The peculiarities of Blastocrithidia triatomae

Parasitology Today,vol. 6. no. I I, I990

The Peculiarities of Blustocrithidia triatomae G.A. Schaub, D. Reduth and M. Pudney Blastocrithidia trlatomae earasitizes triotomine bugs, the vectors of Chagas disease. Co-cultivation with a host-derived cell line permits continuous culture but the host cells are destroyed. Removal of the reduviid cells induces the formation of drought-resistant cysts, bu1 the factors that induce encystment are unknown. Excystment is triggered afier the onset of blood digestion in the insect host, a transition that is associated with unusual ultrastructural alteratrons. Gijnter Schaub, Dagmar Red&h and Mary Pudney believe that B. triatomae is a good candidate for the biological control of Chagas disease, not least because ofits capacii:y to form highly resistant cysts in vitro. The homoxenous tryparlosomatid B/astocrithidia triatomae and Trypanosoma cruzi, the aetiologic agent of Chagas disease, both develop in the intestinal tract and Malpighian tubules of triatomines’-3. Both flagellates are trans.mitted directly between triatomines by cannibalism and coprophagy4,‘. Transmission of 6. triatomae is facilitated by the development of drought-resistant cysts which can survive for at least three years in dry faeces2, and can also be dried (in culture media, physiological saline or water) and stored. When these cysts are ingested by bugs, excystment occurs 12-24 h lai:er in the small intestine’, in contrast to the ingested blood trypomastigotes of T. cruzi, which directly transform in the Ibug’s stomach7. However, epimastigotes of both species multiply in the small intesitine and, in the

rectum, both parasites prefer to colonize the rectal pads8,9. In contrast to 6. tn’atomae, the initial population growth of T. cruzi is more rapid in the rectum than in the small intestine but later, I OO-fold more B. triatomae occur in the small intestine and threefold more in the rectum. In established double infections, 6. triatomae suppresses development of T. cruzi but cannot displace it totally during the course of larval development of the bug (G.A. Schaub and M. Mehl, unpublished). The suppression appears to be caused mainly by competition for attachment sites but differential rates of uptake of essential nutrients may also play a role. Whereas T. cruzi itself appears to be a subpathological stressor of its main vector T. infestans (see Ref. IO for review) infection with 6. triatomae is highly pathogenic for T. infestans and other reduviid vectors of Chagas disease ’’-’2. Infections with 6. triatomae cause a complex sickness syndrome involving retardation of larval development, increase of larval mortality rate, decrease of adult life span and decrease of reproductive rate. The effects of B. triatomae on both T. cruzi and the triatomines open the possibility of using the homoxenous flagellate as a biological control agent. A prerequisite for field trials is the availability of large numbers of cysts, and in vitro culture could provide such a source of material.

flagellate density as compared to the lepidopteran system. When initiated with 0. I ml intestinal contents of infected bugs (containing about 6 X IO6 epimastigotes and IO6 cysts), and after a short lag phase (two days), primary cultures grow exponentially (for about nine days) with a doubling time of 38-41 h, reaching 4 x IO8 organisms in each culture flask (5 ml). The dominant forms are eplmastigotes and the number of cysts increases only in the stationary growth phase, as would be expected from the results from the lepidopteran system. Subcultures on TI-32 cells grow at similar rates as primary cultures, but without any initial lag phase. Morphometry, lectlninduced agglutination and counts of intramembrane particles show no differences between epimastigotes or cysts from the fortieth subculture or the respective bug-derived specimens (D. Reduth, PhD thesis, University of Freiburg, 1986). Wlthin a few hours of inoculation Into the culture flasks, 5. triatomae attaches to the TI-32 cells (Fig. I). The flagellum seems to be pushed into the host cell, causing local destruction of the cell membrane (Fig. 2) and the death of the cell. Rosettes of flagellates form around remnants of TI-32 cells (Fig. 3) but the underlying mechanism for cell killing remains unknown. Other extracellularly developing trypanosomes, grown In association with cultured insect cells,

In Vitro Culture

Fig. I. Scanning electron rmicrograph of a TI-32 cell with an intact, smooth cell membrane invaded by the tip of a jlagellum of B. triatomae (Reproduced, with permission, from Ref: 15.) @ ,990

Elsev,er Scmce

Pubkhers

Ltd. (IJK) 0 I694707/90/802

CO

In vitro, only T. cruzi can be cultivated easily, and initial tests with 5. triatomae in routinely used trypanosomatid media failed. Then Peng and Wallace’3 reported the successful co-cultivation of 5. triatomae with a lepidopteran cell line; however, the parasites needed years before they adapted to culture conditions. No such adaptation is necessary if 5. triatomae is co-cultivated with the J. infestans cell line (TI-32) established by Pudney and Lanar14 (for further details see Ref. IS). This system also produces superior doubling times and higher

fig. 2. Scanning electron micrograph of a U-32 cell in which most ofthe flagellum ofan epimastigote has penetrated. Some parts of the host cell membrane have been destroyed (arrow). (Reproduced, with permission, from Ref: IS.)

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Porosrtology Today,vol. 6, no.

Fig. 3. Established culture of B. triatomae with rosettes around remnants of V-32 cells.

do not destroy the host cells and, surprisingly, even T. cruzi, which invades mammalian culture cells and multiplies intracellularly, does not invade the TI-32 cellQ6. infected T. Within chronically infestons, 5. triatomae are found with their flagella inserted into the cells of the Malpighian tubules and the small intestine, destroying the intestinal cells3,‘7; only insertion however, flagella occasionally occurs, presumably in greatly weakened insects. Intracellular forms have never been observed in VIVO,and rarely in vitro. After infection of T. infestons with culture-derived cysts, no loss of infectivity or virulence has been observed for 6. tn’atomae, although other trypanosomatid species are known to attenuate during culture I5 We have not compared the pathogenicity of cysts produced in vitro with those produced in vivo, since the latter have to be sterilized, a procedure that lyses a high proportion of cysts. After infection of first instar larvae of T. infestans with in vitro-derived cysts from the second subculture, the resulting pathology IS similar to that of insects infected by coprophagy. Encystment and Excystment In the insect host, the encystment rate seems to be increased by starvation of the bug’*. In hosts that are starved after the infectious feed, the percentage of cysts increases from 30x, three weeks after this feeding, to 80x, eight weeks later. It is not yet known what induces encystment, but it is presumably caused by the lack of essential substances. Similarly, the trigger for excystment directly after Initiation of blood digestion in the small intestine of the bug is unknown’. Growing flagella are seen at the anterior end of the small intestine in the partly

digested brown-coloured foodmass; they are never seen in the red contents originatingfrom the stomach. Similarly to the development in the insect, depletion of nutrients, and particularly of the TI-32 cells, induces enhanced rates of encystment in cultures. In cultures with TI-32 cells, all host cells are usually destroyed by 6. triatomae within l-2 weeks, and if no more host cells are added, the development of the flagellates switches from epimastigote replication to cyst production. When 5 X I O7 epimastigotes are inoculated into a culture flask with a scratched culture surface (to provide attachment sites) and the medium (5 ml) is changed weekly, about 2 X IO9 cyst stages can be harvested from each flask within four weeks, although the epimastigote density does not increase15. The first step to encystment is an unequal cell division (D. Reduth, PhD thesis, University of Freiburg, 1986) and in contrast to the normal doubling growth of epimastigotes, cell separation during encystment always starts at the posterior end. Often the smaller encysting cell remains connected to the flagellum of the eplmastigote, thus forming a ‘straphanger’. After at least two more equal cell divisions, a further reduction in size occurs and finally, the cell poles become rounded, and the cell contents condense. The final cysts are only weakly stained by Giemsa. Attempts to induce excystment in vitro by incubation of the cysts in culture medium with or without TI-32 cells and supplemented with bug gut contents have failed (D. Reduth, PhD thesis, University of Freiburg, 1986). Ultrastructural Peculiarities of cysts Transmission electron microscopy reveals that old cysts do not possess an

I 1, I990

outer cyst wall and, instead of the subpellicular microtubuli (which are typical of all trypanosomatids), a ‘homogeneous region’, bordered by a subpellicular layer of granules, ISfound (Fig. 4). No further details of the internal structure can be recognized6,‘9,20. During the initial stages of excystment, ‘net-like’ and ‘labyrinthine’ structures develop that then transform into the kinetoplast and nucleus, respectively6. The peculiar nuclear organization, which sometimes also occurs in epimastigotes, also seems to develop in the sperm of some insects”. When the kinetoplast of excysting specimens shows a normal appearance, the subpellicular microtubuli are also found. Similar ultrastructural alterations have also been described for Blastocrithidia familiaris and Leptomonas lygaei22,23. Freeze-fracture and freeze-etch electron microscopy of cysts from hosts and from in vitro culture reveals that these peculiarities are not artefacts, and that the protoplasmic face resembles that of certain bacteria and not that of eukaryotes24. The ‘homogeneous region’ appears to consist of several rows of closely packed particles (Fig. 5) and is, together with the surface membrane, probably responsible for the resistance to drought’ as well as to short treatments with chaotropic agents and disinfectants25 (E. Schurr, unpublished; D. Reduth, PhD thesis, University of Freiburg, 1986). Prospects for Biological Control Because of its capacity for resistance and the strong pathogenicity, 5. triatomae would appear to be a good candidate for an integrated or biological control programme. Since the chemotherapy of Chagas disease is difficult, the vectors are the main target of control campaigns. Apart from health education and

Fig. 4. Ultrathin section of a cyst of B. triatomae with the subpellicular homogeneous region (arrow). No identification ofinternal organelles is possible. (Reproduced, with permission, from Ref

25.)

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/ I, I990 36,

20 Mehlhorn, H. et al. (I 979) Tropenmed. Paraslt-

Schaub, G.A. and Pretsch, M. (I 98 I ) Trans. R. Sot. Trap. Med. Hyg. 75, I 68- I7 I Zeled6n. R. (I 987) In Chagas’ Disease Vectors (Anatomrc and Physlologrcal Aspects, Vol. I!, (Brenner, R.R. and Stoka, A. de la M.. eds), pp 59-75. CRC Press 8 B6ker, C.A. and Schaub, G.A. (I 984) Z. Parasrtenkd. 70.459-469 9 Schaub, G.A. and Bbker, C.A. (I 986)J. Protoroof. 33,266-270 IO Schaub. G.A. (1989) Paras,tology Today 5.

2 I Schin, K.S. (I 965) Z. Zellforxh. 65, 48 l-5 I3 22 Tieszen, K.L. et al. (I 985) Z. Parasitenkd. 7 I,

Schaub, G.A. 171-175

et al. (1989) J. Protozool.

01. 30. 289-300

185-188

II I2 13 I4 I5 16 I7 I8 19

Schaub. G.A. and Jensen, C. (I 990)]. Invertebr. Pathol. 55, 17-27 Schaub. G.A. 2. Angew. Zoo/. (in press) Peng, P.L-M. and Wallace, F.G. (I 98 I)]. Protozool. 28, I l&l I8 Pudney, M. and Lanar, D. (1977) Ann. Trop. Med. Porasitol. 71, 109-I I8 Reduth, D. et al. (1986) Parasitology 98, 387-393 Lanar, D.E. (I 979) J. Protozool. 26, 457-462 Jensen, C. et of. ( 1990) Parasitology 100, I-9 Reduth, D. ( 1980) Zbl. Bokteriol. Parasitenk. Infektronskr Hyg.. 1. Abt. Ref- 267, 290 Peng, P.L-M. and Wallace, F.G. (I 982)]. Prororool. 29, 464-467

179-188 23 Tieszen. K.L. et al. (1989) Parasrtology 98, 395-400 24 Reduth. D. and Schaub. G.A. (I 988) Parasitol. Res. 74, 30 l-306 25 Schaub, G.A. (1990) Parasitol. Res. 76, 306-3 IO 26 Dlas, J.C.P. (1987) Parasrtology Today 3, 336-34 I 27 Schofield, C.J. (1985) Br. Med. Bull. 41, 187-194 28 Lacey, L.A. et al. (I 989) Buli. Sot. Vector Ecol. 14, 81-86 29 Schaub. G.A. et al. (I 988)J. Microbial. Methods 7. 277-284 Gijnter

Schoub

(ZooIogie),

is at the lnstitut

fiir B,o/ogie I

Albert-Ludwigs-UniversitGt,

Albert-

strasse

2 I a, D-7800

Freiburg, FRG. Dagmar

Reduth

is at the Swiss

Tropical Institute,

Socin-

strasse 57, CH-4002, Basle,Switzerland. Mary Pudney is at the Welcome Research Laboratones, Langley Court, Beckenham BR3 3BS. UK.

Fig. 5. Freeze-fractured cyst of B. triatomae showing the rows of granules in the subpelliculor region (arrow), lipid vesicles (L), the protoplasmic face (PF), and, presumably, a nucleus (N). (Reproduced, with permission, from Ref: 25.)

improvements these the

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standards, based

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houses is common unless insecticides are included Into domestic paints 27,28, but it is unknown whether prolonged exposure to such insecticides affects humans. In addition, this use of chemicals is likely to Increase chances for the development of insecticide resistance. An integrated control programme is thus worth considering, and the first field trials are planned using large numbers of cysts of 13. triotomoe from culture and from bugs after infection with isolated cyst stages25,29. However, costs, efficacies, logistics and so on can only be estimated after preliminary field trials. treated

Acknowledgements We thank Erlka Walter and Theresa Litchfield for their excellent assistance. The support of the Deutsche Forschungsgemelnschaft (Scha 339/I-3 to I-4) is gratefully acknowledged.

Before cloning was perfected, the only way to maintain schistosome strains was by sustaining the entire parasite cycle, ie. by ensuring the regular transmission of the parasite from the snail to the vertebrate, then from the vertebrate to the snail, and so on ad infinitum. joseph jourdane explains that by cloning the parasite in the snail and transplanting the larval parasite from snail to snail, the passage in vertebrates can be avoided indefinite/y. In 1963, Chernin’ showed that daughter sporocysts of Schistosoma mansoni could survive when grafted into a healthy snail. Despite other attempts, it took ten years before a successful microsurgical technique was reported [J. Jourdane ( 1978) Fourth International Congress of Parasitology, Warsaw] that allowed two successive transplantations of cloned daughter sporocysts with production of infective cercariae. In 1980 in our laboratory, 5. mansoni was solely

Schaub, 260-273

G.A.

(1989)

Exp.

Porosrtol.

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Schaub. G.A. (I 983) in Pormitologisches Praktrkum (Bdckeler. W. and Wtilker. W., eds). pp l-6, Verlag Chemie Schaub. G.A. and Schnltker, A. (I 988) Parasltol. Res. 75,88-97 Schaub, G.A. (I 988)Acta Trop. 45, I I-1 9

maintained

in snails and

over

a

year, six successive transplantations were made, which were always followed by the production of infective cercariae*. In I98 I, we established both a male and a female clone of 8. mansoni that have now been maintained through 40 serial transplantations. Likewise, in @1990. Elsewr Sr,ence Publishers Ltd. (UK) 0 l69--1707190/802 00

1980 Nojima and colleagues in Japan’ repot-ted successful serial implantation of larval 8. mansoni by a different technique. CloningTechnique Methodology, The technique described here is that used for the 5. manson+ Biomphalaria sp. model. Explants consisting of I mm3 pieces of the snail’s digestive

gland parasitized

by secondary

Fig. I. Microsurgical transplantation of larva/ schistosome stages into Biomphalaria glabrata The cephalopedal region ofthe snail is kept extended with a microretractor (M) inserted into the male genital opening. Parasite larval stages are implanted into the cephalopeda/ sinus by means of a glass microneedle (GM) attached to a microsyringe.