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.     

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.     

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.     

Electrophoretic Analysis of Endonuclease-Generated Fragments of k-DNA,of Esterase Isoenzymes,and of Surface Proteins as Aids for Species Identification of Insect Trypanosomatids1

In order to verify the applicability of biochemical methods for species identification of Trypanosomatidae, 13 species of monoxenic trypanosomatids plus the heteroxenous Trypanosoma cruzi were comparatively analyzed by three different biochemical methods. Insect trypanosomatids examined were: Crithidia acanthocephali, C. fasciculata (three varieties), C. luciliae luciliae, C. luciliae thermophila, C. deanei, C. oncopelti, Herpetomonas muscarum muscarum, H. megaseliae, H. samuelpessoai, H. mariadeanei, Leptomonas seymouri, L. collosoma, L. samueli, and Blastocrithidia culicis. Also included in the survey were aposymbiotic strains of C. deanei and C. oncopelti. Methods used were: electrophoretic profiling of endonuclease-generated fragments of k-DNA, esterase isoenzymes profiling, and polyacrylamide-gel electrophoresis (SDS-PAGE) of radioiodinated cell surface proteins. Interspecific but not intraspecific differences were detected by all three methods among the 13 monoxenic species examined. Thus, it is concluded that these methods can be successfully used, in addition to classical criteria, for species identification of insect trypanosomatids.     

The Cell Cycle in Crithidia fasciculata. Temporal Relationships between Synthesis of Deoxyribonucleic Acid in the Nucleus and in the Kinetoplast1, 2

SYNOPSIS. Autoradiographic technics with tritium-labeled thymidine have been used to determine G1, S, G2 and D for the kinetoplast and the nucleus of Crithidia fasciculata at 15, 25 and 32 C. The kinetoplast completes division before the nucleus at all 3 temperatures. The S phases of both organelles occur in approximate synchrony and are approximately equal in length but the nucleus begins and completes S before the kinetoplast at the 2 lower temperatures. This relationship is reversed at 32 C. Most of the effect of temperature on generation time is due to its effect on the length of S. The results are compared with similar studies on C. luciliae, Trypanosoma mega, other protozoa and tissue cells in culture. The role of the approximate synchrony of nuclear and kinetoplastic cycles in maintenance of the kinetoplastic condition is discussed and the hypothesis is proposed that this synchrony results from the sharing by nucleus and kinetoplast of the same mechanism for the production of the deoxyribonucleotides used in replication of their respective DNAs.     

Use of glycoconjugates for trypanosomatid taxonomy

Glycoconjugates from five trypanosomatid genera—Crithidia, Herpetomonas, Endotrypanum, Leishmania, and Trypanosoma—were extracted with Triton X-114 and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by periodic acid-Schiff staining. Most of the glycoconjugates were detected in the hydrophobic phase, indicating the presence of anchored glycoconjugates. All the trypanosomatids expressed a glycoconjugate with a low molecular weigh (below 20 kDa) in this phase. In each species, however, a characteristic and specific pattern of glycoconjugates was also observed in both phases. In the hydrophobic phase: 14–29 kDa lycoconjugates in C. guilhermei; 24–70 kDa in C. fasciculata, C. luciliae, E. schaudinni, and T. cruzi Y and G strains; 45–66 kDa in C. oncopelti and H. samuelpessoai; above 36 kDa in T. dionisii; 20–24 kDa, 36–45 kDa, and 70 kDa in L. tarentolae and T. mega. In the hydrophilic phase, typical glycoproteins were observed in some trypanosomatids: 60 kDa in T. mega and T. cruzi Y strain; 70 kDa in H. samuelpessoai; 66 kDa in C. oncopelti; 20–70 kDa in C. luciliae. These findings suggest that Triton X-114-extracted glycoconjugates could be useful markers for trypanosomatid taxonomy.     

Surface charge and hydrophobicity of wild and mutantCrithidia fasciculata

Surface charge of wild-typeCrithidia fasciculata and three drug-resistant mutants (T^R3, TFR^R1, and FU^R11) was studied by direct zeta-potential determination and ultrastructural cytochemistry. Surface tension was also investigated by measurements of the advancing contact angle formed by the protozoa monolayers with drops of liquids of different polarities. The individual zeta potential varies markedly among theC. fasciculata cells. The wild and FU^R11 mutant strains displayed lower negative surface charge (?12.5 and ?9.5 mV, respectively), as compared with the T^R3 (?14.8 mV) and TFR^R1 (?14.7 mV) mutant strains. Binding of cationized ferritin (CF) was observed at the cell surface of wild and mutant strains ofC. fasciculata. Neuraminidase treatment reduced the negative surface charge in the TFR^R1 and T^R3 mutants in about 37 and 29%, respectively, whereas no significant change was observed with the wild and FU^R11 mutant strains. These findings suggest that sialic acid residues are the major anionogenic groups on the surface ofC. fasciculata. The density of sialic acid residues per cell in wild and mutant strains ofC. fasciculata falls in a range of 1.4×10⁴ to 3.6×10⁴. Marked differences of hydrophobicity were also observed. For example, the TFR^R1, FU^R11, and T^R3 drug-resistant mutant strains showed higher contact angle values (55.4, 54.2, and 49.3, respectively) than the wild-type (35.6), as assessed by α-bromonaphtalene.     

Drouetiella epilithica sp. nov. and Drouetiella lurida (Oculatellaceae,Synechococcales) isolated in the Republic of Korea based on the polyphasic approach

Five strains of Drouetiella (ACKU666, 667, 668, 669 and 670) were isolated from gravels in water, stone monument and coastal mudflat in Korea, and were studied using morphological and molecular traits. All five strains had thin and simple trichomes and exhibited false branching. From these strains, four strains (ACKU666, 667, 668 and 669) exhibited similar cell lengths with reddish–brown colored cells such as Drouetiella lurida. The 16S rRNA gene phylogeny showed the four strains formed a clade with Drouetiella lurida, and their DNA similarity was calculated to be 99.1–100%. The color of strain ACKU670 appeared to be in bright blue–green color like Drouetiella fasciculata, and their thylakoids showed a parietal arrangement, which is a characteristic feature of the family Oculatellaceae. Strain ACKU670 turned out to be a sister clade to the D. lurida according to the phylogenetic analysis of the 16S rRNA gene. The 16–23S rRNA internal transcribed spacer secondary folding structure (D1–D1′, Box-B and V3 helices) confirmed the uniqueness of strain ACKU670, therefore indicating differences from the related species. Considering all the results, we described our strain ACKU670 as Drouetiella epilithica sp. nov. in accordance with the International Code of Nomenclature for Algae, Fungi and Plants.     

Parasitism,longevity and progeny production of six indigenous Kenyan trichogrammatid egg parasitoids (Hymenoptera: Trichogrammatidae) at different temperature and relative humidity regimes

Six native Kenyan species/strains of Trichogramma and Trichogrammatoidea, recovered from Helicoverpa armigera were evaluated at six different temperatures (10, 15, 20, 25, 30 and 35°C) and two relative humidity levels (40–50 and 70–80%) with the aim of selecting strains adapted to warmer temperature regimes. The species/strains were collected from low (<700 m), medium (between 700 and 1200 m) and high altitude (>1200 m) locations and were evaluated for parasitism, adult longevity, progeny production and progeny sex ratio at the different environmental regimes. Eggs of the factitious host, Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) were used in the investigations. Temperature and humidity interactions affected parasitism and progeny production. The highest parasitism at the two humidity levels was at 25 and 30°C for all the strains evaluated. Adult longevity was also significantly affected by the interaction of temperature and relative humidity and was longer at the lower than higher relative humidity. Survival followed a type I survivorship curve at lower temperatures and a type III survivorship curve at the higher temperatures. Trichogramma sp. nr. mwanzai from low altitude, Trichogramma sp. nr. mwanzai from medium altitude and Trichogrammatoidea sp. nr. lutea also from medium altitude lived longer than other strains at all the temperatures and relative humidity levels evaluated, including the warmest regimes of 30 and 35°C. These strains appear promising as candidates for augmentative biocontrol of H. armigera in Kenya.     

Survival and Growth of Tetrahymena thermophila in Media that are Conventionally Used for Piscine and Mammalian Cells

The transfer of Tetrahymena thermophila from normosmotic solutions (~20–80 mOsm/kg H₂O) to hyperosmotic solutions (> 290 mOsm/kg H₂O) was investigated. During the first 24 h of transfer from proteose peptone yeast extract (PPYE) to either 10 mM HEPES or PPYE with added NaCl to give ~300 mOsm/kg H₂O, most ciliates died in HEPES but survived in PPYE. Supplementing hyperosmotic HEPES or PPYE with fetal bovine serum (FBS) enhanced survival. When ciliates were transferred from PPYE to a basal medium for vertebrate cells, L‐15 (~320 mOsm/kg H₂O), only a few survived the first 24 h but many survived when the starting cell density at transfer was high (100,000 cells/ml) or FBS was present. These results suggest that nutrients and/or osmolytes in either PPYE or FBS helped ciliates survive the switch to hyperosmotic solutions. FBS also stimulated T. thermophila growth in normosmotic HEPES and PPYE and in hyperosmotic L‐15. In L‐15 with 10% FBS, the ciliates proliferated for several months and could undergo phagocytosis and bacterivory. These cell culture systems and results can be used to explore how some Tetrahymena species function in hyperosmotic hosts and act as opportunistic pathogens of vertebrates.     

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.     

Marinamoeba thermophila, a new marine heterolobosean amoeba growing at 50 °C

Two amoeba strains were isolated from marine sediment taken at the same place with 18 months interval from a region of the sea floor heated by extended submarine hot springs and fumaroles. These thermophilic amoebae grow at temperatures up to 50 °C. Sequences of the internal transcribed spacer demonstrated that the two strains belong to the same species and are different from any genus for which sequences are known. Phylogeny using small subunit ribosomal RNA places the amoeba in the Heterolobosea. Their closest relatives are the hypersaline flagellate Pleurostomum flabellatum and the hypersaline amoeba Tulamoeba peronaphora. The freshwater amoeboflagellate genera Naegleria and Willaertia belong to the same phylogenetic clade in the Vahlkampfiidae. The new marine species does not transform into flagellates. It forms cysts, which are round to ellipsoidal with few pores. Because of their unique place in the molecular phylogenetic tree, and because there is no morphologically identical species found in the literature, these isolates are considered to be a new species and a new genus, Marinamoeba thermophila.     

First description of the environmental niche of the epibenthic dinoflagellate species Coolia palmyrensis,C. malayensis,and C. tropicalis (Dinophyceae) from Eastern Australia

Environmental variables such as temperature, salinity, and irradiance are significant drivers of microalgal growth and distribution. Therefore, understanding how these variables influence fitness of potentially toxic microalgal species is particularly important. In this study, strains of the potentially harmful epibenthic dinoflagellate species Coolia palmyrensis, C. malayensis, and C. tropicalis were isolated from coastal shallow water habitats on the east coast of Australia and identified using the D1‐D3 region of the large subunit (LSU) ribosomal DNA (rDNA). To determine the environmental niche of each taxon, growth was measured across a gradient of temperature (15–30°C), salinity (20–38), and irradiance (10–200 μmol photons · m^?2 · s^?1). Specific growth rates of Coolia tropicalis were highest under warm temperatures (27°C), low salinities (ca. 23), and intermediate irradiance levels (150 μmol photons · m^?2 · s^?1), while C. malayensis showed the highest growth at moderate temperatures (24°C) and irradiance levels (150 μmol photons · m^?2 · s^?1) and growth rates were consistent across the range of salinity levels tested (20–38). Coolia palmyrensis had the highest growth rate of all species tested and favored moderate temperatures (24°C), oceanic salinity (35), and high irradiance (>200 μmol photons · m^?2 · s^?1). This is the first study to characterize the environmental niche of species from the benthic harmful algal bloom genus Coolia and provides important information to help define species distributions and inform risk management.     

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.     

A novel osmolality-shift fermentation strategy for improving acarbose production and concurrently reducing byproduct component C formation by Actinoplanes sp. A56

Component C (Acarviosy-1,4-Glc-1,1-Glc) was a highly structural acarbose analog, which could be largely formed during acarbose fermentation process, resulting in acarbose purification being highly difficult. By choosing osmolality level as the key fermentation parameter of acarbose-producing Actinoplanes sp. A56, this paper successfully established an effective and simplified osmolality-shift strategy to improve acarbose production and concurrently reduce component C formation. Firstly, the effects of various osmolality levels on acarbose fermentation were firstly investigated in a 50-l fermenter. It was found that 400–500 mOsm/kg of osmolality was favorable for acarbose biosynthesis, but would exert a negative influence on the metabolic activity of Actinoplanes sp. A56, resulting in an obviously negative increase of acarbose and a sharp formation of component C during the later stages of fermentation (144–168 h). Based on this fact, an osmolality-shift fermentation strategy (0–48 h: 250–300 mOsm/kg; 49–120 h: 450–500 mOsm/kg; 121–168 h: 250–300 mOsm/kg) was further carried out. Compared with the osmolality-stat (450–500 mOsm/kg) fermentation process, the final accumulation amount of component C was decreased from 498.2 ± 27.1 to 307.2 ± 9.5 mg/l, and the maximum acarbose yield was increased from 3,431.9 ± 107.7 to 4,132.8 ± 111.4 mg/l.     

Growth and sporulation of entomopathogenic Beauveria bassiana,Metarhizium anisopliae,Isaria farinosa and Isaria fumosorosea strains in relation to water activity and temperature interactions

This study examined six strains of Beauveria bassiana s.l. and Isaria farinosa, one strain of Isaria fumosorosea and five strains of Metarhizium anisopliae s.l. to identify the ability for (1) growth and (2) sporulation under interacting environmental factors of water activity (a_w) and temperature stress. Growth on Sabouraud Dextrose Agar (SDA; water activity, a_w = 0.995) or SDA modified with glycerol to 0.98, 0.96 and 0.94 a_w was measured at four different temperatures (25, 30, 35 and 37°C). All M. anisopliae strains grew at 25–35°C and 0.995 a_w while only two strains tolerated extreme water stress at 0.94 a_w.Three strains of B. bassiana were able to grow at 25–37°C and 0.995 a_w. Only one strain of I. farinosa was able to grow at 25–37°C and 0.995 a_w. A_w and temperature interactions resulted in different strain-dependent responses, in terms of growth and sporulation. Only one strain of I. farinosa and three of M. anisopliae grew at 0.94 a_w and none of the B. bassiana strains tolerated such water stress. At 0.96 and 0.94 a_w and 35–37°C, sporulation by all the strains of the three species were significantly affected. Under elevated temperatures and drought stress, very few of these strains of entomopathogenic fungi are able to grow and sporulate. Indeed, the B. bassiana strains were unable to tolerate the extreme conditions examined. Resilience to such abiotic interactions is critical for selecting strains for formulations. Tolerance to water and temperature stress could be good criteria for selection of strains with secondary spread potential for use as part of an integrated pest management system where secondary cycling may be important, especially in sub-tropical and tropical environments.     

Studies on Prolonged Storage of Beauveria bassiana Conidia: Effects of Temperature and Relative Humidity on Conidial Viability and Virulence against Chickpea Borer,Helicoverpa armigera

Unformulated conidia of Beauveria bassiana were stored at five different temperatures (0°, 10°, 20°, 30° and 40°C) at six different relative humidities (RH) (0, 33, 53, 75, 85 and 98%). Conidial viabilities and virulence against third instar larvae of Helicoverpa armigera were determined over a 24‐month period. Conidia survived longest at lower temperatures (0–20°C) and lower RH levels (0–53% RH). At higher temperatures (30–40°C) conidia did not survive. When the temperature was decreased from 30°C to 0°C, at nearly all RH levels the longevity of conidia increased. Conidia remained virulent for third instar larvae of H. armigera under favourable storage conditions for 24 months.     

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     

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.