Dyskinetoplastic cells from both Crithidia fasciculata and Trypanosoma equiperdum lack detectable kinetoplast DNA (kDNA) by conventional staining techniques. Two dyskinetoplastic strains of T. equiperdum, either acriflavine-induced or spontaneously occurring, show normal amounts of kDNA (ρ = 1 · 692g/cm3) in analytical caesium chloride ultracentrifugation. Electron and fluorescence microscopy of the dyskinetoplastic strains of T. equiperdum suggest that the kDNA network is fragmented and dispersed throughout the mitochondrion. The fragmentation and dispersion of the kDNA, rather than a reduction in the amount of kDNA, is the cause of the lack of kinetoplast staining in the dyskinetoplastic strains of T. equiperdum. Acriflavine-treated cultures of C. fasciculata show a decrease in the amount of kDNA (ρ = 1·703 g/cm3) corresponding to the percentage of dyskinetoplastic cells in the cultures. Electron and fluorescence microscopy of acriflavine-treated cultures of C. fasciculata show the loss of the kDNA network in cells which lack Giemsa and Feulgen staining, confirming the hypothesis that the kDNA is lost in dyskinetoplastic trypanosomatids from insects. Possible modes of acriflavine action are considered and a proposed mechanism for acriflavine action in trypanosomes from mammals is presented.

The kinetoplast of the trypanosomatid flagellates is a specialized region of the cell’s single mitochondrion containing 10–20% of the total cellular DNA (Simpson, 1972). The kinetoplast DNA (kDNA) exists as a highly organized network of catenated, covalently closed minicircles (Brack, Delain, Riou & Festy, 1972a; Brack, Delain & Riou, 1972b; Riou & Delain, 1969; Laurent & Steinert, 1970; Renger & Wolstenholme, 1970, 1971, 1972; Simpson, 1972, 1973; Simpson & Simpson, 1974; Simpson & Berliner, 1974; Wolstenholme, Renger, Manning & Fouts, 1974), varying in contour length from 0·3 μm in Leishmania tarentolae (Simpson & da Silva, 1971) to 0·8 μm in Crithidia fasciculata (Renger & Wolstenholme, 1972). Small amounts of large circular DNA, maxicircles, have also been observed in kDNA preparations (Steinert & Van Assel, 1975; Steinert, Van Assel & Steinert, 1976; Borst & Fairlamb, 1976; Kleisen, Weislogel, Fonck & Borst, 1976; Borst, Fase-Fowler, Steinert & Van Assel, 1977; Weislogel et al. Borst & Hoeijmakers, 1978; Fairlamb et al. 1978). In Giemsa and Feulgen preparations of kinetoplastic strains of trypanosomatids the region containing the kDNA network appears as an intensely stained granule at the base of the flagellum. Cells completely lacking kDNA demonstrable by these staining procedures have been termed dyskinetoplastic (Trager & Rudzinska, 1964).

Small percentages of naturally occurring dyskinetoplastic cells have been observed in a variety of species of trypanosomatids from insects and mammals (Cosgrove & McSwain, 1960; Mühlpfordt, 1963). Completely dyskinetoplastic, spontaneously occurring strains of the mammalian flagellates Trypanosoma equiperdum (Tobie, 1951) and Trypanosoma evansi (Hoare & Bennett, 1937) have also been reported.

Treatment of certain bloodstream trypanosomes with acriflavine, a DNA intercalating compound, results in the production of completely dyskinetoplastic strains (Deane & Kloetzel, 1969; Hajduk, 1978; Mühlpfordt, 1963; Stuart, 1971) which are viable in their mammalian host. Both the spontaneously occurring and drug-induced dyskinetoplastic bloodstream trypanosomes have been shown, in analytical ultracentrifugation studies, to retain their kDNA in normal amounts (Renger & Wolstenholme, 1971; Stuart, 1971).

Insect trypanosomatids grown in vitro in the presence of acriflavine show varying percentages of dyskinetoplastic individuals in their populations (Cosgrove, 1966; Hajduk, 1978; Simpson & Simpson, 1974; Simpson, 1968; Steinert & Van Assel, 1967; Trager & Rudzinska, 1964). These dyskinetoplastic cells appear to be incapable of multiplication and completely dyskinetoplastic populations have not been obtained. Analytical ultracentrifugation of DNA isolated from such populations shows a decrease in the amount of kDNA present (Morales, Schaefer, Keller & Meyer, 1972; Renger & Wolstenholme, 1970, 1972; Simpson, 1968; Steinert & Van Assel, 1967).

In this study I report on the dyskinetoplastic phenomena in a spontaneously occurring dyskinetoplastic strain and an acriflavine-induced dyskinetoplastic strain of the bloodstream flagellate T. equiperdum and in acriflavine-induced dyskinetoplastic cultures of the insect flagellate C. fasciculata. I shall also discuss possible mechanisms of acriflavine action in trypanosomatids.

The normal kinetoplastic (ATCC 30019), the spontaneously dyskinetoplastic (ATCC 30023), and the acriflavine-induced dyskinetoplastic strains of T. equiperdum were grown in albino rats infected by intraperitoneal injection of thawed frozen stocks. These stocks were prepared by washing heparinized, infected blood from rats twice with Hanks’ glucose containing 10% glycerol and freezing to —70 °C (Packchanian (personal communication)). The acriflavine-induced dyskinetoplastic strain was produced by repeated subcutaneous injections of neutral acriflavine (Allied Chemicals) into mice infected with the kinetoplastic strain of T. equiperdum. A dosage of 30 μg/g body weight was found most satisfactory. The percentage of dyskinetoplastic cells was monitored by Giemsa staining of each relapse population; injections were continued until a completely dyskinetoplastic strain was produced.

Infected blood was obtained from etherized animals by heart puncture and trypanosomes were separated from blood constituents by Lanham’s (1968) anion-exchange technique. The elute from the exchange column was routinely centrifuged at 3300 g for 10 min at 4 °C and washed 3 times with Lanham’s phosphate saline glucose buffer, pH 8 (PSG). Separated cells in PSG were held on ice until used.

C. fasciculata (ATCC 1 1745) was grown axenically in a yeast extract-sucrose-hemin medium (Cosgrove, 1959) at 25 °C. Dyskinetoplastic populations were produced by addition of neutral acriflavine (30 μg/ml) to early log-phase cultures. The percentage of dyskinetoplastic cells was determined by Giemsa staining.

Fluorescence microscopy

4,6-diamidino-2-phenylinole (DAPI) was obtained as a gift from Dr O. Dann. Cells were labelled with DAPI and examined by fluorescence microscopy as described previously (Hajduk, 1976).

Electron microscopy

Cells were fixed in 1 % glutaraldehyde in 0 ·1 M cacodylate buffer, pH 7 · 4 for 1 h at 4 °C, centrifuged at 3300 g for 10 min and washed 3 times with 0 ·1 M cacodylate buffer. Cells were then postfixed for 1 h at room temperature with 1 % osmium tetroxide in the same buffer. Following postfixation the cells were washed 3 times and the final pellet broken into small pieces and dehydrated through graded ethanols and propylene oxide, embedded in Epon-Araldite and polymerized at 60 °C for 48 h. Silver sections were stained with uranyl acetate and lead citrate and examined in a Philips 200 electron microscope operating at 80 kV.

Chemical determinations of cellular DNA content

Chemical determinations of total cellular DNA were done by the method of Martin & Donohue (1972). Cell numbers were estimated with a Coulter counter.

DNA extraction and analytical ultracentrifugation

Total cellular DNA was isolated from SDS-lysed cells by the procedure of Fouts, Manning & Wolstenholme (1975). Analytical neutral caesium chloride equilibrium density gradient centrifugations were performed according to Meselson, Stahl & Vinograd (1957) in a Beckman Model E analytical ultracentrifuge using an An-D rotor. Centrifugations were carried out at 44 770 rev/min for 24 h at 20°C. Ultraviolet photographs were scanned at 800 nm with a Gilford Model 240 spectrophotometer coupled to a Linear Instruments Model 232 chart recorder. The relative amount of kDNA in the normal and dyskinetoplastic strains was estimated by determining the area under the DNA peaks in the resulting tracings with a Ladd graphical digitizer. Buoyant densities were calculated as described by Schildkraut, Marmur & Doty (1962) using Micrococcus iysodeikticus DNA = 1 · 732 g/cm3) as a reference.

Giemsa staining of the normal strains of both T. equiperdum (Fig. 1a) and C. fasciculata (Fig. 1d) confirms the presence of a kinetoplast. The kinetoplast appears as an intensely stained granule at the base of the flagellum, posterior to the nucleus in T. equiperdum and anterior to the nucleus in C. fasciculata. No such granule can be seen in the dyskinetoplastic strains of T. equiperdum (Fig. 1b, c); the nucleus stains normally and the spontaneously occurring and acriflavine-induced dyskinetoplastic strains are indistinguishable. Giemsa preparations of acriflavine-treated cultures of C. fasciculata show varying percentages of cells lacking a detectable kinetoplast (Fig. 1C): the nucleus stains normally in the dyskinetoplastic cells and dividing dyskinetoplastic cells; cells with 2 nuclei and no kinetoplast, are observed (Fig. 1g). Dividing cells with 2 nuclei and 1 kinetoplast are also seen (Fig. 1f); completion of division of these cells would result in 1 kinetoplastic and 1 dyskinetoplastic cell. In cells retaining kinetoplast staining there is no reduction in the size of the kinetoplast.

Fig. 1.

Giemsa-stained smears of normal and dyskinetoplastic C. fasciculata and T. equiperdum. N, nucleus; k, kinetoplast. × 5000 approx, (a) The kinetoplastic strain of T. equiperdum showing a nucleus and kinetoplast. (b) The spontaneously dyskinetoplastic strain of T. equiperdum showing only nuclear staining, (c) The acriflavine-induced dyskinetoplastic strain of T. equiperdum also showing only nuclear staining, (d) Untreated C. fasciculata showing a nucleus and kinetoplast. (e-g) Acriflavine-treated C. fasciculata.

Fig. 1.

Giemsa-stained smears of normal and dyskinetoplastic C. fasciculata and T. equiperdum. N, nucleus; k, kinetoplast. × 5000 approx, (a) The kinetoplastic strain of T. equiperdum showing a nucleus and kinetoplast. (b) The spontaneously dyskinetoplastic strain of T. equiperdum showing only nuclear staining, (c) The acriflavine-induced dyskinetoplastic strain of T. equiperdum also showing only nuclear staining, (d) Untreated C. fasciculata showing a nucleus and kinetoplast. (e-g) Acriflavine-treated C. fasciculata.

Table 1 shows the frequency of cell types in cultures of untreated, acriflavine-treated, and acriflavine + hydroxyurea-treated C. fasciculata. The frequency of cells with 2 nuclei and i kinetoplast (2N/1K) increases only in cultures treated with acriflavine alone. The combined treatment with acriflavine and hydroxyurea inhibits the occurrence of cells with 2 nuclei and one kinetoplast and prevents the production of dyskinetoplasty. Hydroxyurea stops both nuclear and kinetoplast DNA synthesis and cell division of C. luciliae (Steinert, 1969Z,) and C. fasciculata (W. B. Cosgrove, M. J. Skeen & S. L. Hajduk, unpublished observations).

Table 1.

Comparison of cell type frequencies in drug-treated and untreated cultures of C. fasciculate

Comparison of cell type frequencies in drug-treated and untreated cultures of C. fasciculate
Comparison of cell type frequencies in drug-treated and untreated cultures of C. fasciculate

The graph in Fig. 2 shows the effect of acriflavine on the growth and dyskinetoplasty of C. fasciculata. The maximum frequency of dyskinetoplastic cells (0 · 546) occurs shortly before cell division stops in the culture. Although this figure shows the maximum percentage of dyskinetoplastic individuals as being about 50%, cultures containing up to 80% dyskinetoplastic cells have been obtained using the treatment shown here. The rate of cell division in the culture prior to acriflavine treatment appears to influence the percentage of dyskinetoplastic cells obtainable.

Fig. 2.

The effect of acriflavine on the growth (•) and dyskinetoplasty (○) of C. fasciculata. Addition of filtration-sterilized acriflavine (30 μ g/ml) is indicated by the arrow.

Fig. 2.

The effect of acriflavine on the growth (•) and dyskinetoplasty (○) of C. fasciculata. Addition of filtration-sterilized acriflavine (30 μ g/ml) is indicated by the arrow.

The effect of acriflavine treatment on the growth and dyskinetoplasty of T. equiperdum in white mice is shown in Fig. 3. During the decline in trypanosome population a small percentage of dyskinetoplastic cells is observed. A relapse strain generally occurs which has a very high percentage of dyskinetoplasty; elimination of the remaining kinetoplastic cells requires repeated acriflavine injections.

Fig. 3.

The effect of acriflavine on the growth (•) and dyskinetoplasty (○) of the kinetoplastic strain of T. equiperdum (left-hand side) and of relapse strain (righthand side). Subcutaneous injection of acriflavine (30 μg/g mouse body weight) is indicated by the arrow.

Fig. 3.

The effect of acriflavine on the growth (•) and dyskinetoplasty (○) of the kinetoplastic strain of T. equiperdum (left-hand side) and of relapse strain (righthand side). Subcutaneous injection of acriflavine (30 μg/g mouse body weight) is indicated by the arrow.

Fig. 4 is a summary of the results of CsCl analytical ultracentrifugation of DNA from the normal, spontaneously dyskinetoplastic and acriflavine-induced dyskinetoplastic strains of T. equiperdum. The kDNA appears as a rapidly banding satellite at ρ = 1 · 692, the main band nuclear DNA at ρ = 1 · 707, and a shoulder on the nuclear DNA peak at ρ = 1 · 702, of unknown origin, is also present. Analysis of the relative amounts of kDNA in the 3 strains (Table 2) shows no significant decrease in the amount of the ρ = 1 · 692 satellite band in the 2 dyskinetoplastic strains. No difference in buoyant densities was detectable in the banding patterns from the three strains.

Table 2.

Analysis of DNA in the organisms studied

Analysis of DNA in the organisms studied
Analysis of DNA in the organisms studied
Fig. 4.

Spectrophotometer tracings of ultraviolet photographs of caesium chloride buoyant density gradients of DNA isolated from whois cells of the normal kinetoplastic (A), the spontaneously dyskinetoplastic (B) and an acriflavine-induced dyskinetoplastic (C) strains of T. equiperdum.

Fig. 4.

Spectrophotometer tracings of ultraviolet photographs of caesium chloride buoyant density gradients of DNA isolated from whois cells of the normal kinetoplastic (A), the spontaneously dyskinetoplastic (B) and an acriflavine-induced dyskinetoplastic (C) strains of T. equiperdum.

Analytical ultracentrifugation of DNA from the normal, 54% dyskinetoplastic and 77% dyskinetoplastic populations of C. fasciculata (Fig. 5) shows the kDNA banding at ρ = 1 · 703. Again no buoyant density changes were detectable in the banding patterns of DNA from the 3 populations. However, there is a reduction in the amount of kDNA in the 2 partially dyskinetoplastic populations. This decrease in kDNA correlates with the increase in the percentage of dyskinetoplastic cells in the cultures as determined by Giemsa staining (Table 2).

Fig. 5.

Spectrophotometer tracings of ultraviolet photographs of caesium chloride buoyant density gradients of DNA from whole cells of C. fasciculata grown under normal conditions (A) and in the presence of acriflavine, the latter 54 and 77 % dyskinetoplastic (B and C, respectively). The reference band (ρ = 1·731 g/cm3) is native DNA of Micrococcus lysodeikticus.

Fig. 5.

Spectrophotometer tracings of ultraviolet photographs of caesium chloride buoyant density gradients of DNA from whole cells of C. fasciculata grown under normal conditions (A) and in the presence of acriflavine, the latter 54 and 77 % dyskinetoplastic (B and C, respectively). The reference band (ρ = 1·731 g/cm3) is native DNA of Micrococcus lysodeikticus.

Table 2 also presents the values obtained from chemical determinations of DNA content in normal and dyskinetoplastic strains of T. equiperdum and C. fasciculata. The mean values show no significant decrease in the amount of total cellular DNA in the dyskinetoplastic strains of T. equiperdum. Chemical determinations of total cellular DNA from an 80% dyskinetoplastic population of C. fasciculata show a reduction of 23 · 2% in the amount of DNA.

In thin sections the kinetoplast of the normal strains of T. equiperdum (Fig. 6) and C. fasciculata (Fig. 13) appears as an expanded portion of the cell’s single mitochondria containing a highly organized, electron-dense fibrous band of kDNA in a volume of finely granular material surrounded by the double limiting mitochondrial membrane. Numerous cristae occur in the mitochondrion of C. fasciculata, while the mitochondrion of T. equiperdum has few. The fibrous band of kDNA is absent in the dyskinetoplastic strains of T. equiperdum and clumps of electron-dense material are seen; these clumps, though located predominantly in the kinetoplast region of the mitochondrion (Figs. 9, 10), may also occur along its length (Figs. 7, 8, 11, 12). Some mitochondrial swelling was observed in a few cells of both dyskinetoplastic strains but no other structural alterations were observed in the dyskinetoplastic strains of T. equiperdum and the 2 dyskinetoplastic strains were indistinguishable at the ultrastructural level.

Fig. 6.

Section through the kinetoplast region of the mitochondrion (m) of the normal kinetoplastic strain of T. equiperdum. The kDNA appears as an electron-dense fibrous band bounded by the double mitochondrial membrane. Note that the kinetoplast is adjacent to the basal body (bb) in the normal strain, × 45 800.

Fig. 6.

Section through the kinetoplast region of the mitochondrion (m) of the normal kinetoplastic strain of T. equiperdum. The kDNA appears as an electron-dense fibrous band bounded by the double mitochondrial membrane. Note that the kinetoplast is adjacent to the basal body (bb) in the normal strain, × 45 800.

Fig. 7.

Longitudinal section through the mitochondrion (m) of the spontaneously dyskinetoplastic strain of T. equiperdum showing a pair of electron-dense clumps of altered kDNA (ak) in regions of the mitochondrion not at the base of the flagellum. × 18800.

Fig. 7.

Longitudinal section through the mitochondrion (m) of the spontaneously dyskinetoplastic strain of T. equiperdum showing a pair of electron-dense clumps of altered kDNA (ak) in regions of the mitochondrion not at the base of the flagellum. × 18800.

Fig. 8.

Cross-section through a portion of mitochondrion in the spontaneously dyskinetoplastic strain of T. equiperdum showing clumps of altered kDNA (ak). ×25500.

Fig. 8.

Cross-section through a portion of mitochondrion in the spontaneously dyskinetoplastic strain of T. equiperdum showing clumps of altered kDNA (ak). ×25500.

Fig. 9.

Section through the kinetoplast region of the mitochondrion at the base of the flagellum (f) in the spontaneously dyskinetoplastic strain of T. equiperdum. Numerous electron-dense clumps of altered kDNA (ak) are observed in this micrograph. × 45 800.

Fig. 9.

Section through the kinetoplast region of the mitochondrion at the base of the flagellum (f) in the spontaneously dyskinetoplastic strain of T. equiperdum. Numerous electron-dense clumps of altered kDNA (ak) are observed in this micrograph. × 45 800.

Fig. 10.

Longitudinal section through the kinetoplast of a cell from the acriflavine-induced dyskinetoplastic strain of T. equiperdum. This section shows clumps of altered kDNA (ab) in the portion of the mitochondrion (pi) adjacent to the basal body (bb). × 21000.

Fig. 10.

Longitudinal section through the kinetoplast of a cell from the acriflavine-induced dyskinetoplastic strain of T. equiperdum. This section shows clumps of altered kDNA (ab) in the portion of the mitochondrion (pi) adjacent to the basal body (bb). × 21000.

Fig. 11.

A cell from the acriflavine-induced dyskinetoplastic strain of T. equiperdum sectioned longitudinally showing a clump of altered kDNA (ak) in a region of the mitochondrion (m) near the nucleus (n). × 18 800.

Fig. 11.

A cell from the acriflavine-induced dyskinetoplastic strain of T. equiperdum sectioned longitudinally showing a clump of altered kDNA (ak) in a region of the mitochondrion (m) near the nucleus (n). × 18 800.

Fig. 12.

Section showing a large region of mitochondrion (m) and clumps of altered kDNA (ak) spread along it in a cell from the acriflavine-induced dyskinetoplastic strain of T. equiperdum. × 18800.

Fig. 12.

Section showing a large region of mitochondrion (m) and clumps of altered kDNA (ak) spread along it in a cell from the acriflavine-induced dyskinetoplastic strain of T. equiperdum. × 18800.

Fig. 13.

Section through the kinetoplast of the normal strain of C. fasciculata showing a large, highly organized kDNA band located in a region of the mitochondrion (m) adjacent to the basal body (bb) at the base of the flagella pocket (Jp). × 34500.

Fig. 13.

Section through the kinetoplast of the normal strain of C. fasciculata showing a large, highly organized kDNA band located in a region of the mitochondrion (m) adjacent to the basal body (bb) at the base of the flagella pocket (Jp). × 34500.

Electron microscopy of an acriflavine-treated population of C. fasciculata containing 54 · 4% dyskinetoplastic individuals showed 2 kinds of alterations in the kinetoplast (Figs. 14-17). Of the several hundred individual cells examined only 50 were sectioned longitudinally through the flagellum and kinetoplast; of these 28 contained a single large clump of electron-dense material in the kinetoplast region of the mitochondria (Figs. 14, 15). The remaining 22 lacked any structure in the mitochondria at the base of the flagellum (Figs. 16, 17). No clumps of electron-dense material were seen in other regions of the mitochondria as in the dyskinetoplastic strains of T. equiperdum. The mitochondrial cristae in the cells with the clumps of material in the kinetoplast appear slightly swollen but are present in normal numbers. The mitochondria of cells lacking any structure in the kinetoplast have few cristae and those which are present are swollen.

Fig. 14.

Cell from an acriflavine-treated culture of C. fasciculata sectioned longitudinally through the flagellum (f) and the kinetoplast region of the mitochondrion (m) showing a large clump of altered kDNA (ab). × 34500.

Fig. 14.

Cell from an acriflavine-treated culture of C. fasciculata sectioned longitudinally through the flagellum (f) and the kinetoplast region of the mitochondrion (m) showing a large clump of altered kDNA (ab). × 34500.

Fig. 15.

Electron micrograph of an acriflavine-treated cell from a culture of C. fasciculata showing a large single clump of altered kDNA (ak) at the base of the flagellum (f). × 53400.

Fig. 15.

Electron micrograph of an acriflavine-treated cell from a culture of C. fasciculata showing a large single clump of altered kDNA (ak) at the base of the flagellum (f). × 53400.

Fig. 16.

Longitudinal section through the flagellum (f) and mitochondrion (m) of a cell from an acriflavine-treated culture of C. fasciculata. This cell lacks any structure in the mitochondria at the base of the flagellum, × 25 500.

Fig. 16.

Longitudinal section through the flagellum (f) and mitochondrion (m) of a cell from an acriflavine-treated culture of C. fasciculata. This cell lacks any structure in the mitochondria at the base of the flagellum, × 25 500.

Fig. 17.

High-magnification electron micrograph of a section through the kinetoplast region of the mitochondrion, at the base of the flagellum (f) in a cell from an acriflavine-treated culture of C. fasciculata. × 68600.

Fig. 17.

High-magnification electron micrograph of a section through the kinetoplast region of the mitochondrion, at the base of the flagellum (f) in a cell from an acriflavine-treated culture of C. fasciculata. × 68600.

Fluorescence microscopy of the normal strain of T. equiperdum shows fluorescence restricted to the nucleus and kinetoplast (Fig. 18a-c). The nucleus appears as a large, blue fluorescent structure in approximately the centre of the cell; the kinetoplast appears either as a small fluorescent body (Fig. 18b, c) or as a pair of fluorescent bodies following kinetoplast division (Fig. 18a). Fluorescence in both the spontaneously occurring and acriflavine-induced dyskinetoplastic strains of T. equiperdum is not limited to the nucleus and kinetoplast regions of the cell (Fig. 18d-g). Small fluorescent bodies of varying size and intensity occur in the kinetoplast region and throughout the cell, generally arranged as a row of particles.

Fig. 18. Fluorescence photographs of DAP1-labelled cells taken using an XBO high-pressure xenon lamp, Zeiss UG1 and BG32 exciter filters and a Zeiss GG400 barrier filter. × 5100. (a-c)Kinetoplaβtic strain of T. equiperdum showing a kinetoplast (arrow) and a nucleus (N). (d, e) Spontaneously dyskinetoplastic strain of T. equiperdum with fluorescent cytoplasmic granules (arrow) and nucleus (N). (f, g) Acriflavine-induced dyskinetoplastic strain of T. equiperdum also showing cytoplasmic (arrow) and nuclear (N) fluorescence, (h) Untreated C. fasciculata showing nucleus (N) and kineto-plast (arrow), (i) Acriflavine-treated C. fasciculata showing lack of kinetoplast fluorescence in all but one cell (arrow), (j) Acriflavine-treated C. fasciculata with 2 nuclei and a single kinetoplast (arrow), (k) Acriflavine-treated C. fasciculata with 2 nuclei (N) and no kinetoplast.

Fluorescence microscopy of the normal strain of C. fasciculata labelled with DAPI also shows fluorescence restricted to the nucleus and kinetoplast (Fig. 18h). Nuclear fluorescence is much fainter than the fluorescence of the kinetoplast, which suggests that kDNA binds more DAPI than nuclear DNA, perhaps because of the higher A-T content of kDNA (Williamson & Fennell, 1975). Fluorescence microscopy of cells from an acriflavine-treated culture of C. fasciculata (Fig. 18i-k) showed the presence of DNA-DAPI complexes in the nuclear region of all cells. Of 415 cells examined 45·9% also showed fluorescence in the region of the kinetoplast, while 54 · 1% showed fluorescence only in the nuclear region, in good agreement with the 54 · 4% dyskinetoplasty found in Giemsa preparations.

My results show that the dyskinetoplastic condition is quite different in T. equiperdum and C. fasciculata in spite of its apparent similarity as judged by conventional staining criteria. In T. equiperdum, dyskinetoplastic cells contain the normal amount of kDNA of normal base composition but the organization of the kDNA is altered, resulting in dispersion of small, condensed pieces of the network throughout the mitochondrion. In C. fasciculata, dyskinetoplastic cells lack kDNA.

The presence of kDNA in dyskinetoplastic cells of T. equiperdum is consistent with the results of Renger & Wolstenholme (1971) and with those of Stuart (1971) suggesting the retention of kDNA in dyskinetoplastic bloodstream trypanosomes. My results support the hypothesis held by several workers (Hajduk, 1976; Renger & Wolstenholme, 1971; Strauss, 1972; Vickerman, 1977) that the failure of the kinetoplast, of dyskinetoplastic trypanosomes from mammals, to stain with Giemsa or Feulgen is due to dispersion of the kDNA and not to a reduction in the amount of kDNA. Although this hypothesis is supported by results obtained from several species of bloodstream trypanosomes, recent studies (Opperdoes, Borst & de Rijke, 1976; Borst & Fairlamb, 1976; Borst & Hoeijmakers, 1978) using other strains of these bloodstream trypanosomes suggest that the status of the kDNA may be quite variable and even completely absent in at least one case.

Loss of kDNA in dyskinetoplastic cells of C. fasciculata is in agreement with results obtained by others using acriflavine (Cosgrove, 1966; Simpson, 1968; Steinert & Van Assel, 1967; Stuart & Hanson, 1967) and ethidium bromide (Steinert, 1969a; Riou & Pautrizel, 1967) to induce dyskinetoplasty in a variety of insect trypanosomatids. Cosgrove (1966) suggested that the appearance of dyskinetoplastic individuals in cultures of C. fasciculata treated with acriflavine is the result of the failure of the kDNA to duplicate prior to cell divisions; my results support this view. In contrast to the observations of Simpson (1968) and others (Deane & Kloetzel, 1969; Steinert & Van Assel, 1967; Steinert, 1969 a) on other insect trypanosomatids, there is no fragmentation of the kDNA network as a result of acriflavine treatment in C. fasciculata.

Clearly, from the evidence I have presented, it is necessary to be cautious in classification of cells as dyskinetoplastic and in making generalizations about dyskinetoplasty in trypanosomatids. The common method of classification, on the basis of Giemsa or Feulgen staining, should be maintained but generalizations concerning loss or retention of kDNA based on these staining characteristics must be avoided.

In acriflavine-treated cultures of C. fasciculata dyskinetoplasty appears to be caused by steric interference with kDNB replication (Cosgrove, 1966). This steric interference is introduced by intercalation (Lerman, 1961) of acriflavine into the kDNA and results in blockage or delay of kDNA replication. Since the time of kinetoplast division in C. fasciculata precedes cell division by only 5–7 min even a slight delay in kDNA replication would result in a dyskinetoplastic cell being formed. Although cell division prior to kDNA replication is probably the principle mode of production of dyskinetoplasty in C. fasciculata the observed frequency of cells with no kinetoplast and 2 nuclei, in acriflavine-treated cultures, suggests that the dyskinetoplastic cells are capable of division. Attempts to subculture dyskinetoplastic cells from these cultures have been unsuccessful, suggesting that the ability of the dyskinetoplastic cells to divide is limited.

The mechanism of acriflavine action on the kDNA of T. equiperdum is not as straightforward. Blockage of kDNA replication by acriflavine is unlikely to produce viable dyskinetoplastic strains of T. equiperdum since such cells would lack kDNA and it has been shown that the kDNA is present in normal amounts in these strains of T. equiperdum. It is possible that a small number of cells in the kinetoplastic strain of T. equiperdum are resistant to acriflavine and that these cells are dyskinetoplastic in appearance. This possibility seems unlikely since both spontaneously occurring and acriflavine-induced dyskinetoplastic strains of T. equiperdum are sensitive to acriflavine at the dosage used (unpublished data). I propose the following explanation for the difference in dyskinetoplastic bloodstream and cultured trypanosomatids.

The immediate consequence of exposure to acriflavine, quaternary alterations in the kDNA resulting in inhibition of kDNA replication, is the same for both C. fasciculata and T. equiperdum. In acriflavine-treated cultures of C. fasciculata, a cell division then produces a daughter cell containing normal amounts of kDNA, which is phenotypically kinetoplastic and viable, and one which lacks kDNA, is phenotypically dyskinetoplastic and not viable under standard conditions of culture. In the phenotypically kinetoplastic daughter, the kDNA remains clumped but does not become fragmented. In T. equiperdum, a cell division also produces a daughter cell lacking kDNA, phenotypically dyskinetoplastic and not viable. Their nonviability results in their rapid destruction by the host. The other product of a cell division is a daughter cell containing the normal amount of kDNA but in the ‘clumped’ configuration. As drug concentration decreases in the host’s blood kDNA replication resumes and viable dyskinetoplastic cells containing normal amounts of kDNA appear; their dyskinetoplastic phenotype is the result of fragmentation and dispersion of the kDNA throughout the mitochondrion. Thus the major difference in the effect of acriflavine on the organization of the kDNA in C. fasciculata and T. equiperdum appears to be the dispersion of the kDNA in T. equiperdum following drug treatment.

I wish to thank the director, Dr Walter Humphrys, and the staff of the Central Electron Microscope Laboratory at the University of Georgia for the use of their excellent facilities. I would also like to thank Dr Terry Thomas, Dr Allen Clarkson, Mr Ben Spurlock, Dr Bill Henk and Professor Keith Vickerman for helpful discussions and technical advice and especially Dr William Cosgrove for his encouragement, guidance and support during this research. Professor O. Dann’s generous gifts of DAPI are gratefully acknowledged.

Borst
,
P.
&
Fairlamr
,
A. H.
(
1976
).
DNA of parasites, with special reference to kinetoplast DNA
.
In The Biochemistry of Parasites and Host-Parasite Relationships
(ed.
H.
Van Den Bossche
), pp.
169
191
.
Amsterdam
:
North-Holland
.
Borst
,
P.
,
Fase-Fowler
,
F.
,
Steinert
,
M.
&
Van Assel
,
S.
(
1977
).
Maxi-circles in the kinetoplast DNA of Trypanosoma mega
.
Expl Cell Res
. no,
167
173
.
Borst
,
P.
&
Hoeijmakers
,
J. H. J.
(
1978
).
Kinetoplast DNA
.
Plasmid
(in press).
Brack
,
C.
,
Delain
,
E.
,
Riou
,
G.
&
Festy
,
B.
(
1972a
).
Molecular organization of kinetoplast DNA of Trypanosoma cruzi treated with berenil, a DNA interacting drug
.
J. Ultrastruct. Res
.
39
,
569
579
.
Brack
,
C.
,
Delain
,
E.
&
Riou
,
G.
(
1972b
).
Replication of covalently closed circular DNA from kinetoplast of Trypanosoma cruzi
.
Proc. natn. Acad. Sci. U. S. A
.
69
,
1642
1646
.
Cosgrove
,
W. B.
(
1959
).
Utilization of carbohydrates by the mosquito flagellate, Crithidia fasciculata
.
Can. J. Microbiol
.
5
,
573
578
.
Cosgrove
,
W. B.
(
1966
).
Acriflavine-induced akinetoplasty in Crithidia fasciculata
.
Acta protozoal
.
4
,
155
161
.
Cosgrove
,
W. B.
&
Mcswain
,
M.
(
1960
).
Absence of the kinetoplast in trypanosomids of insects
.
Anat. Rec
.
138
,
341
.
Deane
,
M. P.
&
Kloetzel
,
J. K.
(
1969
).
Differentiation and multiplication of dyskinetoplastic Trypanosoma cruzi in tissue culture and in the mammalian host
.
J. Protozool
.
16
,
121126
.
Fairlamb
,
A. H.
,
Weislogel
,
P. O.
,
Hoeijmakers
,
J. H. J.
&
Borst
,
P.
(
1978
).
Isolation and characterization of kinetoplast DNA from bloodstream form of Trypanosoma brucei
.
J. Cell Biol
.
76
,
293
309
.
Fouts
,
D. L.
,
Manning
,
J. E.
&
Wolstenholme
,
D. R.
(
1975
).
Physiochemical properties of kinetoplast DNA from Crithidia acanthocephali, Crithidia luciliae, and Trypanosoma letcisi
.
J. Cell Biol
.
67
,
378
399
.
Hajduk
,
S. L.
(
1973
).
Comparison of the kinetoplasts of normal, spontaneously dyskinetoplastic, and dye-induced dyskinetoplastic strains of Trypanosoma equiperdum
.
Progress in Protozool
.
168
.
Hajduk
,
S. L.
(
1976
).
Demonstration of kinetoplast DNA in dyskinetoplastic strains of Trypanosoma equiperdum
.
Science, N. Y
.
191
,
858
859
.
Hajduk
,
S. L.
(
1978
).
Influence of DNA complexing compounds on the kinetoplast of trypanosomatids
.
In Progress in Molecular and Subcellular Biology
, vol.
6
(ed.
F. E.
Hahn
), pp.
158
200
.
Heidelberg
:
Springer-Verlag
.
Hoare
,
C. A.
&
Bennett
,
S. C. J.
(
1937
).
Morphological and taxonomic studies on mammalian trypanosomes. III. Spontaneous occurrence of strains of Trypanosoma evansi devoid of kinetonucleus
.
Parasitology
29
,
43
56
.
Kleisen
,
C. M.
,
Weislogel
,
P. O.
,
Fonck
,
K.
&
Borst
,
P.
(
1976
).
The structure of kinetoplast DNA. II. Characterization of a novel component of high complexity present in the kinetoplast DNA network of Crithidia luciliae
.
Eur. F. Biochem
.
64
,
153
160
.
Lanham
,
S. M.
(
1968
).
Separation of trypanosomes from the blood of infected rats and mice by anion-exchangers
.
Nature, Lond
.
218
,
1273
1274
.
Laurent
,
M.
&
Steinert
,
M.
(
1970
).
Electron microscopy of kinetoplast DNA from Trypano soma mega
.
Proc. natn. Acad. Sci. U. S. A
.
66
,
419
424
.
Lerman
,
L. S.
(
1961
).
Structural considerations in the interaction of DNA and acridines
.
J. molec. Biol
.
3
,
18
30
.
Martin
,
R. F.
&
Donohue
,
D. C.
(
1972
).
New analytical procedure for the estimation of DNA with p-nitrophenylhydrazine
.
Analyt. Biochem
.
47
,
562
574
.
Meselson
,
M.
,
Stahl
,
F.
&
Vinograd
,
J.
(
1957
).
Equilibrium sedimenation of macromolecules in density gradients
.
Proc. natn. Acad. Sci. U. S. A
.
43
,
581
588
.
Morales
,
N. M.
,
Schaefer
,
F. W.
,
Keller
,
S. J.
&
Meyer
,
R. R.
(
1972
).
Effects of ethidium bromide and several acridine dyes on the kinetoplast DNA of Leishmania tropica
.
J. Protozool
.
19
,
667
672
.
Mohlpfordt
,
H.
(
1963
).
Über die Bedeutung und Feinstruktur des Blepharoplasts bei parasitischen Flagellatan. I. Teil
.
Z. Tropenmed. Parasit
.
14
,
357
398
.
Opperdoes
,
F. R.
,
Borst
,
P.
&
De Rijke
,
D.
(
1976
).
Oligomycin sensitivity’ of the mitochondrial ATPase as a marker for fly transmissability and the presence of functional kinetoplast DNA in African trypanosomes
.
Comp. Biochem. Physiol
.
55B
25
30
.
Renger
,
H. C.
&
Wolstenholme
,
D. R.
(
1970
).
Kinetoplast deoxyribonucleic acid of the hemoflagellate Trypanosoma leveisi
.
J. Cell Biol
.
47
,
689
-702.
Renger
,
H. C.
&
Wolstenholme
,
D. R.
(
1971
).
Kinetoplast and other satellite DNAs of kinetoplastic and dyskinetoplastic strains of Trypanosoma
.
J. Cell Biol
.
50
,
533
540
.
Renger
,
H. C.
&
Wolstenholme
,
D. R.
(
1972
).
Form and structure of kinetoplast DNA of Crithidia
.
J. Cell Biol
.
54
,
346
364
.
Riou
,
G.
&
Delain
,
E.
(
1969
).
Electron microscopy of the circular kinetoplast DNA from Trypanosoma cruzi: occurrence of catenated forms
.
Proc. natn. Acad. Sci. U. S. A
.
62
,
210
217
.
Riou
,
G.
&
Pautrizel
,
R.
(
1967
).
Fractionnement et caracterisation de deux bandes satellites de DNA chez Trypanosoma gambiense
.
C. r. hebd. Séanc. Acad. Sci., Paris
265
,
61
63
.
Schildkraut
,
C. L.
,
Marmur
,
J.
&
Doty
,
P.
(
1962
).
Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl._7
.
molec. Biol
.
4
,
430
443
.
Simpson
,
A. M.
&
Simpson
,
L.
(
1974
).
Isolation and characterization of kinetoplast DNA networks and minicircles from Crithidia fasciculata
.
J. Protozoal
.
21
,
774
781
.
Simpson
,
L.
(
1968
).
Effect of acriflavin on the kinetoplast of Leishmania tarentolae. Mode of action and physiological correlates of the loss of kinetoplast DNA
.
F. Cell Biol
.
37
,
660
682
.
Simpson
,
L.
(
1972
).
The kinetoplast of the hemoflagellates
.
Int. Rev. Cytol
.
32
,
139
207
.
Simpson
,
L.
(
1973
).
Structure and function of kinetoplast DNA
.
J. Protozoal
.
20
,
2
8
.
Simpson
,
L.
&
Berliner
,
J.
(
1974
).
Isolation of the kinetoplast DNA of Leishmania tarentolae in the form of a network
.
J. Protozool
.
21
,
382
393
.
Simpson
,
L.
&
Da Silva
,
A.
(
1971
).
Isolation and characterization of kinetoplast DNA from Leishmania tarentolae
.
J. molec. Biol
.
56
,
443
473
.
Steinert
,
M.
(
1969a
).
Specific loss of kinetoplast DNA in trypanosomatidae treated with ethidium bromide
.
Expl Cell Res
.
96
,
406
409
.
Steinert
,
M.
(
1969b
).
Reversible inhibition of the division of C. luciliae by hydroxyurea and its use for obtaining synchronized cultures
.
FEBS Letters, Amsterdam
5
,
29
.
Steinert
,
M.
&
Van Assel
,
S.
(
1967
).
The loss of kinetoplast DNA in two species of trypanosomatidae treated with acriflavine
.
J. Cell Biol
.
34
,
489
503
.
Steinert
,
M.
&
Van Assel
,
S.
(
1975
).
Large circular mitochondrial DNA in Crithidia luciliae
.
Expl Cell Res
.
96
,
406
409
.
Steinert
,
M.
, VAN
Assel
,
S.
&
Steinert
,
G.
(
1976
).
Minicircular and non-minicircular components of kinetoplast DNA
.
In The Biochemistry of Parasites and Host-Parasite Relationships
(ed.
H.
Van Den Bossche
), pp.
193
209
.
Amsterdam
:
North-Holland
.
Strauss
,
P. R.
(
1972
).
Acriflavin resistance in the hemoflagellate, Leishmania tarentolae
.
J. Cell Biol
.
53
,
312
334
.
Stuart
,
K. D.
(
1971
).
Evidence for the retention of kinetoplast DNA in an acriflavine-induced dyskinetoplastic strain of Trypanosoma brucei which replicates the altered central element of the kinetoplast
.
J. Cell Biol
.
49
,
189
195
.
Stuart
,
K. D.
&
Hanson
,
E. D.
(
1967
).
Acriflavine induction of dyskinetoplasty in Leptomonas kary ophilus
.
J. Protozool
.
14
,
39
43
.
Tobie
,
E. J.
(
1951
).
Loss of the kinetoplast in a strain of Trypanosoma equiperdum
.
Trans. Am. microsc. Soc
.
70
,
251
254
.
Trager
,
W.
&
Rudzinska
,
M. A.
(
1964
).
The riboflavin requirements and the effects of acriflavin on the fine structure of the kinetoplast of Leishmania tarentolae
.
J. Protozool
.
11
,
133
145
.
Vickerman
,
K.
(
1977
).
The dyskinetoplasty mutation in Trypanosoma evansi and other flagellates
.
Protozoology
3
,
57
69
.
Weislogel
,
P. O.
,
Hoeijmakers
,
J. H. J.
,
Fairlamb
,
A. H.
,
Kleisen
,
C. M.
&
Borst
,
P.
(
1977
).
Characterization of kinetoplast DNA networks from the insect trypanosome Crithidia luciliae
.
BioMm. biophys. Acta
478
,
167
179
.
Williamson
,
D. H.
&
Fennell
,
D. J.
(
1975
).
The use of a fluorescent DNA-binding agent for detecting and separating yeast mitochondrial DNA
.
Meth. Cell Biol
.
12
,
335
351
.
Wolstenholme
,
D. R.
,
Renger
,
H. C.
,
Manning
,
J. E.
&
Fouts
,
D. L.
(
1974
).
Kinetoplast DNA of Crithidia
.
J. Protozool
.
21
,
622
631
.