the infective potentialities of the insect trypanosomatids of their areas for the vectors of Trypanosoma and Leishmania along with the possible association in nature of these vectors with insects which might harbor insect-limited Trypanosomatidae.     

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.     

Trypanosomatid protozoa: survey of acetylornithinase and ornithine acetyltransferase

Trypanosomatid protozoa (Crithidia deanei, C. deanei aposymbiotic, C. oncopelti, C. fasciculata, C. acanthocephali, Leptomonas seymouri, L. collosoma, L. samueli, Herpetomonas samuelpessoai, H. sp., H. megaseliae, H. muscarum muscarum, Leishmania donovani, L. braziliensis, Trypanosoma cruzi, T. conorhini and T. mega) were examined for the presence of acetylornithinase (EC 3.5.1.16) and ornithine acetyltransferase (EC 2.3.1.35). As a rule, species of the genus Crithidia presented one of the two enzymes for the conversion of acetylornithine into ornithine. Crithidia fasciculata and C. acanthocephali presented acetylornithinase, while C. deanei and C. oncopelti, species harboring symbionts, presented ornithine acetyltransferase. The enzyme was absent in the aposymbiotic strain of C. deanei, which suggests that the enzyme belongs to the symbiont. Among the other trypanosomatids examined only Herpetomonas samuelpessoai presented acetylomithinase. The participation of acetylornithinase and ornithine acetyltransferase in the metabolism of trypanosomatids is discussed in the light of their nutritional requirements and possession of enzymes of the arginineornithine metabolism.     

Effect of Temperature and Osmolarity on Growth of Crithidia fasciculata,C. hutneri,C. luciliae thermophila,and Herpetomonas samuelpessoai1

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.     

Physicochemical properties of kinetoplast DNA from Crithidia acanthocephali. Crithidia luciliae, and Trypanosoma lewisi

The protozoa Crithidia and Trypanosoma contain within a mitochondrion a mass of DNA known as kinetoplast DNA (kDNA) which consists mainly of an association of thousands of small circular molecules of similar size held together by topological interlocking. Using kDNA from Crithidia acanthocephali, Crithidia luciliae, and Trypanosoma lewisi, physicochemical studies have been carried out with intact associations and with fractions of covalently closed single circular molecules, and of open single circular and unit length linear molecules obtained from kDNA associations by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium centrifugation. Buoyant density analyses failed to provide evidence for base composition heterogeneity among kDNA molecules within a species. The complementary nucleotide strands of kDNA molecules of all three species had distinct buoyant densities in both alkaline and neutral cesium chloride. For C. acanthocephali kDNA, these buoyant density differences were shown to be a reflection of differences in base composition between the complementary nucleotide strands. The molar ratios of adenine: thymine:guanine:cytosine, obtained from deoxyribonucleotide analyses were 16.8:41.0:28.1:14.1 for the heavy strand and 41.6:16.6:12.8:29.0 for the light strand. Covalently closed single circular molecules of C. acanthocephali (as well as intact kDNA associations of C. acanthocephali and T. lewisi) formed a single band in alkaline cesium chloride gradients, indicating their component nucleotide strands to be alkaline insensitive. Data from buoyant density, base composition, and thermal melting analyses suggested that minor bases are either rare or absent in Crithidia kDNA. The kinetics of renaturation of 32P labeled C. acanthocephali kDNA measured using hydroxyapatite chromatography were consistent with at least 70% of the circular molecules of this DNA having the same nucleotide sequence. Evidence for sequence homologies among the kDNAs of all three species was obtained from buoyant density analyses of DNA in annealed mixtures containing one component kDNA strand from each of two species.     

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.     

Growth of an Insect Trypanosomatid at 37 C in a Defined Medium*

SYNOPSIS. A defined medium for an insect trypanosomatid, “Leptomonas pessoai” (probably a member of the genus Herpetomonas) isolated from the reduviid Zelus leucogrammus, allows growth up to 37 C. No marked differences were evident for growth at 37 C. The requirements for amino acids, vitamins, purine, and hemin, and pH range were like those established for Crithidia fasciculata. Again like C. fasciculata, it remains alive at 4 C for at least 3 months in a çlycerolated defined medium.     

Immunologic and Electrophoretic Characteristics of Two Species of Crithidia*†

SYNOPSIS. Crithidia harmosa and Crithidia fasciculata were compared immunologically by the indirect fluorescent antibody (FA) method and by agglutination. The FA technic yielded more specific results with cellular-debris than with whole-cell antigens. The immune sera collected from chickens 9 days after the last inoculations with whole-cell antigens had higher homologous titers than those collected after 4 days. Major antigenic differences between the 2 species were revealed by both methods. The electrophoretic patterns of C. harmosa and C. fasciculata obtained by polyacrylamide gel slab electrophoresis differed in the numbers and relative mobilities of their component bands.     

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.     

Honey bee and bumblebee trypanosomatids: specificity and potential for transmission

Abstract 1. Experimental studies of multihost parasite dynamics are scarce. Understanding the transmission dynamics of parasites in these systems is a key task in developing better models of parasite evolution and to make more accurate predictions of disease dynamics. 2. Bumblebee species (Bombus spp.) host the trypanosomatid parasite, Crithidia bombi. Its transmission in the field occurs through the shared use of flowers. Flowers are a perfect scenario for inter‐taxa transmission of diseases because they are used by a wide range of animals. 3. Honey bees host a poorly studied trypanosomatid, Crithidia mellificae. In this study, five questions have been experimentally addressed: (a) Can C. bombi infect honey bees? (b) Can C. mellificae infect bumblebees? (c) Can the honey bee act as a vector for C. bombi? (d) Are C. bombi cells present in honey‐bee faeces? (e) Does C. bombi have an effect on the mortality of honey bees after ingestion? 4. While both parasites were found to be specific to their hosts at the genus level, results suggest that honey bees may play a role in the epidemiology of C. bombi transmission.     

Molecular Divergence Defines Two Distinct Lineages of Crithidia bombi (Trypanosomatidae), Parasites of Bumblebees

ABSTRACT. This study provides, for the first time, sequence data for the protozoan flagellates Crithidia bombi and Crithidia mellificae (Kinetoplastea: Trypanosomatidae). We amplified the partial sequences of the small subunit ribosomal RNA (SSU rRNA), glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH), cytochrome b (Cyt b), and the complete internal transcribed spacer region 1 (ITS1) of the ribosomal RNA gene region for 66 clones of C. bombi from Switzerland and Alaska. Furthermore, we sequenced the same stretch of SSU rRNA and gGAPDH for one isolate of C. mellificae from Switzerland. All four molecular markers classified the C. bombi samples into two distinct lineages A and B. Both lineages were found in the two sampling locations. Variation within lineages was small or non‐existing. Sequence differences between lineages were 1.64% for SSU rRNA, 4.36% for gGAPDH, and 12.02% for Cyt b. The ITS1‐sequences of lineages A and B have diverged so much that no alignment was possible. With regard to ITS1, we additionally found fragment length polymorphism (variation in microsatellite repeat numbers) as well as nucleotide diversity within each lineage. Furthermore, the sequences of SSU rRNA and gGAPDH of C. mellificae were different from both lineages of C. bombi. The separation of lineages A and B, based on sequence differences and phylogenetic reconstruction, is so pronounced as to characterize two species of “C. bombi.” We propose to retain C. bombi for the more common lineage A and suggest the name Crithidia expoeki n. sp. for lineage B.     

Identification of C3 Acceptors Responsible for Complement Activation in Crithidia fasciculata1

Crithidia fasciculata, an insect trypanosomatid is readily lysed by normal human serum at concentrations as low as 3%. Lysis occurs in the presence of Mg⁺²-EGTA and is antibody independent, indicating that the alternative pathway of complement activation is involved. Analysis of [¹³¹I]C3 deposition on C. fasciculata cells using C8-deficient serum, revealed that about 4 times 10⁵ C3 molecules bound to each cell. Most of the C3 was bound to cells as C3b, part of it forming high molecular weight complexes, which could be dissociated by methylamine treatment at alkaline pH. To characterize the C3 acceptors on C. fasciculata, surface-iodinated cells were incubated with C8D or heat-inactivated serum, extracted and immunoprecipitated with anti-C3 or anti-arabinogalactan antisera. Analysis of the immunoprecipitated material on SDS gels showed high-molecular weight components, which disappeared after methylamine treatment, giving rise to a component of 200 kDa molecular size. This 200-kDa component corresponded to a purified arabinogalactan complex, which was immunoprecipitated from labeled cell extracts, without incubation with C8D, using anti-arabinogalactan antibodies. These results suggest that the arabinogalactan glycoconjugate is a C3 acceptor in C. fasciculata during complement activation. Purified arabinogalactan complexes were able to inactivate C3 in vitro. Solubilization in KOH to cleave the peptide moiety rendered it unable to inactivate C3. Apparently, the aggregated state of the purified arabinogalactan component at the cell surface is important for C3 deposition and activation.     

Crithidia spp.: Structural comparison of polysaccharides for taxonomic significance

The chemical structures of polysaccharide components of cells of several Crithidia species have been partially elucidated. The structures have been used as criteria to evaluate evolutionary lines previously proposed for species of Crithidia and Herpetomonas and for Trypanosoma cruzi. In accord with the suggestion that Crithidia and Herpetomonas are closely interrelated, all species investigated synthesize a linear (1→2)-linked β-d-mannopyranan and a heteropolysaccharide. These differ from T. cruzi polysaccharide, which contains α-d-mannopyranosyl structures and likely indicates a separate evolutionary route for this flagellate. Crithidia fasciculata, Crithidia harmosae, and Crithidia luciliae form a closely knit group since they form arabinogalactans with related structures. The similarity is particularly close between arabinogalactans of C. fasciculata and C. harmosae whose ¹³C nuclear magnetic resonance spectra show a high degree of resemblance. An unnamed Crithidia sp. contains polysaccharides with fucose and xylose units, intermediate between those of Crithidia deanei, which gave glucose and fucose on hydrolysis, and Herpetomnas samuelpessoai, which gave glucuronic acid and xylose.     

Surface Anionic Groups in Symbiote-Bearing and Symbiote-Free Strains of Crithidia deanei1

The surface anionic groups of symbiote-bearing and symbiote-free strains of Crithidia deanei were compared by determining cellular electrophoretic mobility, by ultrastructural cytochemistry, and by identification of sialic acids by thin-layer and gasliquid chromatography. Symbiote-free Crithidia deanei has a highly negative surface charge (-0.9984 μm?s^-1? V^-1? cm), which is slightly reduced (-0.8527 μm?s^-1? V^-1? cm) by the presence of the endosymbiote. Treatment of both strains of C. deanei with neuraminidase decreased significantly the electrophoretic mobility of cells toward the cathodic pole, indicating the existence of exposed sialic acid residues responsible for the negative charge on the protozoan cell surface. Thin-layer and gas-liquid chromatography showed that N-glycolyl- and N-acetylneuraminic acids were present in both strains of C. deanei.     

Evidence for a partial RNA transcript of the small circular component of kinetoplast DNA of Crithidia acanthocephali.

The major component of kinetoplast DNA (kDNA) in the protozoan Crithidia acanthocephali is an association of approximately 27,000, 0.8 micrometers (1.58 x 10(6) dalton) circular molecules apparently held together in a particular structural configuration by topological interlocking. We have carried out hybridization experiments between kDNA samples containing one or the other of the two complementary (H and L) strands of purified 0.8 micrometers molecules derived from mechanically disrupted associations and RNA samples prepared either from whole C. acanthocephali cells or from a mitochondrion-enriched fraction. The results of experiments involving cesium sulfate buoyant density centrifugation indicate that whole cell RNA contains a component(s) complementary to all kDNA H strands, but none complementary to kDNA L strands. Similar results were obtained using mitochondrion-associated RNA. Digestion of RNA/DNA hybrids and suitable controls with the single-strand-specific nuclease S1 indicated that 10% of the kDNA H strand is involved in hybrid formation. Visualization of RNA/DNA hybrids stained with bacteriophage T4 gene 32 protein revealed that hybridation involves a single region of each kDNA H strand, equal to approximately 10% of the molecule length. These data suggest that at least 10% of the small circular component of kDNA of Crithidia acanthocephali is transcribed.     

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.     

Purine Metabolism in Trypanosomatids*

SYNOPSIS. Purine nucleotide biosynthesis was studied in culture forms of Trypanosoma cruzi strain Y, Crithidia deanei (a reduviid trypanosomatid with an endosymbiote) and an aposymbiotic strain of C. deanei (obtained by curing C. deanei with chloramphenicol). Trypanosoma cruzi was found to synthesize purine nucleotides only from the preformed bases adenine and guanine (“salvage” pathway), adenine being incorporated into both adenine and guanine nucleotides. Similar results were obtained with guanine, indicating that this flagellate has a system for the interconversion of purine nucleotides. Crithidia deanei was able to synthesize purine and pyrimidine nucleotides from glycine (“de novo” pathway) and purine nucleotides from adenine and guanine (“salvage” pathway). Adenine was incorporated into both adenine and guanine nucleotides, while guanine was incorporated into guanine nucleotides only, indicating the presence of a metabolic block at the level of GMP reducaase. The aposymbiotic C. deanei strain was unable to utilize glycine for the synthesis of purine nucleotides, although glycine was utilized for synthesizing pyrimidine nucleotides. These results suggest that the endosymbiote is implicated in the de novo purine nucleotide pathway of the C. deanei-endosymbiote complex. The incorporation of adenine and guanine by aposymbiotic C. deanei strain followed a pattern similar to that observed for C. deanei.     

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.     

Kinetoplast DNA in the insect trypanosomes Crithidia luciliae and Crithidia fasciculata: I. Sequence evolution and transcription of the maxicircle

Kinetoplast DNA from the insect trypanosome Crithidia luciliae contains a maxicircle of 22 × 10⁶ D. We have cleaved this DNA with endonucleases PstI, XbaI, XhoI, SstI, HaeIII, SalII, HindIII, EcoRI, and HapII and constructed a physical map of the 31 cleavage sites. The maxicircle segments hybridizing with total cellular RNA are clustered on one-half of the maxicircle; the genes for the 9 and 12 S mitochondrial (r)RNAs are located on a 1.7-kb segment. Restriction enzyme analysis indicates a sequence homology of more than 96% between the maxicircles of C. luciliae and C. fasciculata, which is not lower than that found between maxicircles of individual Trypanosoma brucei stocks. We conclude therefore that C. luciliae and C. fasciculata are one species and propose to name this species C. fasciculata and to rename C. luciliae as C. fasciculata, var. luciliae. Furthermore we show that the overall maxicircle genome organization of Crithidia resembles that of Trypanosoma.