Theileria annulata and Babesia ovis: Ultracytochemical lactic dehydrogenase activity of sporozoites in salivary glands of female ticks, Hyalomma anatolicum excavatum and Rhipicephalus bursa

EXPERIMENTAL

53, 326-334 (1982)

PARASITOLOGY

Theileria annulata and Babesia ovis: Ultracytochemical Lactic Dehydrogenase Activity of Sporozoites in Salivary Glands of Female Ticks, Hyalomma anatolicum excavatum and Rhipicephalus bursa G~NTER Institut

fiir

Parasitologie

der Tierarztlichen

WEBER

Hochschule,

Btinteweg

17, D-3000

Hannover

71, West Germany

(Accepted for publication 6 May 1981) WEBER, G. 1982. Theileria annulata and Babesia ovis: Ultracytochemical lactic dehydrogenase activity of sporozoites in salivary glands of female ticks, Hyalomma anatolicum excavatum and Rhipicephalus bursa. Experimental Parasitology 53, 326-334. The occurrence of a marker enzyme of glycolysis, lactic dehydrogenase (LDH) (EC 1.1.1.27.), was studied ultracytochemically in sporozoites of Babesia ovis in the tick Rhipicephalus bursa and in sporozoites of Theileria annulata in the tick Hyalomma anatolicum excavatum. Female ticks infected transstadially were fed on rabbits and dissected 3-5 days post infestationem. The salivary glands were removed and incubated in the cytochemical medium unfixed or after fixation in buffered paraformaldehyde solution. A modified ferricyanide medium adjusted to pH 6.5 was used for incubation. Controls were performed by preincubating specimens in 10m3 M iodine or by omitting NAD+ or lactate in the incubation medium. Following incubation, the specimens were fixed in buffered solutions of paraformaldehyde or glutaraldehyde, postfixed in osmium tetroxide, and embedded in Durcupan ACM. Mature “schizonts” consisting of an abundant number of sporozoites were examined in both piroplasmean species. In sporozoites of B. ovis the final enzymic product was deposited within the nucleus. No cytoplasmic reaction was observed. However, the membrane delimiting the schizont from the adjacent glandular cells was distinctly reactive. In T. annulata reactivity was usually confined to the cytoplasm. Sometimes, a reaction within mitochondria could be observed. The reaction product had formed aggregations which often appeared to be attached to micronemes. There was no nuclear reactivity in this species. The results suggest the existence of a glycolytic metabolic pathway with different subcellular localizations in sporozoites of the two piroplasmean species. INDEX DESCRIPTORS: Theileria annulata; Babesia ovis; Protozoa, parasitic; Hyalomma anatolicum excavatum; Rhipicephalus bursa ; Ticks; Lactic dehydrogenase (EC 1.1.1.27.); Electron microscopy; Enzyme ultracytochemistry; Tick salivary glands; Piroplasms; Glycolytic pathway.

INTRODUCTION

Little is known about the metabolism of piroplasms in the tick. Biochemical data on this subject are not available and will not be as long as the technical problem of supplying pure parasite preparations in sufficient quantities is not solved. On the other hand, certain metabolic potencies of these parasite stages can be estimated by the demonstration of metabolically active components, especially enzymes, with cytochemical techniques. In the first study of this kind succinic dehydrogenase (SDH) activity was

demonstrated by light microscopy in schizonts of Babesia ovis in tick salivary glands (Weber, 1974). The term “schizont” is used here in a general sense for a developmental stage producing multiple parasites (Levine 1971.) Recently, SDH and cytochrome oxidase activities could be localized ultrastructurally in sporozoites of B. ovis and Theileria annulata (Weber 1980b). Ultracytochemical demonstration of cytochrome oxidase in kinetes of B. ovis and of acid phosphatase and acid esterase in kinetes of B. ovis and B. bigemina was reported (Weber 1978a, 1980a). Martins

326 0014-4894/82/030326-09$02,00/O Copyright All rights

0 1982 by Academic Press. Inc. of reproduction in any form reserved.

Theiferia

anndata

AND Babesia ovis: LACTIC

(1976) looked for activities of alkaline phosphatase and some acid hydrolases in sporozoites of T. parva under the light microscope but obtained negative results. The energy metabolism of piroplasmean sporozoites is of special interest because those stages have to survive in both an arthropod and a mammalian host. As indicated by cytochemical enzyme activities, the sporozoites of B. ovis and T. annulata have respiratory potency (Weber 1978b, 1980b). In the present study the occurrence of a marker enzyme of glycolysis, lactic dehydrogenase (LDH) (EC 1.1.1.27.), was studied in these stages. Salivary glands of Rhipicephalus bursa ticks infected with B. ovis, and those of Hyalomma anatolicum excavatum infected with T. annulata were incubated for ultracytochemical activities and examined by electron microscopy.

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specimens were then immediately incubated unfixed. The others were fixed in 2 or 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer for 15 min at 4 C or 8 min at room temperature. Subsequently, they were washed in phosphate buffer for at least 3 hr. The ferricyanide medium by KerpelFronius and Haj6s (1968) as modified by Hanker et al. (1973) was used for the LDH experiments. The composition of the medium was the following: 0.5 M sodium potassium- tartrate, 6 ml; 0.1 M sodium phosphate buffer, pH 7.2, 1.6 ml; 0.3 M CuSO,, 0.7 ml; 24 mg L-lactate, or 48 mg DL-lactate (lithium salt), dissolved in 0.025 ml 0.3 M CuSO,; nicotinamide adenine dinucleotide (NAD+), 5 mg; 0.05 M potassium ferricyanide, 0.3 ml. The final pH was 6.5. Dimethyl sulfoxide, 1.5 ml, was added to the medium used in the studies of Theileria. InMATERIALS AND METHODS cubations were carried out for 40-60 min Rhipicephalus bursa Canestrini et Fan- at 37 C. zago, 1877, infected with Babesia ovis, is The specificity of the reaction was maintained in Hannover, West Germany, checked either by preincubation of specias strain “Ankara 1959” (Friedhoff and mens in 10e3 M iodine followed by short Scholtyseck 1968). The ticks of the species rinsing in distilled water, or by omitting Hyalomma anatolicum excavatum Koch, lactate or NAD+ in the incubation medium. All control specimens with T. annulata 1844, were derived from the strain “Syrien 1964” established and maintained by Dr. E. were negative (Fig. 10). Those with B. ovis Schein at the Institut fiir Parasitologie, were negative, as well, except for speciFreie Universitst Berlin (Schein et al. mens incubated in substrate-free medium. 1975). This strain was temporarily propa- In these preparations a slight deposition of gated on calves and rabbits in Hannover. an electron-dense product had taken place B. ovis (BabCs 1892) Starcovici,l893, within the parasite nuclei (Fig. 3). These had been derived from the tick strain deposits, however, do not impair the specificity of the test reactions. They were “Ankara 1959” and described as “Hanprobably caused by the tartrate incorponover 1959” (Friedhoff and Scholtyseck rated in the incubation medium. This com1968). Theileria annulata Dschunkowsky and Luhs, 1904, had been isolated from a pound, like lactate, is an a-hydroxy acid substrate of naturally infected calf in Turkey and estab- and, therefore, a potential lished by Dr. E. Schein (Berlin) in the LDH (Meister 1950). Following incubation the specimens were Hyalomma strain “Syrien 1964” (Schein et washed in phosphate buffer for 20 min and al. 1975). Adult ticks were fed on rabbits. Females then fixed in 2.5% glutaraldehyde or 4% were removed from the host 3 to 5 days PFA in phosphate buffer for various peripost infestationem and dissected submerged ods of time. Subsequently, they were in cold PBS or 0.15 M NaCl. Some of the thoroughly washed in buffer and postfixed

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in OsOl in cacodylate buffer, pH 7.1, at room temperature. They were then dehydrated in acetone and embedded in Durcupan ACM (Fluka, Switzerland). Ultrathin sections were examined unstained or stained with a vanadyl-molybdate solution (Callahan and Horner 1964). Microscopy was carried out with a Siemens Elmiskop I A or a Zeiss EM 10 A operated at 40 or 60 kV. RESULTS

The structural preservation of the tick salivary glands was good and provided adequate morphological reference. The appearance of the parasites resembled that in morphologic pictures. The majority of Babesia ovis in ultrathin sections showed a positive reaction (Fig. 1). The electron-dense final product of enzymic activity was present in the nucleus, only. Deposits had accumulated alongside the nuclear membrane and in the peripheral zone of the nucleus. The central part of the nucleus was usually free of deposits (Fig. 2). No other site of reactivity was observed in the sporozoites of this species. However, the membrane delimiting the babesial

WEBER

schizont from the adjacent tissue cells was distinctly reactive (Figs. 4, 5). This membrane showed enlargements of its surface in the shape of folds or microvilli-like protrusions (Fig. 5). Host nuclei never displayed deposits of the final enzymic product (Fig. 1). Some deposits of the final enzymic product were present in the cytoplasm of both parasitized and nonparasitized alveolar cells. Frequently, they appeared accumulated in the vicinity of or alongside the plasmalemma of ‘cells (Fig, 6). No reaction product was observed in nuclei of salivary gland cells (Fig. 1). The reaction product had formed aggregations of coarse granules in Theiferia annulatu sporozoites (Fig. 7). These agglomerations were associated with structures which could not be definitely identified; probably they were micronemes (Fig. 8). Rhoptries did not show reactivity or reactive material attached to them (Fig. 8). In some parasites the reaction product was observed within mitochondria where it appeared deposited alongside the mitochondrial wall (Fig. 9). No reaction product was visible outside of the parasites in the alveolar cells.

FIGS. l-6. Electron micrographs of stained sections. Scale represents 1 pm. FIG. 1. Babesia ovzs. Electron microscopic aspect of a schizont in a tick’s salivary gland incubated for LDH activity: Reactivity of parasite nuclei (some indicated by arrows, bottom). Note the absence of elctron-dense deposits in the nucleus (NH) of an adjacent glandular cell. ~4,000. Inset: Light microscopic aspect of a schizont in a histological section stained after Giemsa. NH, host nucleus. x 1200. FIG. 2. Babesia ovis. LDH in sporozoite nuclei (large arrows). Some nuclei are negative (small arrows). The “spherical body” of the parasite is negative, as well (asterisk). x 10,000. FIG. 3. Babesia wk. Sporozoites after substrate-free control incubation. Some nuclei show fine deposits at their margin (large arrows), others appear completely free of reactivity (small arrows). x 9800. FIG. 4. Babesia ovis. LDH activity of the cellular membrane surrounding a schizont (light small arrows). Reactive (light large arrows) and nonreactive (small arrows) nuclei are visible in the schizont. x 9000. FIG. 5. Babesia ovis. Protrusions of the LDH active perischizontal membrane into the cytoplasm of an adjacent glandular cell (small light arrows). Above an enzymically active nucleus of a sporozoite (large light arrow). x 17,500. FIG. 6. Babesia ovis. Cytoplasmic LDH activity in a glandular cell adjacent to a parasitized one (large arrows). The schizont shows several positive parasite nuclei (medium-sized arrows) and a positive limiting membrane (small arrows). x6500.

Theileria

annulata

AND

Babesia

ovis:

LACTIC

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AND Babesia ovis: LACTIC

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of cytoplasmic LDH to dislocate in tissue specimens. Up-to-date knowledge on miThe purpose of the present study was to cronemes does not offer clues that these ordetect activity of a representative glycolytic ganelles are sites of glycolytic processes enzyme in the piroplasmean sporozoites in (For references see de Souza and Soutoorder to obtain information on the para- Padr6n 1978). On the other hand, Akao sites’ possible ability to utilize glycolysis in and Matsubayashi (1966) and Akao (1967) their metabolism. Though it is understood using autoradiographic techniques obthat cytochemical activity of a single en- served eventual accumulation of 3Hzyme does not prove its incorporation in a marked glucose in micronemes of T. gondii. might in functional sequence, ultrastructural dem- This suggests that micronemes onstration of LDH activity suggests exis- some way or other be involved in carbohydrate metabolism processes. In this context tence of a glycolytic pathway. The marked differences in intensity of the reactions ob- it is worthwhile mentioning that light miserved in Babesia ovis, in particular (Figs. croscopic cytochemical LDH activity was 2, 4, 6), do not necessarily reflect different observed in the anterior part of sporozoites metabolic activities of the parasites. They of Eimeria tenella. This portion of the paramight rather be due to possible uneven pen- site is almost completely occupied by mietration of the electron acceptor, fer- cronemes (Ryley 1969). Sporozoites of E. ricyanide, included in the incubation tenella would be a suitable object for medium (see also Weber 1980b). The differstudying ultrastructural localization of ence between the two piroplasmean species LDH in a motile apicomplexian stage. regarding the sites of reactivity is surprisDifferences between the sporozoites of ing. Especially, the nuclear activity ob- the two piroplasmean species in the potenserved in B. ovis instead of a cytoplasmic tial role of glycolysis are indicated not only one is an unusual cytochemical feature of by different sites of LDH activity in the LDH. Although eukaryotic nuclei are parasites themselves. They are also reknown to contain glycolytic enzymes flected by the presence of enzymic activity (Dounce and Beyer 1948) or might even in the “perischizontal” membrane obpossess a complete glycolytic system served exclusively in glandular cells inof (Siebert 1961), ultracytochemical LDH ac- fected with B. ovis. The plasmalemma tivity in cell nuclei has not been reported. nonparasitized glandular cells never reIts significance in the sporozoites of B. ovis acted for LDH. According to Friedhoff et cannot be explained at present. It is note- al. (1972) the schizonts of B. ovis in salivary worthy that light microscopic LDH activity glands of female ticks are delimited only by was demonstrated in the nuclear area of the plasmalemma of the host cell. Therefore it is assumed that the membraneous encoccidian developmental stages. Thus, Frandsen (1970) detected LDH in Eimeria zymic activity around the schizonts of B. stiedai where the activity was located in ovis has been induced by the parasitism, front of the refractile body. Beyer et al. possibly due to enhanced uptake of nutri(1977) observed LDH activity in the peri- ents by the host cell (see Cohn 1966). This interpretation is supported by the observed nuclear zone of zoites of Toxoplasma enlargements in the surface of this delimitgondii. The cytoplasmic structures, with which ing membrane (Figs. 4, 5). In previous studies activities of succinic the final product of LDH in sporozoites of dehydrogenase and cytochrome oxidase Theileria annulata was associated, could were demonstrated in sporozoites of B. ovis not be identified with certainty; probably and T. annufata (Weber 1978b, 1980b). they were micronemes. This location of the reaction product has to be looked at with Those and the present results suggest the possible occurrence of both an oxidative some reservation because of the tendency DISCUSSION

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7- 10. Electron micrographs of unstained sections. Scale represents 0.1 pm. 7. Theileria annulara. Sporozoite showing cytoplasmic activity of LDH (arrows). The mitochondrion (m) is free of any reactive product. n, nucleus. ~48,000. FIG. 8. Theileria annulata. Sporozoite with cytoplasmic LDH activity (arrows). The deposits of the enzymic product seem to be attached to micronemes. n, nucleus. ~47,300. FIG. 9. Theileria annulara. LDH activity in the mitochondrion (arrows). The enzymic product appears associated with the wall. mn, micronemes. x48,000. FIG. 10. Theileria annulara. Three sporozoites in a tick’s salivary gland incubated substrate free for LDH. No reactivity. m, mitochondrion; mn, micronemes; n, nucleus. ~32,500. FIGS. FIG.

and a glycolytic energy metabolism in these parasite stages. One can only speculate about possible preference of either of these pathways during the parasite’s life cycle. Due to ultracytochemical activities of suc-

cinic dehydrogenase and cytochrome oxidase in sporozoites of T. annulata and B. ovis it can be assumed that respiratory activity is more pronounced in the former (Weber 1980). This may also apply to eryth-

Theileria

annulata

AND Babesia ovis: LACTIC

rocytic stages of T. annulata; they did not reveal LDH activity when run in starch gel electrophoresis (Melrose and Brown 1979). In contrast, LDH could be detected in erythrocytic stages of various Babesia species in rodents (Momen et al. 1979). Unfortunately, nothing is known about respiratory activities in erythrocytic stages of Theileria and Babesia, although ultracytochemical activity of cytochrome oxidase was demonstrated in B. ovis (Weber 1980). These data, though fragmentary, suggest differences in the energy metabolism of the piroplasms depending on the genus, species, or developmental stage. It seems most likely that a “mixed” pattern of energy metabolism in sporozoites reflects an adaptation to the prospective change of this stage from one host to another. Mack and Vanderberg (1978) studying sporozoites of Plasmodium berghei with the aid of physiological techniques found indications that these stages metabolize both aerobically and anaerobically. They ascribed this fact to the need of surviving in both an invertebrate and a mammalian host. Such an explanation seems feasible, but on the other hand it has been shown that Toxoplasma gondii, which does not inhabit hosts of different classes, has oxidative and glycolytic enzymes as well (Beyer et al. 1977. In further studies comparative cytochemical and biochemical data on relevant enzymes should be gathered in motile stages of various species of Apicomplexa. This would help to obtain a better idea of the metabolism and structure-function relationship of these stages. ACKNOWLEDGMENTS I am grateful to Dr. E. Schein, Institut fiir Veterin&-parasitologie und Tropenveterin&medizin, Freie Universitat, Berlin (West), and Dr. K. T. Friedhoff, Institut fiir Parasitologie, Tiertiztliche Hochschule, Hannover, for supplying the infected ticks. Thanks are also due to Miss Dorothee Zimmermann for expert technical assistence and Miss Gabriele Klinckmann for linguistic help in preparation of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft.

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