The Effects of Trypanosomatids on Insects

The Effects of Trypanosomatids on Insects GUNTERA. SCHAUB

Department of Special Zoology and Parasitology, Ruhr University, 0-4630 Bochum, Germany I. Introduction ............................

11. Parasitogenic Alterations of Host Behaviour

111.

IV.

V.

VI.

VII. VIII.

A. Reduction of fitness . . . . . . . . . . . . . . . . . B. Modification of vector feeding behaviour ........................ Disturbances in Organ Systems . A. Disturbances of the digestive tract B. Disturbances of the Malpighi C. Effects on the haemolymph .................................. D. Effects on the cuticle .................. E. Other affected organ systems ......................................... Effects on Pre-adult Development and Mortality A. Trypanosoma infections of Triatominae ................................ B. Blastocrirhidia rriafomae infections of Triatominae ...................... C. Homoxenous trypanosomatids in Hymenoptera and Diptera Effects on Adult Life Span and Reproduction Rate . . . . . . . . . . . . A. Leishmania and Trypanosoma ................ B. Homoxenous trypanosomatids ........................................ Synergistic Effects of Trypanosomatids and other Stressors . . . . . . . . . . . . . . . . . . A. Sensitivity to insecticides B. Starvation resistance ................................................. C. Sensitivity to isolation and overcrowding .............................. Mechanisms of Pathogenicity . . . . . . . . . . . . . . Acknowledgements .......... References ..............................................................

I.

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275 281 282 284 291 294 295 296 297 298 304 304

INTRODUCTION

The trypanosomatids in insects can be divided according to their life cycles and their genus-specific developmental stages into two groups, both to be considered in this review. Members of the genera of the first group, the heteroxenous trypanosomatids, are transmitted by insects to vertebrates (Leishmania, Trypanosoma, Endotrypanum) or plants (Phytomonas) and are ADVANCES IN PARASITOLOGY VOL. 31 ISBN 0-12-031731-1

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causative agents of important diseases (Hoare, 1972; Molyneux, 1977). Those of the second group, the homoxenous (entomophilic) trypanosomatids, have only a single host, an arthropod (Herpetomonas, Crithidia, Rhynchoidomonas, Blastocrithidia) or sometimes another invertebrate (Leptomonas) (Wallace, 1966, 1979; Molyneux, 1977).* Usually they colonize the intestinal tract of the insects, but some species of both groups also invade the haemocoele (summarized by Molyneux et al., 1986b). In most cases the heteroxenous trypanosomatids do not affect their vectors; some exceptions (Leishmania spp., Tryp. cruzi, Tryp. lewisi, Tryp. rangeli, a bat and a bird trypanosome) are mentioned by Kramer (1963), Jenkins (1964), Brooks (1974) and Molyneux (1977, 1981, 1983). Only Tryp. rangeli has been the object of detailed studies. Like Tryp. cruzi, the causative agent of Chagas disease, it colonizes the intestinal tract of triatomines, but only Tryp. rangeli additionally invades the haemocoele and salivary glands and multiplies intra- and extracellularly in the haemocoele (D’Alessandro. 1976). Investigations with Tryp. rangeli are complicated by the high variability of strains: also rapid attenuation during culture in vitro occurs, indicated by loss of the ability to invade the haemocoele. Therefore, some workers have inoculated the parasite directly into the haemocoele. The pathology of this trypanosome has been reviewed by D’Alessandro (1 976), but additional interesting aspects were investigated later (e.g. by Aiiez, 1982, 1983, 1984; Aiiez and East, 1984; Schwarzenbach, 1987). Since insects are important pests, any parasite of insects such as the homoxenous trypanosomatids should be considered as a possible agent for biological control. However, textbooks or reviews on insect pathology usually emphasize the importance of viruses, bacteria and fungi (Steinhaus, 1949, 1963a,b; Weiser, 1977). Of the Protozoa, the Sporozoa are always considered, and most diseased insects are infected by Microsporidia and Gregarina and only rarely by Flagellata (Lipa and Steinhaus, 1962; McLaughlin, 1973). The opinion of Sweetman (1958) that trypanosomatids “do not seem to seriously interfere with reproduction or produce serious epizootics among their hosts” is shared by most protozoologists. In later reviews of insect pathology or trypanosomatids, some pathogenic effects of entomophilic trypanosomatids are mentioned. Flagellatoses due to homoxenous trypanosomatids have been reported, for Lept. pyrrhocoris, H. muscarum, H . swainei ahd B. caliroa (Lipa, 1963; Brooks, 1974; Molyneux, 1977, 1980b; Wallace, 1979; Henry, 1981). * Based on the Greek term xenos = host, the term “heteroxenous” should be used for trypanosomatids which develop in different groups of hosts and “homoxenous” for those developing in related species; “monoxenous” should be used only for those developing in a single species. The terms “monogenetic” and “digenetic”, which are sometimes used, are misleading-specially the latter (see “Lexikon Biologie”, published by Herder Verlag, Freiburg).

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In 1 9 7 6 f i v e years after the first description-Haberkorn first presented observations showing that B. triatomae is pathogenic for Triatoma infestans, the most important vector of Chagas disease. At his invitation, his system has been taken over by me for standardization and the inclusion of further vectors of Chagas disease. Since then, B. triatomae has been the object of detailed studies to elucidate its possible application for biological control of triatomines (results are summarized by Schaub, 1988e, 1990c; Schaub et al., 1990a). Like Tryp. cruzi and Tryp. rangeli, B. triatomae colonizes the intestinal tract and the Malpighian tubules, but only B. triatomae develops drought-resistant cysts with peculiar ultrastructural adaptations (Schaub and Pretsch, 1981; Schaub, 1983; Reduth, 1986; Reduth and Schaub, 1988; Schaub and Losch, 1988). Interestingly, B. triatomae and Tryp. rangeli are pathogenic or non-pathogenic to different species of triatomines; specifically, Tryp. rangeli affects species of the genus Rhodnius only. More and more effects of trypanosomes have been recognized in other systems in the last 10 years, making it possible to write this present review which is concerned, for the first time, solely with pathological effects of trypanosomatids on insects.* Instead of describing the effects in each system, I have grouped them into five topics: behavioural alterations, disturbances of organ systems, effects on pre-adult developmental times and mortality rates, effects on adult life span and reproduction rate, and synergistic effects of trypanosomatids and other stressors. This review is also intended to direct the reader to a phenomenon which is indicated only by minor effects and needs to receive more attention, the subpathogenic stressing of the insect hosts. Since natural populations of ihsects rarely live under optimum conditions, the subpathogenic stressor trypanosomatid might act synergistically with other biotic or abiotic stressors and thus harm the insect host.

IT. PARASITWENIC ALTERATIONS OF HOSTBEHAVIOUR Effects of infections on host behaviour, which improve parasite transmission and which have been elucidated in recent years in many parasite-host systems (reviewed by Schaub, 1989c; Hurd, 1990), also occur in trypanosomatid-insect systems. Some insects are only weakened by the infection while others, bloodsucking insects, attack their hosts more frequently. As this is the first review summarizing publications concerning the influence of trypanosomatids on insects, it is possible that I have missed some publications. If any reader knows of any such missing reports I should very much appreciate the information, so as to be able to include them in a later review.

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REDUCTION OF FITNESS

So far, no investigation has studied the predation rate of insects infected with trypanosomatids, but this rate should be increased if the insects are weakened. Such non-specific effects on behaviour, i.e. sluggish movements, have been reported for Rhodnius prolixus infected with Tryp. rangeli (Grewal, 1957, 1969), Pyrrhocoris apterus infected with Lept. pyrrhocoris (Lipa, 1963), Tri. infestans infected with B. triatomae (Schaub and Schnitker, 1988), and advanced infections of Hippelates pusio with H . muscarum (Bailey and Brooks, 1972a). Most recently, Shykoff and Schmid-Hempel (1991b) found that worker bumble bees naturally infected with C. bombi are less likely to forage for pollen. The only quantitative investigation of endurance has been undertaken by Arnqvist and Maki (1990): male water striders, naturally infected with B. gerridis and/or C.Jlexonema, do not skate as intensively against the current in a circular stream channel as uninfected specimens. Skating endurance is negatively correlated with the intensity of the trypanosomatid infection, and may adversely affect predation of food and mating. Such an adverse effect on the ability of males to acquire mates occurs in natural populations. Whereas light and moderate infections lower the mate acquisition ability only to some extent, heavy infections drastically reduce the mating success of infected males. This effect is caused by the reduced fitness, since the search for females and the precopulative struggle with females, which are reluctant to mate, is energy-intensive (G. Arnqvist, personal communication). t

B.

1.

MODIFICATION OF VECTOR FEEDING BEHAVIOUR

General

Parasitogenic alterations of behaviour of many bloodsucking insects by infections with trypanosomatids are not so spectacular as those induced by helminths in their intermediate hosts, e.g. they do not include the death of the invertebrate host, but they seem to be very efficient (Schaub, 1989~). There are two possible mechanisms by which the number of attacks on blood donors by bloodsucking insects could be increased. (i) Trypanosomatids and the insect host compete for metabolites in the ingested blood, and the depletion leads to a new attempt by the insect to ingest blood. This possibility seems to occur in phlebotomines which are presumed to be infected with the bat trypanosome Tryp. leonidasdeani (Williams, 1976) (see Section V.A), and it may also be relevant in infected bugs (see Section II.B.4). (ii) The trypanosomes interfere with the ingestion process. These

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effects on bloodsucking insects are connected with disturbances of the digestive tract, especially the foregut and the anterior midgut (see Section II1.A). Ingestion of blood by infected sandflies, tsetse flies and bugs is often delayed and ceases if the host makes repulsive actions. These vectors may then attack another host. Additionally, the infected vectors often take no, or only a small, bloodmeal and therefore become hungry earlier and attack a new host, enhancing the chances of parasite transmission (reviewed by Molyneux and Jefferies, 1986). The mechanisms of these effects seem to differ slightly in the different trypanosomatid-vector systems. 2. Sandflies infected with Leishmania In several Leishmania-sandfly systems the parasites initially colonize the midgut and then the foregut, which can be covered by a “carpet” of attached flagellates (Warburg et al., 1986; Kaddu et al., 1988; Killick-Kendrick et al., 1988). The infectious stages then detach and remain lying on the “carpet” or migrate forward. At least in infections with one species, Leish. donovani, the pharynx of the sandfly Phlebotomus argentipes can be blocked for its entire length with a plug of parasites (Shortt et al., 1926), similar to the development of Bacillus pestis in the rat flea (Bacot and Martin, 1914; Holdenried, 1952). Nearly 22% of the infected flies possess blocked foreguts, and in other specimens the lumen of the foregut is significantly narrowed by the parasites (Smith et al., 1940). Sandflies with a partially blocked foregut can take up only minute quantities of blood, and complete blockage excludes a further blood meal. However, all continue trying to obtain a blood meal (Smith et al., 1940), sometimes at different locations, but in an extreme case for 18 min at one location (Smith et al., 1941). Probing without subsequent uptake of blood increases the chance of transmission of the parasites compared to infected flies which successfully engorge (Killick-Kendrick et al., 1977b). Five out of 16 sandflies infected with Leish. mexicana amazonensis probed repeatedly but took no blood, and a further eight flies ingested only a small meal (Killick-Kendrick et al., 1977b). This behaviour occurs also in phlebotomines infected with Leish. major, thereby explaining the occurrence of 11 separate, closely adjacent lesions in humans after 11 probings of one sandfly (Beach et al., 1984). A single infected sandfly probed 26 times in an area 2 cm in diameter on the arm of a volunteer; 11 small cutaneous lesions resulted, indicating transmission of parasites (Killick-Kendrick et al., 1985). Individual evaluation of the number of probings, the occurrence of blood ingestion and the colonization of the different intestinal regions by Leishmania showed that uninfected sandflies and those infected with Leish. major, in which the infection was limited to the midgut, engorged in less than 10min after the first or second probing. Sandflies in which the established infection had

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proceeded to the cibarium region of the foregut probed at least three timesin most cases more often-and took only a little or no blood during a period of 15 min or more (Beach e f af., 1985). The mechanism of the action of the parasites is still unknown. The theory of blockage of the foregut has been called in question by Killick-Kendrick et a f .(1977b). They emphasized that “the blockage is probably more apparent than real, since the powerful dilator muscles of the cibarium and pharynx would easily widen the canal” and suggested that parasites might interfere with sensilla in the cibarium. At that time such sensilla were known only from other bloodsucking insects, but later they were indeed described in the proboscises of uninfected sandflies (Killick-Kendrick and Molyneux, 198 1) and their presence in the labrum and the cibarium was suggested by Lewis ( 1 984). In a detailed scanning electron microscopical investigation, Jefferies (1987) described in the cibarium two to five trichoid sensilla with a tapering hair that were not chemoreceptors, but were perhaps mechanoreceptors. In addition to the blockage and the sensilla theory, calculations of the fluid mechanisms of blood flow suggest a third possible mechanism, as they indicate that the attached parasites, especially those in the pharynx, are likely to impair flow (Jefferies et af.,1986). Perhaps this results in an indirect feedback effect on receptor functions in the anterior foregut. In a later publication Killick-Kendrick et a f . ( 1988) described a pharynx blocked by Leish. major and indicated the importance of a gel around the parasites, possibly an excreted factor. This report supports the blockage theory (Killick-Kendrick and Molyneux, 1990).

3.

Tsetse .pies infected with Trypanosoma

Development of the salivarian trypanosomes in tsetse flies varies subgenusspecifically. Some of these species, which are the causative agents of nagana and sleeping sickness, e.g. Tryp. vivax, colonize the mouthparts only, while others develop in the midgut and salivary glands (Hoare, 1972; Molyneux and Ashford, 1983). Results of investigations of the feeding behaviour of tsetse flies are contradictory. Compared to uninfected flies, fewer flies infected with Tryp. hrucei fed at the first probe and about two to three more probes occurred before blood ingestion. In addition, the infected flies seemed to be more voracious (Jenni et al., 1980). Also, tsetse flies infected with Tryp. congolense probed significantly more times than uninfected flies, and-as in the case of Leishmania infections-probing alone was sufficient to infect mammals (Roberts, 1981). These results seem to explain the observation that natural infection rates are much lower in tsetse flies than in mammals. However, in the studies by Moloo’s group most aspects of feeding (e.g. number of probes, ingestion

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rate, volume of ingested blood) of Glossina morsitans morsitans, G . m. centralis or G . palpalis gambiensis fed on mice, rabbits or goats were not affected by infections with Tryp. congolense, Tryp. vivax or Tryp. brucei (Moloo, 1983; Moloo and Dar, 1985; Makumi and Moloo, 1991). Searching for explanations for these effects, the early studies of sandflies infected with Leishmania prompted similar studies in tsetse flies. Scanning electron microscopy demonstrated heavy colonization of the labrum and a close association of Tryp. congolense with mechanoreceptive sensilla which act as fluid flow meters (Molyneux et al., 1979). In addition, Tryp. brucei and Tryp. vivax also attach to the bases of the sensilla, and sensilla hairs are entangled in rosettes of flagellates, but the colonization density of Tryp. brucei in the labrum is remarkably low compared with that of the other two species (Molyneux, 1980a; Molyneux and Jenni, 1981) (Fig. la,b). Comparing development of laboratory and natural infections with Tryp. congolense and Tryp. vivax in the cibarium of Glossina, Tryp. congolense infections also tended to be heavier (Jefferies et al., 1987). A compact layer of Tryp. congolense in the labrum was also evident by light microscopy (Ladikpo and Seureau, 1988) and transmission electron microscopy (ThCvenaz and Hecker, 1980). In the latter study, hemidesmosome-like plaques in the enlargements of flagella at the bases of the receptors indicated firm attachment of parasites (Fig. Ic). Calculation of the effect of the colonization on blood flow in labrum and hypopharynx indicated that the reduced diameter would strongly affect the blood flow and increase the pressure required to expel saliva (Molyneux, 1980a). The frequency or capacity of the cibarial pump cannot be increased without limit, since increased viscosity of the blood meal in membrane feeding experiments reduced the rate of feeding (Jenni et al., 1980). The reduced rate of feeding can be compensated by a longer feeding period. This has been observed in tsetse flies infected with Tryp. congolense (Roberts, 1981). Since mammals normally attempt to repel a probing tsetse fly, the increase of feeding time increases the chance of feeding being interrupted. At first the data indicated that the discrepancies between investigations of the effects on feeding behaviour could be due to different colonization densities. As stated by Molyneux and Jefferies (l986), Jefferies also found no effects in his PhD thesis research, but only small areas of the labrum were colonized by Tryp. congolense and Tryp. vivax and not the region with the sensilla. 'However, in the most recent investigation by Moloo's group, rosettes of Tryp. vivax were reported to be present in the labrum (Makumi and Moloo, 1991). Further detailed studies with natural infections or fresh strains of parasite and insect host are necessary, including determination of the colonization densities in different regions of the foregut. The mechanism of action of salivarian trypanosomes could thus be further elucidated. So far, the dense

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colonization of the foregut and/or the interference with the sensilla seem to be responsible for the altered feeding behaviour of infected flies (Livesey et al., 1980). However, pathological effects on the salivary glands should also be considered. Such effects occur in infected Gfossina(see Section 1II.E) and it has been suggested that they are responsible for affecting the feeding behaviour of mosquitoes infected with malaria (Rossignol et af., 1986). 4.

Triatornines infected with Trypanosoma

Effects on feeding behaviour are also known to occur with triatomines after infection with Tryp. cruzi or Tryp. rangeli (D’Alessandro and Mandel, 1969; Aiiez and East, 1984). If uninfected larvae and adults and those which are naturally infected with Tryp. rangeli and/or Tryp. cruzi were given an opportunity to feed on mice, infected larvae fed less frequently (the difference was statistically significant) than uninfected larvae (D’Alessandro and Mandel, 1969). This phenomenon was also evident with infected adults, but the difference was not statistically significant from those infected with Tryp. cruzi. Probing behaviour of R. robustus and R . prolixus infected with Tryp. rangefi was also affected (Aiiez and East, 1984): whereas uninfected bugs probed on average twice before engorging (range 1-5 probes), infected bugs probed on average 13 times (range 2-28 probes) and for longer periods than uninfected ones. Some of the infected bugs ingested only small amounts or no blood at all, one of them even after 28 probes. The mechanisms of these disturbances, e.g. the colonization of the foregut, have not been elucidated. Effects on the salivary gland have to be considered, because their cells can be damaged or destroyed (Schwarzenbach, 1987) (see Section 1II.E). In addition, more features have to be included to elucidate the action of the trypanosomatids on bugs. In a series of investigations of the effects of starvation on trypanosomatid-triatomine interactions (Schaub and Boker, 1986b; Schaub, 1988b, 1990d, 1991; Schaub and Losch, 1989; Schaub et al., 1989a), we had the impression that prolonged starvation affected the volume of ingested blood. In our most recent study of the feeding behaviour of FIG. 1. Sensilla (arrow heads) in the labrum of Glossina morsitans morsitans associated with Trypanosoma parasites (P) (a, b: scanning electron micrographs; c: transmission electron micrograph). (a) Trypanosoma (Trypanozoon) brucei. Bar = 5 pm. (b, c) Trypanosoma (Nannomonas) congolense. (b) Bar = 2 pm. (c) Hemidesmosomal plaques (arrowheads) are present in the attachment zone of parasites to the cuticle (C) of the labrum, and to the basal cup (B) and stalk (S) of a sensillum. Bar = I pm. (Fig. la, b reproduced by permission from Molyneux and Jenni, 1981, Transactions of the Royal Society of Tropical Medicine and Hygiene 75, 160-163, and Fig. lc reproduced by permission from Thevenaz and Hecker, 1980, Acta Tropica 37, 163-175.)

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uninfected first instars of Tri. infestans with different starvation periods, young and old first instar bugs probed more often, and especially after longterm starvation they ingested less or no blood (G. A. Schaub, unpublished observations). Therefore, the effects described with infected bugs can be explained by a competition of trypanosomatids and insect host for the ingested food and an earlier hunger response of infected bugs. This effect could be elucidated by providing a constant food supply and making a nonstop videotape recording of behaviour. 111. DISTURBANCES IN ORGAN A.

SYSTEMS

DISTURBANCES OF THE DIGESTIVE TRACT

In many trypanosomatid-insect systems, e.g. phlebotomines infected with Leishmania, different regions of the host’s digestive tract are colonized by different species (Molyneux and Ashford, 1983). Other flagellates like Tryp. melophagium, Tryp. cruzi or B. triatomae are prevalent in all regions of the intestine (Molyneux, 1975; Molyneux et al., 1978; Schaub and Boker, 1986a; Schaub, 1989a; Jensen et al., 1990; Schaub et al., in press b). Often “carpets” of flagellates cover the intestinal wall (e.g. see Paillot, 1933; Anderson, J. R. and Ayala, 1968; Hoare, 1972; Molyneux et al., 1978; Mehlhorn et al., 1979; Jensen et al., 1990; Schaub et al., in press b) (Fig. 2). A presumed trypanosome of bats (Williams, 1976) and Leish. donovanican even completely block the lumen of the posterior intestine or the pharynx, respectively, with a solid plug of parasites, thereby also distending the oesophagus (see Section 1I.B). The intense colonization of the intestinal tract must, presumably, interfere with the normal function of this organ system. However, even invasion of the haemocoele does not necessarily affect the function (Smirnoff and Lipa, 1970); larvae of the jack pine sawfly, Neodiprion swainei, naturally infected with H. swainei, are normal with respect to movement, appetite and digestion. Only in isolated cases have trypanosomatid-induced disturbances of digestion been observed: in wild-caught sandflies, in which cardia, stomach and hindgut are colonized by different species of trypanosomatids, e.g. of toads and lizards, tbe period of blood digestion in the stomach is sometimes increased (Ayala, 1971, 1973). The opposite effect, more rapid digestion, seemed to occur in phlebotomines which were naturally infected, presumably with a bat trypanosome (Williams, 1976). In the experimental vector Aedes aegypti, infections with Tryp. avium may slightly accelerate the rate of erythrocyte breakdown, thereby favouring the development of the parasite which multiplies only in the digested, initially peripheral regions of the blood meal (Bennett, 1970b). Perhaps one of these two phenomena explains the

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observation that large, round, refractile globules or granules in the cytoplasm of the midgut wall of wild-caught sandflies indicated infections with Leishmania (Johnson et al., 1963). A homoxenous trypanosomatid, Lept. pyrrhocoris, attaches to the intestinal wall of the hemipteran Pyr. upterus and sometimes has been found in the salivary glands and the haemocoele. If the parasites are restricted to the gut, fluid faeces are deposited (Lipa, 1963). In tsetse flies heavily parasitized by Tryp. congolense, the gut seems to be affected, since it broke more easily during dissection than it did in uninfected flies (Kaddu and Mutinga, 1983).

FIG. 2. Transmission electron micrographs showing dense colonization of the intestinal tract of Triatoma infestans by Blastocrithidia triatomae. Bar = 2 pm. (a) Small intestine. Flagellopodia (arrow) or flagella (arrowhead) anchor the parasites in the microvillar border. (Reproduced by permission of Gustav Fischer Verlag from Schaub et al., in press b, European Journal of Protistology.) (b) Hindgut. Flagellar enlargements (arrow) anchor the parasites to the cuticular lining.

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1. Efects in the foregut

Whereas in different trypanosomatid-vector systems the feeding behaviour is affected by the colonization of the foregut (see Section II.B), ultrastructural alterations of the foregut have been reported for Phl. papatasi infected with Leish. major only (Schlein et al., 1991). In an established infection the parasites concentrate in the cardiac valve region which loses its cuticular lining. The cuticle seems to be digested by enzymes which are secreted by the parasites: in cultures in vitro three Leishmania spp. secreted two enzymes, chitinase and N-acetylglucosaminidase.In addition to the cuticle the underlying epithelial cells also appeared to be damaged (Schlein et al., 1991). 2. Efects in the midgut An effect on digestive enzymes has been reported by Schlein’s group only (Schlein and Romano, 1986; Borovsky and Schlein, 1987). The initially reduced proteolytic activity of gut homogenates from Phl. papatasi infected with Leish. major indicates that the flagellates may inhibit enzyme production of the sandflies, presumably by the release of glycoconjugates which are also released into the supernatant of cultures in vitro. If such glycoconjugates are fed to sandflies they delay the digestion of infective meals (Schlein et al., 1990). Modulation of the digestive enzymes seems to be an important mechanism, determining the susceptibility of a sandfly for a species of Leishmania. Whereas Tryp. cruzi does not affect haemoglobin crystallization in the stomach (Pick, 1952), two other trypanosomatids of triatomines, Tryp. rangeli and B. triatomae, strongly affect the midgut. Several effects are evident in R . prolixus infected with Tryp. rangeli. After penetration of the gut wall, parasites invade the gut muscles to multiply and haemocytes accumulate at these sites (Watkins, 1971a). Depending on the intensity of infection, only some or many parasitized muscle cells degenerate, and eventually the gut cells are lysed. Thereby, in moderate infections-indicated by only a slight increase of haemolymph-bugs continue to suck blood, but the gut of larvae, not of adults, may burst. In heavy infections with a great increase of haemolymph, gut peristalsis is reduced or absent, perhaps because of insufficient stimuli from damaged nerves. These bugs neither excrete nor feed. Most of these effects also occur in larvae with blocked abdominal spiracles (Watkins, 1971a) (see Section VII). More obvious effects can be observed in several species of triatomine bugs infected with B. triatomae. Beakers containing infected Tri. infestans, Tri. sordida and Dipetalogaster maxima often contain blood-red faecal drops,

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compared with the normal white, yellow or dark brown drops (Schaub, 1988a; Schaub and Breger, 1988; Schaub and Meiser, 1990; Jensen et al., 1990). Whereas in uninfected bugs the onset of digestion of haemoglobin at the anterior end of the small intestine coincides with a colour change to brown, infected bugs regularly have red contents in the dilated small intestine (Schaub and Meiser, 1990). Interestingly, none of the bugs which die of starvation has red intestinal contents (Schaub and Losch, 1989). Occasionally haemolymph of Tri. infestans infected with B. triatomae is a light red colour (Schaub, 1988a, 1990a; Schaub and Meiser, 1990; Jensen et al., 1990). Similar effects in sheep keds infected with Tryp. melophagium were later shown to be caused by experimental conditions resulting in blockage of the spiracles (Nelson, 1956, 1981; Hoare, 1972). Whereas red rectal fluid is usually deposited by healthy Anopheles stephensi (Briegel and Rezzonico, 1985), in triatomines both reddening phenomena indicate a disturbance of the function of the intestine. By starch gel electrophoresis, the posterior intestines and the haemolymph of these bugs were shown to possess proteins with the same migration behaviour as marker haemoglobin (Schaub and Meiser, 1990). In additional photometric measurements of the contents of different regions of the intestine, absorption spectra of red stomach contents of infected and uninfected Tri. infestans, and also of the red contents of the posterior small intestine of infected bugs, showed the two typical haemoglobin maxima, whereas brown contents showed neither of these maxima (G. A. Schaub, unpublished observations). These data support the interpretation that ingested blood is not fully digested in bugs infected with B. triatomae. What is the mechanism of these disturbances in bugs infected with B. triatomae? Valuable indications are offered by an ultrastructural study in which we detected sequential steps of the damage process to the functional subunits of the midgut, which are the extracellular membrane layers (acting like the peritrophic membranes in other insects), the microvilli and the epithelial cells (Figs2a, 3). These subunits are also affected by other trypanosomatids. ( a ) Membrane systems. Usually peritrophic membranes or extracellular membrane layers act as a barrier to parasites and provide microenvironments for different digestive enzymes (Peters, W., 1982). Some salivarian trypanosomes can penetrate the peritrophic membranes (Evans and Ellis, 1983), and in tsetse flies infected with Tryp. congolense, and also in sandflies infected with Leish. aethiopica, the ultrastructure of the peritrophic membranes seems to be disturbed (Kaddu and Mutinga, 1981, 1983). Whereas in uninfected Phl. papatasi the peritrophic membranes disintegrate at the posterior end, in specimens infected with Leish. major the chitin layer is also

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lysed in the anterior region (Schlein et al., 1991). These observations might be explained by the secretion of the two enzymes, chitinase and Nacetylglucosaminidase, which are secreted in cultures in vitro by different trypanosomatids (Schlein et al., 1991).

(4

(b)

FIG. 3. Transmission electron micrographs of sections of small intestine of Triatoma infestans infected with Blastocrithidia triatomae, showing different types of pathology. Bar = 2 p.(a) Cell with reduced microvilli. (b) Lysed epithelial cell with parasites. The cell on the basal lamina (arrowhead) is vacuolated. (Reproduced by permission of Cambridge University Press from Jensen et al., 1990, Parasitology 100, 1-9.)

The barrier function of the peritrophic membranes is evident.inthe speciesdependent establishment of Leishmania in phlebotomines, in which the peritrophic membranes either disintegrate or remain intact, thus allowing or preventing colonization (Feng, 1951), and also in their role in determining whether early infections of other trypanosomatids are restricted to the endoperitrophic space (e.g. Mungomba et al., 1989). Whereas Lept. lygaei in the bug Lygaeus pandurus is closely associated with the extracellular membrane layers, but not attached to the midgut epithelium, B. familiaris in the

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same host attaches only to microvilli free of extracellular membrane layers (Tieszen et al., 1986, 1989). In contrast, in Tri. infestans infected with B. triatomae, flagella can cross the extracellular membrane layers and reach the microvilli, and often these layers are lacking (Mehlhorn et al., 1979; Jensen et al., 1990; Schaub er al., in press b) (Fig. 2a). Their absence is not caused by the feeding state. Whereas in starving adult R. prolixus extracellular membrane layers are not present on the microvilli but develop after a blood meal (Billingsley and Downe, 1986; Billingsley, 1990), in the larvae of R. prolixus and Tri. infestans the developmental phases of the extracellular membrane layers are not so strictly separated (Bauer, 1981; Jensen et al., 1990). In contrast to uninfected bugs, in the intestines of those infected with B. triatomae, fed and unfed, the extent of regions without extracellular membrane layers is increased (Jensen et al., 1990). Because of the variation in the production of the extracellular membrane layers, we cannot ascertain if the layers are destroyed by B. triatomae or if their production is disrupted. The precursor, the double apical membrane, is developed below attached flagella, a phenomenon not observed in B. familiaris (Tieszen et al., 1986). Since these layers and membranes normally serve to keep separate the different digestive enzymes (Billingsley and Downe, 1988; Ferreira et al., 1988), digestion of haemoglobin is likely to be disturbed. ( b ) Microvilli. The apical microvilli, the second functional subunit of the midgut, are also affected by different trypanosomatids. Their height seems to be reduced in phlebotomines infected with Leish. amazonensis (Molyneux er al., 1986a). Microvilli in the midgut of G. pallidipes infected with Tryp. congolense are poorly developed or can be totally reduced (Kaddu and Mutinga, 1983), as is also found in water-striders infected with B. gerridis (Tieszen et al., 1983). Progressive reduction in height and number of microvilli occurs in the small intestine and the stomach of Tri. infestans infected with B. triatomae (Jensen et al., 1990; Schaub et al., in press b) (Fig. 3a). These effects are not caused by the direct attachment of the parasites, since densely colonized regions can possess well-developed microvilli, and in some microvilli-free regions no parasites are attached. ( c ) Intestinal cells. Not only the apical microvilli but also the body of the intestin'al cells can be affected by the flagellates. One group of trypanosomatids frequently destroys the cells if they are invaded for intracellular multiplication. For example, only a mere membrane remains from the stomach cells of the rat flea invaded by Tryp. lewisi after multiplication of the trypanosome (Wenyon, 1926). Members of a second group of trypanosomatids penetrate the midgut cellS

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only during invasion of the haemocoele. The penetration of Tryp. rangeli does not occur intercellularly, but only via an intracellular route, even through the nucleus of a cell of the intestinal wall (Schwarzenbach, 1987; Hecker et al., 1990). The intracellular parasites are surrounded by a membrane, presumably derived from the host, which remains around the parasite together with a portion of extruded cytoplasm as the trypanosome penetrates the basal lamina. The penetration pores in the cell membrane and the basal lamina are repaired, but can be recognized by their unstructured cytoplasm (Hecker et al., 1990). There are no ultrastructural differences between infected and uninfected cells of infected bugs or intestinal cells of uninfected bugs, even with high parasite densities in single cells. No ultrastructural data are available for other flagellates of this group. In the third group of trypanosomatids, the parasites normally insert only their flagella into the epithelial cells, sometimes occur intracellularly and occasionally invade the haemocoele. All three phenomena have been observed in different species of Leishmania in phlebotomines, both experimentally and in natural infections (Adler and Theodor, 1929; Adler and Ber, 1941; Killick-Kendrick et al., 1974, 1977a; Molyneux et al., 1975; Kaddu and Mutinga, 1981; Molyneux and Killick-Kendrick, 1987), but it is unknown whether or not the parasites are killed after invasion (see Section 1II.C.I). There sqems to be an effect on the host cell in sandflies infected with Leish. amazonensis (Killick-Kendrick et al., 1974), and midgut cells of Ornithomyia avicularia invaded by Tryp. corvi have vesiculated endoplasmic reticulum (Mungomba er al., 1989). The host cells are also affected in tsetse flies infected with Tryp. congolense (Kaddu and Mutinga, 1983). However, in a morphometric study of the midgut of tsetse flies infected with Tryp. brucei only one of 12 features (relative volume of lysosomes) was significantly affected, and therefore the authors stated that “cellular functions do not seem to be strongly impaired” (Hecker and Moloo, 1981). This third group also contains homoxenous trypanosomatids. Sometimes Lept. pyraustae invades the haemocoele of corn borer larvae, but the light colour of the intestinal epithelium when observed under the microscope is normal (Paillot, 1933). In larvae of eye gnats (Diptera) infected with H. muscarum, invasion of the haemocoele occurs in about half of the host population. Ultrastructural appearance of organelles indicates that the cells penetrated by parasites are not affected. The penetration results in a bacterial septicaemia which kills the flagellates and the host larvae. Whereas midgut epithelium is relatively intact in heavy infections of the intestinal tract, its degeneration is evident after development of heavy haemocoelic infections (Bailey and Brooks, 1972a). In Muscidae, H. muscarum colonizes the intestine (Wallace, 1979) and is usually non-pathogenic. However, in moribund and dead Musca domestica larvae, large numbers of flagellates

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may occur in the haemocoele, indicating that this trypanosomatid might also be pathogenic to this host under certain conditions (Kramer, 1961). Only insertion of the flagellum occurs in water striders infected with B. gerridis (Tieszen et al., 1983) and in triatomines infected with B. triatomae (Jensen et a[., 1990; Schaub et al., in press b). B. triatomae also inserts its flagellum into the epithelial cells of the Malpighian tubules (Schaub and Schnitker, 1988) and into host cells co-cultivated in vitro (Reduth et al., 1989). Penetration of the intestinal wall of R . prolixus has been postulated by Peng (1979), based on haemocoele infection in five of 16 bugs after experimental rectal infection. However, artefactual damage to the intestinal wall cannot be excluded and, in seven of 16 bugs, B. triatomae was found in the haemocoele 2-1 6 weeks after inoculation into the haemocoele. After infection by coprophagy or feeding in vitro through a membrane, we found no flagellates in the haemocoele of about 50 Tri. infestans (G. A. Schaub and C . Jensen, unpublished observations). Whereas the other trypanosomatids of this group rarely affect the intestinal cells, the cells of midguts colonized by B. triatomae are often vacuolated or lysed (Jensen et al., 1990) (Fig. 3). Thus, the basal lamina is freely accessible to the intestinal contents, and cannot be a barrier to the passage of haemoglobin into the haemolymph. Perhaps the very rare penetration by Tryp. corvi and Leish. major, cited above, is restricted to degenerating cells in weakened insect hosts, i.e. cells which had perhaps been affected before invasion (Mungomba et al., 1989). This could also be the explanation for the intracellular development of Tryp. cruzi in cells of the bug’s intestinal wall (Gomes de Faria and Cruz, 1927) or its penetration and infection of the coelomic cavity (Lacombe, 1980). Also the phenomenon that bacteria were found only in G. m. morsitans infected with Tryp. brucei (see Hecker and Moloo, 1981) might be due to parasitogenic weakening of the insect. The importance of the fitness of the host is also shown by Herpetomonas sp. in Drosophila melanogaster (Lushbaugh et al., 1976): the trypanosome normally develops in the lumen of the intestinal tract, but penetrates the cells of the intestinal wall and multiplies intracellularly if the insect has a concomitant infection with a yeast-like fungus. 3. EfSects in the hindgut The German term “Schorf” [scab] indicates a reaction of bees to infection with C . mellificae (syn. Lept. apis), occurring in the dorsal part of the pylorus and only at its end. However, Lotmar (1946) and Fyg (1954) suggested that this scab material was of flagellate origin. The low colonization density also argues against pathological effects. Number and size of the scabs was affected by the type of food but not by a concomitant infection with Nosema apis (Bahrmann, 1967).

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Another flagellate species in bees-and also other species in different insects such as fleas, water striders or bugs-may cover the hindgut cuticle like a “carpet” (Fyg, 1954; Molyneux and Ashford, 1975; Molyneux et al., 1981; Zeledon ef al., 1977, 1984, 1988; Boker and Schaub, 1984; Tieszen et al.. 1986; Zimmermann et al., 1987; Schaub et al., 1989b; Tieszen and Molyneux, 1989). Trypanosomatids of toads and lizards, and some presumed to infect bats, can multiply so intensively that the hindgut and/or rectal ampulla becomes noticeably distended (Anderson, J. R. and Ayala, 1968; Christensen and Telford, 1972; Williams, 1976), and masses of Tryp. lewisi practically block the hindgut of the flea (Garnham, 1955). However, no cytopathological effect on the rectal ampullae of fleas infected with Leptomonas was observed (Molyneux et al., 1981); only a slight reaction of the host at the attachment site seems to occur (Molyneux and Ashford, 1975). Often the parasites prefer a specialized region, the so-called rectal glands or rectal pads (Fig. 4a). Investigating the course of colonization in the rectum of triatomines by Tryp. cruzi and B. triatomae with the scanning electron microscope, we found that this region is preferred by both species after initiation of the rectal infection, and it continues to be more densely colonized (Boker and Schaub, 1984; Schaub and Boker, 1986a,b, 1987; Schaub and Losch, 1988). In B. triatomae infections, about five interdigitated layers of flagellates cover the rectal pads (Fig.4b). Even after longterm starvation, parasites first detach from the other regions of the rectum and last-shortly before and after death of the host-from the rectal pads (Schaub and Boker, 1986b; Schaub and Losch, 1989). In a variety of different insects the rectal pads seem to be involved in water uptake, but amino acids are also absorbed from the rectal lumen (Wall and Oschmann. 1975). The intense colonization must presumably interfere with the function of the rectum or the rectal pads, a theory already proposed by Lipa (1963) and by Molyneux and Ashford (1975) for infections of fleas, and by Laugi and Nishioka (1977) for Lept. oncopelti infections of lygaeid bugs. In the latter, some slight damage seems to occur since detached flagellates carry the outer part of the epicuticle of the rectal pads with them, necessitating constant replenishment of the epicuticle. This might be explained by the secretion of a chitinase and N-acetylglucosaminidase which were found in supernatants of Leptomonas, Herpetomonas and Crithidia cultures in vitro (Schlein et a!., 1991), all species which attach to the rectal cuticle. B.

DISTURBANCES OF THE MALPIGHIAN TUBULES

Malpighian tubules are colonized by a large number of species of heteroxenous and homoxenous trypanosomatids, e.g. Leishmania and Tryp. theca-

FIG.4. Scanning electron micrographs of the anterior rectal wall of Triatoma infestans. Bar = 100 pm. (a) The rectum of an uninfected bug shows the different cuticular structure of regions A-D. Region A is located around the exit of the midgut/ hindgut from which the processes of the ampullae cells are extended into the rectal lumen. The rectal pads (zone B) are clearly separated from the narrow region C and from region D, the main part of the rectal wall. (Reproduced by permission of Springer Verlag, Heidelberg, from Boker and Schaub, 1984, Zeitschrijt fiir Purasitenkunde 70,459469.) (b) In an established infection of Blastocrithidia triatomae the cuticle is totally covered by a “carpet of flagellates”. (Reproduced by permission of the Society of Protozoologists from Schaub and Boker, 1986, Journal of Protozoology 33, 266270.)

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dactyli in sandflies (Christensen and Telford, 1972; Kaddu and Mutinga, 1984; Range1 et al., 1985), Tryp. cruzi in triatomines (summarized by Schaub and Losch, 1988), H. ampelophilae in Drosophila (Rowton et al., 1981), Lept. pulexsimulantis in fleas (Beard et al., 1989), nearly all species of Rhynchoidomonas in Diptera (Wallace, 1966, 1979), and C.Jlexonema in water striders (Tieszen and Molyneux, 1989). This last species invades the host cells without apparent effects (Tieszen and Molyneux, 1989), whereas Tryp. avium appears to destroy the tubules of tabanids (Bennett, 1970b). In many parasite-insect systems the Malpighian tubules are sometimes slightly hypertrophied; additionally, Malpighian tubules in corn borer larvae infected with Lept. pyraustae are greyish in colour (Paillot, 1927), and in sandflies infected with Endotrypanum schaudinni and Tri. infestans infected with B. triatomae they are highly refractile (Shaw, 1981; Schaub and Schnitker, 1988). Occasionally, in R . prolixus infected with Tryp. rangeli, the diameter of localized areas of the Malpighian tubules is reduced to less than half the normal, while other areas lpve expanded to twice their normal diameter (Watkins, 1971b). Only in the last two trypanosomatid-triatomine systems and in Tri. sordida and D . maxima infected with B. triatomae has an increased volume of haemolymph and/or a reduced excretion rate been observed (Grewal, 1957, 1969; Watkins, 1971b; Schaub, 1988a, 1990a; Schaub and Breger, 1988; Schaub and Schnitker, 1988; Schnitker et al., 1988); Tryp. rangeli and B. triatomae infections have been studied in detail. In R . prolixus infected with Tryp. rangeli, gut infections affect the excretion of various developmental stages differently (Watkins, 1971a,b). One month after infection the excretion rate of fifth instar larvae is reduced by 27%, and after a further month of infection the reduction in females and males is 53% and 6%, respectively. However, 2 months after haemocoelic inoculation the excretion rate of females and males is reduced by 58% and 99%, respectively. Light microscopy demonstrates effects on the apical microvillar border and the basal lamina and sometimes blockage of the lumen of the Malpighian tubules by the uratic spheres. Compared to uninfected bugs, the cytoplasm is coarsely granular and often contains necrotic areas. Watkins (1971b) measured the diuresis in vitro of the entire preparation of Malpighian tubules (free of tracheoles) and the hindgut. In some experiments mesometathoracic ganglia were added (Watkins, 1969) as a source of the diuretic hormone (Maddrell, 1980). Experiments with various combinations of haemolymph and Malpighian tubules of infected and uninfected bugs showed that tissue damage caused by Tryp. rangeli resulted in a reduced secretion rate, even with normal haemolymph. In addition, the ganglia from infected bugs did not contain enough diuretic hormone to increase the secretion in Malpighian tubules from uninfected bugs. This lack of diuretic hormone, or the presence of a chemical inhibitor in the haemo-

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lymph, decreased the secretion of Malpighian tubules of uninfected bugs maintained in the haemolymph of infected specimens. The effect of B. triatomae on bugs is shown by a swollen abdomen, even some days after bloodsucking. During the first 24 h after feeding, fifth instars of Tri. infestans infected with B. triatomae excreted approximately 2.5 times less urine (Schnitker et al., 1988). Even during dissection of long-term infected bugs alterations are conspicuous in the upper region of the Malpighian tubules, which are quite rigid and slightly widened, sometimes having localized conspicuous swellings. The cells are filled with white concretions and strong autofluorescence is evident with the fluorescence microscope. Using the transmission electron microscope the cells of the slightly widened region can be seen to possess many more concretions than normal; the extremely swollen parts of the tubules show a reduction in the number of basal cell interdigitations, mitochondria and microvilli, and the concretions are much larger (Fig. 5). Normally, mitochondria and microvilli are essential structures for fluid secretion by Malpighian tubules (Bradley, 1983). To clarify whether or not the ultrastructural alterations, especially the increased concretions, could be responsible for the dysfunction, we measured the secretion rate of isolated Malpighian tubules. Surprisingly, secretion rates of all isolated tubules were nearly identical. In addition, the storage and release of diuretic hormone in infected bugs was sufficient to induce normal secretion rates by Malpighian tubules of uninfected bugs. These tubules also secreted normally when maintained in the haemolymph of infected bugs and, therefore, no chemical inhibitor can be present. What then might be the cause of disturbed excretion in infected bugs? One possibility is that infection affects the ampullae or rectal reabsorption. However, I emphasize the reduced tracheal system in infected bugs. During measurements of isolated tubules in vitro, oxygen supply is guaranteed, but this is probably not so in vivo (Schaub and Schnitker, 1988). The importance of oxygen supply is also indicated by the experiments of Watkins (1971b) using bugs with blocked spiracles. Pathological effects were very similar to those observed in bugs infected with Tryp. rangeli (see Section VII). Whereas Tryp. rangeli develops inside the tracheal cells and destroys them (Watkins, 1971a,b; Schwarzenbach, 1987), B. triatomae could act only indirectly on the development of the trachea. C.

EFFECTS ON THE HAEMOLYMPH

Obvious effects of parasitization by trypanosomatids on the haemolymph are only rarely reported. In R. prolixus infected with Tryp. rangeli, the haemolymph is whitish and more copious in heavily infected bugs (Grewal, 1969), and in Pyr. apterus infected with Lept. pyrrhocoris the haemolymph is thicker and whitish instead of the normal light green (Lipa, 1963).

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FIG. 5. Diagrammatic representation of the Malpighian tubule wall of Triatoma infestans. Bar = 2pm. (a) Uninfected tubule; (b) slightly swollen region and (c) extremely swollen region of tubules of bugs infected with Blastocrithidia triatomae. b, Basal lamina; c, concretions; i, electron dense inclusions; I, lumen of tubule; m, mitochondria; v, layered vesicles. (Reproduced by permission of Springer Verlag, Heidelberg, from Schaub and Schnitker, 1988, Parasitology Research 75, 88-97.)

1.

Eflects on the immune response

Insects possess a cellular and a humoral immune response. The inoculation of non-virulent bacteria or fungi into the haemocoele induces a strong humoral response, protecting against subsequent inoculations with virulent species (Gotz and Boman, 1985). Immune proteins also appear in the haemolymph of tsetse flies inoculated with bacteria, but not after inoculation of Tryp. hrucei, which additionally does not induce phagocytosis or encapsulation by haemocytes (Kaaya et al., 1986). However, an anti-trypanosomal factor is already present in the haemolymph before inoculation, which specifically destroys Tryp. congolense, Tryp. vivax and Tryp. brucei but not Leish. hertigi or C . fasciculata (reviewed by Molyneux et al., 1986b; Kaaya, 1989). The haemolymph of locusts and cockroaches, used as model systems, aggluti-

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nates Tryp. brucei and Leish. hertigi in vitro, and the agglutinin titres are increased by a prior inoculation of either trypanosomatid into the haemocoele (Ingram et al., 1984). After injection of Tryp. rangeli into the haemocoele of Tri. infestans or R . prolixus, the number of phagocytic cells increases greatly (Zeledon and de Monge, 1966). Uninfected Tri. infestans already possess more than twice as many haemocytes than R. prolixus, and thereby Tri. infestans can overcome the infection. Some strains can also be controlled by R. prolixus and nearly all by Tri. infestans (Zeledon and Blanco, 1965; D’Alessandro, 1976). Whereas Tryp. rangeli multiplies inside the phagocytic haemocytes after inoculation into the haemocoele, Tryp. cruzi is killed by the haemocytes of R. prolixus (Tobie, 1968, 1970). Initially, the number of haemocytes increases in R. prolixus, but to differing extents for the various haemocytic cells (Gomez, 1967). Since all types of haemocytes are parasitized by Tryp. rangeli (Schwarzenbach, 1987), their number is considerably reduced in old and heavy infections (Grewal, 1957). The prophenoloxidase system is not activated by Tryp. rangeli in R . prolixus or in Tri. infestans. Since the intensity of this immune response is lowered if the parasites are incubated together with a microbially derived molecule, which normally activates the prophenoloxidase system, it was suggested that the susceptibility of R. prolixus might be explained, at least in part, by immune suppression. In the tissues of the refractile Tri. infestans, agglutinating and trypanolytic factors seem to be more widely distributed than in those of R. prolixus (Gregorio and Ratcliffe, 1991a,b). The haemolymph of bugs infected with Tryp. cruzi has a normal appearance, but implantation experiments indicate a strongly reduced cellular immune response of infected Tri. infestans (Bitkowska et al., 1982). Since the fluid from cultures in vitro caused similar effects, the authors suggested that some parasites may develop in the haemocoele after suppression of the host’s immune reactions. However, in our Tryp. cruzi-Tri. infestans system the cellular encapsulation of pieces of nylon thread seemed to be identical in infected and uninfected larvae (G. A. Schaub, unpublished observations), but in bugs infected with B. triatomae, the cellular encapsulation and melanization reactions were almost totally inhibited (G. A. Schaub, unpublished observations). The latter effect may be due to the decreased concentration of amino acids used for melanization (see Section 1II.D). 2. Eflects on chemical composition

The effects of trypanosomes on metabolites in the insect’s haemolymph have been investigated only for triatomines infected with Tryp. cruzi, Tryp. rangeli and B. triatomae (Zeledon and de Monge, 1966; Ormerod, 1967; Watkins,

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1969; Donandt, 1982; Schaub et al., 1990b). In the investigation by Zeledon and de Monge (1966), the total concentration of free amino acids decreased by 27% at 5-6 days after inoculation of Tryp. rangeli into the haemocoele of R. prolixus, while it increased by 66% in uninfected bugs. Concentrations of total proteins and carbohydrates also decreased in these infected bugs. In Tri. infestans slight alterations occurred (Zeledon and de Monge, 1966). In all the other studies cited above, concentrations of individual amino acids were determined. Watkins (1969) and Donandt (1982) used the semiquantitative thin layer chromatography technique, whereas Ormerod ( 1967) and Schaub et al. (l990b) used ion-exchange chromatography; only the last method allowed analyses of the haemolymph from individual bugs by a sensitive fluorescence detection system after post-column derivatization of amino acids with o-phthaldialdehyde. Ormerod (1967) compared the effects of a slightly and a highly virulent strain of Tryp. rangeli on R . prolixus. Despite the difficulties of comparing the data for infected and uninfected bugs (discussed by Schaub et al., 1990b), some results were obvious. The less virulent strain produced a large increase in the concentration per bug of aspartic acid and taurine in short- and longterm infected bugs, and a 10-fold increase of isoleucine in those long-term infected bugs which survived but did not moult. In infections with the lethal strain, concentrations of alanine, glycine and isoleucine increased 10-fold, and those of taurine and aspartate 100- and 500-fold, respectively. Furthermore, concentrations of tyrosine, phenylalanine and lysine were below the level of detection. Gut infections of R . prolixus with Tryp. cruzi greatly decreased the concentrations of cysteic acid and histidine in the haemolymph of late instar larvae (Watkins, 1969). Infections with Tryp. rangeli also decreased the concentrations of leucine, phenylalanine and serine. In female bugs, concentrations of arginine and tyrosine were decreased by Tryp. ranxeli infection and additionally, that of proline was increased by Tryp. cruzi. After inoculation of Tryp. cruzi into the haemocoele of R . prolixus, no effect was evident in infected larvae, but decreased concentrations of leucine and valine and increased concentrations of proline, serine and tyrosine were found in adults. Infections with Tryp. rangeli greatly increased concentrations of arginine and proline in larvae and adultsi and decreased those of nearly all the remaining amino acids. Tryp. rangeli develops in the haemocoele of Rhodnius, but is killed $by haemocytes in the haemocoele of other bugs; hence, Tri. phyllosoma, studied by Donandt (1982), is not likely to be greatly affected by Tryp. rangeli. The investigation by Donandt (1982) of short-term effects (up to 4 weeks) found only slight differences between bugs infected with Tryp. cruzi and uninfected bugs, and for most amino acids the infection-induced alterations were slightly greater in bugs infected with Tryp. rangeli. During the first week

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concentrations of alanine, glutamate, leucine/isoleucine, lysine, phenylalanine and serine were slightly reduced in infected bugs, but thereafter they were mainly higher than in uninfected bugs. Concentrations of arginine, asparagine and tyrosine were nearly always higher. No consistent trend can be recognized in these three studies on bugs infected with Tryp. cruzi or Tryp. rangeli, and only the decrease of the concentration of tyrosine in the studies by Ormerod (1967) and Watkins (1969) is noteworthy. In our study we investigated the free amino acids in the haemolymph of uninfected fourth instar larvae of Tri. infestans and in fifth instars 1 day after ecdysis, and of those infected with B. triatomae (Schaub et al., 1990b). About 15 and 21 weeks after infection, concentrations of the majority of amino acids in infected fourth instar larvae were lower than those in the respective uninfected bugs--40-80% lower for methionine, serine, threonine and tyrosine. In fifth instar larvae a similar decrease was obvious for alanine, arginine, histidine and tyrosine, and concentrations of aspartate, cystine/ cysteine and lysine were increased markedly by 130%, 380% and 150%, respectively. The differences between infected and uninfected fifth instars, which were also statistically significant in fourth instars, were undoubtedly due to the effects of B. triatomae the lower values of alanine, arginine, histidine and tyrosine. However, the major alteration induced by infection with B. triatomae was the occurrence of p-alanine in infected fifth instars. instars.

D.

EFFECTS ON THE CUTICLE

Many parasites affect the colour of the cuticle of their hosts. In R. prolixus, Pyr. apterus and Tri. infestans infected with trypanosomatids the cuticle is often paler (Grewal, 1957; Lipa, 1963; Watkins, 1969, 1971a; Schaub, 1988a; Schaub et al., 1990b). However, C . cimbexi, which develops in the haemocoele of the hymenopteran host larvae, causes no apparent alterations of external appearance (or behaviour) of the host larvae (Lipa and Smirnoff, 1971). The translucent and pale cuticle of R. prolixus infected with Tryp. rangeli seems to be caused by the parasite’s multiplication in the epidermal cells (Watkins, 1971a). The pigment granules disappear in heavy haemocoelic infections, and periodically orange-coloured urine is excreted (Watkins, 1969). An effect on pigmentation seems also to be evident in the eyes of infected R. prolixus. However, the fact that about 50% of infected adults have white eyes, while only 0.2% of uninfected adults do so (Watkins, 1969), might also be explained by a survival of tolerant or refractory bugs if the presence of white eyes is a genetic marker.

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In infections with B. triatomae tanning of the cuticle can be totally inhibited (Fig. 6 ) . Usually the resulting pale pink colour is only transiently observed during the first 15 min after moulting, but the infected bug shown in Fig.6 was photographed 2 weeks after the moult. In populations of infected Tri. infestans, all grades from pale to the normal dark brown colour occur. On dissecting infected bugs, we often found that the cuticle was softer (Schaub et al., 1990b).

FIG. 6. Male Triatoma infestans infected with Blastocrithidia triatomae (left) and uninfected (right) 2 weeks after ecdysis. (Reproduced by permission of Pergamon Press from Schaub et al., 1990, Journal of Insect Physiology 36, 843-853.)

In bugs infected with B. triatomae, indirect effects due to intoxication or direct effects on the substrate, enzymes or hormones involved in development of the new cuticle are possible causes of this phenomenon. N-Acetyldopamine, which is made from tyrosine, plays an important role in the process of tanning; therefore, determining the concentration of free amino acids in the haemolymph can indicate whether or not the substrate for tanning is limited in infected bugs (Schaub et al., 1990b) (see Section 1II.C). Such measurement; on haemolymph from individual bugs show great variations in concentrations. However, the greatest variation is found in those five amino acids which are specifically used for development of the new ckicle, e.g. a standard deviation of 84% of the mean value of tyrosine in infected fourth instars. Despite this variation, differences are often statistically significant (including those for tyrosine) if the concentration of amino acids before and after moulting in infected and uninfected bugs is compared.

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Infected bugs before moulting contain lower levels of the amino acids which are incorporated into the cuticle. Unfortunately, this effect can also be caused by retarded development, which is seen normally in infected bugs. Including data from bugs which had not changed their metabolism in preparation for the development of the new cuticle presumably lowered the mean values obtained for infected bugs, e.g. for tyrosine. However, three pieces of indirect evidence support the theory that a lower concentration of tyrosine occurs and is responsible for the reduced tanning. (i) Amino acid analysis of cultures of B. triatomae in vitro indicates that the flagellates may compete with the bug for essential amino acids in the food. (ii) The fat body, which presumably makes or stores most of the amino acids needed for the new cuticle, is greatly reduced in bugs infected with B. triatomae (see Section 1II.E). (iii) The most important indication is that after the moult we could find detectable concentrations of p-alanine and an accumulation of its precursor, aspartate, in infected bugs only. There is increasing evidence that not only N-acetyl-dopamine but also N-P-alanyl-dopamine plays an important role in sclerotization, tanning and melanization. In mutants of Diptera, Lepidoptera and Coleoptera, inhibition of the incorporation of p-alanine prevents tanning and causes intense melanization (discussed by Schaub et al., 1990b). Why did this melanization not occur in our bugs? The failure of tanning seems to occur only if the substrate for melanization is not available. This also is dopamine, which is made from tyrosine. Together, these results strongly indicate that it is a reduced concentration of tyrosine that is responsible for the reduced tanning in infected bugs, and not a reduced oxygen supply due to the reduced tracheal system (see Section 1II.B). However, possible actions on enzymes and .hormones involved in sclerotization and tanning cannot at present be ruled out. E.

OTHER AFFECTED ORGAN SYSTEMS

The outer appearance, but not the tanning of the cuticle, of the hymenopteran Caliroa cerasi is affected by an infection (Carl, 1976; Lipa et al., 1977). The yellow spots on so-called “slug larvae” infected with B. caliroa are caused by an effect on the mucous coating, which dries up and peels off. The cause of the change of the colour of the larvae to dark brown or blackish brown was not identified, but the colour indicates a disruption of the gut during penetration of the flagellate into the haemocoele. An obvious effect on colouration also occurs in the salivary glands of R. prolixus; they are normally pink and become whitish in bugs infected with Tryp. rangeli (Grewal, 1956). This might be caused by the parasites penetrating the cells on their way from the haemocoele into the lumen of the

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G. A. SCHAUB

gland. In cases of severe infection the tissue is damaged and the basal lamina is detached from the gland cells (Schwarzenbach, 1987; Hecker et al., 1990). An opposite effect on colouration occurs with salivary glands of infected tsetse flies, which normally have a chalky appearance, but become brown to black in flies with very old natural infections of Tryp. brucei (Burtt, 1942, 1950), a phenomenon reported only from the Amani region of Tanzania. In experimentally infected flies, the host membrane of the microvilli in the salivary gland shows a clear reaction at the attachment site of Tryp. brucei, a clustered arrangement of intermembranous particles (Vickerman et al., 1988). Salivary glands of uninfected G. m. morsitans when dissected into saline display sinuous motility, which is not seen with glands heavily infected by Tryp. brucei (Golder et al., 1987). These changes coincide with considerable alterations of the composition of the secretion, e.g. reduced cholinesterase activity (Patel et al., 1982; Golder et al., 1987), which might be the cause of the reduced feeding behaviour of infected flies (see Section II.B.3). In three host-parasite systems in which the parasite is highly virulent, the fat body is considerably reduced (Smirnoff and Lipa, 1970; Watkins, 1971a; Schaub et al., 1990b). This might explain the retarded development of sawfly larvae infected with H. swainei, bugs infected with B. triatomae and R . prolixus infected with Tryp. rangeli. Because the concentration of metabolites concerned with moulting does not increase above the critical level, the hormonal induction of moult is not initiated (see Section 1II.D). Since Tryp. rangeli invades the haemocoele and develops intracellularly in all organs, they are all affected by the flagellate. In addition to the gut cells, Malpighian tubules, haemocytes, cuticle, tracheal and epidermal cells, salivary glands and fat body, all discussed in earlier sections, Tryp. rangeli damages the nervous system of R. prolixus (Watkins, 1969, 1971b; D’Alessandro, 1976).

ON Iv. EFFECTS A.

,

PRE-ADULT

DEVELOPMENT AND MORTALITY

TR YPANOSOMA INFECTIONS OF TRIATOMINAE

There is only one report of adverse effects of Tryp. cruzi on the larval developmental times of the pre-adult stages of triatomines (Reis dos Santos and Lacombe, 1985). However, the retarded development of infected bugs might be explained by their having been maintained in isolation (see Section V1.C) or it might have been unique to the Tryp. cruzi-bug system used in that study (reviewed by Schaub, 1989b). Such effects did not occur in my system (Schaub, 1988c,d), and Juarez (1970) also reported no adverse effect

EFFECTS OF TRYPANOSOMATIDS ON INSECTS

283

of Tryp. cruzi. Mortality rates also seemed to be unaffected (Schaub, 1988~). In a contrary example, cited by Kramer (1963), the single dead bug in which the body fluid contained numerous Tryp. cruzi (Wood, 1942) was presumably not killed by the flagellate alone. This bug possessed a swollen abdomen, a phenomenon known to occur in aposymbiotic bugs (see Section VII). Tryp. cruzi seems to act as a subpathological stressor, leading to adverse effects only if a second synergistic stressor is present (Schaub, 1989b). Under optimum feeding conditions the metabolite losses to the parasite seem to be compensated by an increase in the number of blood meals and/or the volume of blood ingested (Juarez, 1970). In contrast to Tryp. cruzi, Tryp. rangefi is pathogenic to the vector and not to the human host. It is even more deadly to Cimex than to triatomines, killing more than 80% before they reached the adult stage (Grewal, 1957). In the reduviid bugs R. prolixus and R. robustus, but not in Tri. infestans, infections with Tryp. rangefi cause retardation of larval development (Grewal, 1956, 1957; Tobie, 1965; Aiiez, 1984). Developmental retardation has rarely been measured in this system. Whereas uninfected first and second instar larvae of R. prolixus needed at least 7 days after feeding before they moulted, and third, fourth and fifth instar larvae needed 9, 10 and 16 days, respectively, infected bugs needed 8, 9, 10, 12 and 21 days (Tobie, 1965). After infection of 30 bugs of each instar, most groups needed 10-40% more time to reach the adult stage than the uninfected groups (Aiiez et af., 1987). The mortality rate data from laboratory studies with Tryp. rangefi are summarized in Table 1. The first investigation of the virulence of Tryp. rangeli-unfortunately without control groups-demonstrated a clear dose dependency, at least in the first instar (Grewal, 1957). The infective dose given to group X2 and group X3 was about five and ten times higher, respectively, than that of group XI. Only G6mez (1967) used a mixture of culture stages and blood, with a high concentration of Tryp. rangefi, instead of a living host for the infection of the first instars, which might explain the extremely high mortality rate of his infected group (B in Table 1). However, the control group also had a high mortality rate. Perhaps handling stress caused these increased mortalities, a factor to which bugs react very sensitively (summarized by Schaub, 1988a; Schaub and Breger, 1988). Data in Table I indicate that the first, second and fifth instar larvae react more sensitively to Tryp. rangefi than do the other instars. However, after infection of 30 or 35 bugs of each instar, comparison of their instar-specific mortality rates showed a statistically significantly higher mortality rate in the fourth instar only (Aiiez et af., 1987). This aspect should be investigated again with more bugs, since in all groups the number of deaths per instar was very low, between 0 and 4. A further interesting phenomenon which needs reinvestigation is the observation by Tobie (1965) that in the unin-

284

G. A. SCHAUB

fected groups more females than males developed, but after infection of first instar larvae the numbers of both sexes were identical. Sex also influences salivary gland infections; they arise in a higher proportion of males than females. TABLE1 Instar-specific rates of mortality' in groups of Rhodnius uninfected and infected with Trypanosoma rangeli Uninfected controls'

154

Instarb

L1 L2 L3

3 5 4 0 6

L4 L5

16

LI-LSd

CI

C2

D

100 20

25

10

34

49

74 100 100 170 105 30

0 0 0 0 0

12 3 0 3 7

22 8 3 3 12

29 13 64 8 1 3 1 9 1 6 1 1 0 -' 3 4 6 10 57

0

24

41

-

B

A

Infected groups'

1 1 1

3 2

38

9 1 0

0 5 5

0 0 0

0 0 8 0 4 12

X I X2 X3 A Initial number of bugs

34

B

94

CI

C2

12 6 3 8 4 1 0 4 1 2 14 21 39

D

10 4 8 4 9

46 30

aMortality rate (YO)calculated for each instar from the number of dead larvae in the respective instar and the number which entered that instar. LI, L2, etc., first, second, etc. instar larvae. 'The same capital letter marks control and infected group data originating from one investigation, as follows: A, Tobie (1965), R. prolixus; B, Gomez (1967), R. prolixus; C, Aiiez (1984). CI, R. prolixus, C2, R. robustus; D, Aiiez er al. (1987), R. prolixus; X, Grewal (1957). R. prolixus. X I , X2. X3, increasing infection rates. Total mortality rate. Observations discontinued.

B.

BLASTOCRITHIDIA TRIA TOMAE INFECTIONS OF TRIATOMINAE

In our detailed investigations with E. triatomae, two modes of infection were used, natural and in vitro infection. Only the first mode of infection gives information as to whether or not the parasite may be transmitted in natural populations. In our initial studies we infected the bugs naturally solely by maintaining uninfected and infected animals together. In such groups direct transmission of E . triatomae between bugs occurs, regularly by coprophagy, but cannibalism is not excluded (Schaub et af., 1989a). Coprophagy seems to occur after feeding, and the rate of coprophagic transmission of trypanosomatids is greatly reduced in populations which have been starved for a long time (Schaub, 1988b, 1990d; Schaub et af., 1989a). Dry faeces has to be redissolved by fresh faeces before infection is possible (Schaub et af., 1989a; Schaub and Jensen, 1990).

TABLE 2 Instar-specij5c rates of mortality' and total infection rate in uninfected groups of triatomines and in groups exposed to Blastocrithidia triatomae infection by coprophagy

B. triatomae coprophagy'

Uninfected controls' Instarb L1 L2 L3 L4 L5 Ll-LSd Infection rate

A

B

C

D

3

3

E

F

G

H

A

0

3 f3

- 16

16

7 f 5 0 +O

1 f2

f0

f5

28 f 4

f 2

-

1 f l

3 f4

5 f 5

3 +2

1 f2

3 f3

f7

8

10 f 9

12 f 4 11

fO

0

1

0

5

f 7

4 f 3

0

5 f 9

f 9

2 +3

2 f2

f5

6 fll

20 f 16

21 f12

6 f6

fO

3 f2

22 f14

40

f 9

24 flO

25 f3

22 f 17

f3

3

f5

5 +2

10

8

f5 8

+

2 f 3 1 f 2

f2

f4

+o

6

46

f 18

f4

4 f4 13

B 8

f8 9

f7

2

C

D

E

F

G

4 fll

4 f4

0 f0

2 +3

f3

f5

5 2 1 f26

8 f 13

1 f2

f5

54 *37

2 +5 1 f l 4 f 3

3 f2

1 f2

0 1

1 5-2 1

f11

4

f8 1

f5 22

f7

5 +4

f2

25 f14

8 f7

f7

f20

f16

75 21 f17 f l l

38 f21

68 f24

f15

41 f20 49 f27

59 f10

f13

f7

69 f16

f15

1

14

85 flO 93 f6

f2

4

11 57

f12

36

85

30

83

7

fO

f2

94

16 f 15

11 f4

48

34 f 6

f7

5

H 11 4

f2

17 2

f3

a Mortality rate (%) calculated for each instar from the number of dead larvae in the respective instar and the number which entered that instar; mean and standard deviation of three to five groups, initially each consisting of 20-45 first instar larvae. LI, L2, etc., first, second, etc. instar larvae. 'The same capital letter marks control and infected group data originating from one investigation, as follows: A, Schaub (1988a) Triafoma infesfans;B, Schaub and Breger (1988) Tri. sordida; C, Schaub and Breger (1988), Tri. pallidipennis; D, G . A. Schaub (unpublished data), Tri. spinolai; E, Schaub and Breger (1988), D. maxima; F, Schaub (1988a), R.prolixus; G, G. A. Schaub (unpublished data), R. robustus; H, G . A. Schaub (unpublished data), R . neglectus. Total mortality rate.

286

G. A. SCHAUB

The results from groups exposed to coprophagic infection (Table 2) show that some species react sensitively, i.e. development is retarded and mortality rate increased (Schaub, 1988a, 1990d; Schaub and Breger, 1988; Schaub and Jensen, 1990; G. A. Schaub, unpublished observations). Such retarded development is less apparent during the first three instars, and in the fourth and fifth instar the first animals moult at the same time in all groups. However, in most infected groups, larval development is greatly retarded in later instars. In one of these investigations, 50% of uninfected fifth instar bugs moulted to the adult stage at 16 weeks after the first feed of the first instars, whereas the same proportion of infected bugs needed 22 weeks (Fig. 7) (Schaub and Jensen, 1990). 100

-s-3 v)

c

U

50

c VI

c 3

Bugs in control group:

r"

Bugs in coprophagy groups uninfected: o infected: 0

10

0

15

25 Weeks after first feeding

20

29 34

FIG.7. Cumulative percentage of moults to adults plotted against age for Triatoma infestans in uninfected populations and those infected with Blastocrithidia triatomae. (Reproduced from Schaub and Jensen, 1990, Journal of Invertebrate Pathology 55, 17-27.)

Instar-specific mortality rate varies in the different species (Table 2). The infection rate of Tri. infestuns and--clearly correlated therewith-the mortality rate, is higher in groups given more infected bugs (Schaub and Jensen, 1990). In all sensitive species the final larval instar shows the highest mortality rates (Table 2). This is similar to the situation with Rhodnius spp. infected with Tryp. rangeli. Bugs often die during ecdysis in both systems. However, this increase of mortality during ecdysis is not specific to B. triatomae, but is correlated with the higher mortality (Schaub and Jensen, 1990), an aspect which has not been considered in the Tryp. rungeli infections.

EFFECTS OF TRYPANOSOMATIDS ON INSECTS

287

In another study we excluded the possibility that the pathological effects of B. triatomae were due to our experimental design. Whereas some groups of primarily uninfected first instar larvae had only infected late instar larvae introduced, infected and uninfected larvae were added to other groups. Since the pathological effects in the different groups were very similar, either the bugs did not discriminate between faeces from infected and uninfected bugs or they did not reject the infectious faeces (Schaub and Jensen, 1990). In the series of investigations on coprophagic infection, the infection rates varied greatly between the different species (Table 2). It remains to be investigated whether the relatively low infection rate of Tri. pallidipennis was caused by different coprophagic behaviour or whether the infected bugs which were added to the primarily uninfected first instars excreted only low numbers of cysts of B. triatomae. Only some adults of R . neglectus and R . robustus were infected. This could be due to differences in coprophagic behaviour and/or susceptibility, or to an infection in late instars. A further important result is that only the groups of R . prolixus had relatively high infection rates but remained unaffected. This suggested a tolerance to B. rriaromae infections, or else a late infection. Unequivocal verification would need infections in vitro in which the exact time of infection and infection doses were known. After development of isolation procedures to obtain the infectious stage of B. triatomae-the drought-resistant cysts-bugs were infected in vitro by feeding a mixture of blood and cyst stages through artificial membranes (Schaub et al., 1988; Schaub, 1990a). While investigating the number of cysts necessary to infect all bugs, different percentages of bugs of the various groups became infected. As in the coprophagic infection experiments, the correlation of infection rate and mortality rate was also evident after infection in vitro (Schaub et al., in press a). We varied different experimental features to exclude the possibility that we had wrongly attributed synergistic effects solely to B. triatomae. Using different infection doses ( 104-108 cysts per ml blood) for infection of first instars-all doses were sufficient to infect all bugs-the effects were very similar in the different groups (G. A. Schaub and B. Rohr, unpublished observations). Presumably, this similarity was caused by high division rates of B. triatomae resulting in a similar level of parasites 4 weeks after infection, whether infected with lo4 or lo8 cysts per ml. Also long-lasting starvation of bugs did hot affect the virulence of B. triatomae (Schaub, 1991). Infections of different instars of Tri. infestans clearly show the existence of a time-lag before pathological effects are observed (G. A. Schaub and S. Wolf, unpublished observations). After infection of bugs of the first, second, third or fourth instar, developmental retardation was first evident at the moults of the third, fourth, fifth and fifth instars, respectively, but retardation was

288

C . A. SCHAUB

almost undetectable after infection of fifth instars. Variation of maintenance temperature also showed that B. triatomae needed some time before effects were evident. Normally maintenance was at 26°C; lower temperatures retarded the development of Tri. infestans and increased the pathological effects of B. triatomae. Maintenance at higher temperatures shortened the developmental times, and more bugs reached the adult stage. Variations in relative humidity did not influence the pathological effects (G. A. Schaub, unpublished observations). Crowding stress acted synergistically with the B. triatomae infection only at higher population densities than those we usually used (Schaub, 1990b) (see Section V1.C). Therefore, the pathological effects were caused by B. triatomae alone, and the sensitivity of species and strains of triatomines could be compared. In these infections in vitro, in which all first instars ingested the same number of cysts, effects on sensitive species were similar to those obtained after coprophagic infection (Table 3). The results clearly demonstrated that all three species of Rhodnius are tolerant and that the Triatoma spp. and D . maxima are susceptible and sensitive. Interestingly, exactly the opposite groups of triatomines are affected by B. triatomae or Tryp. rangeli; the homoxenous flagellate is pathogenic to bugs of the genus Triatoma and Dipetalogaster, but not to Rhodnius (D’Alessandro, 1976; Aiiez, 1984; Schaub, 1988a; Schaub and Breger, 1988; G . A. Schaub, unpublished observations). This difference is not strain-specific; using six strains of Tri. iqfestans (old laboratory strains or strains in the first or third laboratory generation) and three strains of R. prolixus, only Tri. infestans was affected by B. triatomae (Schaub, 1988a, 1990d; Schaub and Jensen, 1990; G. A. Schaub, unpublished observations). I should emphasize that B. triatomae affects sensitive bug species much more severely than Tryp. rangeli does its insect hosts. Theoretically, the tolerance of the Rhodnius spp. could be caused by the brief larval developmental period, since mainly late instars are affected in sensitive species. Since bugs usually need only one adequate blood meal to induce development to the next larval instar, insufficient amount of blood or longer starvation periods after the moult prolong the total developmental time of larvae. Therefore, the influence of starvation was studied with R. prolixus infected in vitro, but here again the infected and uninfected groups did not differ in developmental times or mortality rates (G. A. Schaub, unpublished observations). Long-lasting starvation also did not alter the effects of B. triatomae on Tri. infestans infected in vitro, although this species of bugs is affected (Schaub, 1991). In natural populations selection phenomena occur. Theoretically, the tolerance of R . prolixus to B. triatomae could also appear in “wild” populations of the Triatoma spp. Therefore, in four generations of offspring

TABLE3 Instar-specific rates of mortality' in groups

of

triatomines uninfected and infected with Blastocrithidia triatomae

B. triatomae infection

Uninfected con troIs Instarb

LI L2 L3 L4 L5 LI-LSd

A

B

C

D

E

F

G

H

17 16 f l l f16 7 5 f6 f7 3 2 f6 f3 0 1 fO f2 1 0 f2 fO

7 f5 0

7 10 3 f2 +7 f3 f2 1 6 1 1 3 +1 f7 f4 f2 2 7 2 1 2 +3 f12 f4 f4 0 3 8 1 1 fO f3 f6 f9 1 0 2 2 2 0 fO f 7 f16 f2

3 f3 2 f3 7 f6

4 f6

17 25 f 7 f16

1

20 f23

44

f6

40 f18

5

f5 0 fO

22 f17

D

E

F

G

H

25 f14 33 f15 22 f5

10 f3 2 f2 0

10 f8 6 f7

8 f5

1 f2 3 f2

17 12 6 +I f2 flO f l 27 15 8 10 f7 f5 f 7 f12 23 12 23 5 f7 fll f 7 +I0 24 17 22 38 f5 f 7 f16 f18 79 62 36 61 f20 f25 f14 f20

14 f3

f19

fO 4

f3

A

B

C

3

84

86 f12

66

f8

82 f15

44 f29 80 f26 97 f3

4

f2

f3 3 f6 1 f2 3 f4

14 18 f 6 f14

17 f6

fO 1 f2 2 f2

1 f2 0

f0 1

a Mortality rate (%) calculated for each instar from the number of dead larvae in the respective instar and the number which entered the respective instar; mean and standard deviation of three to five groups, initially each consisting of 2 M 5 first instar larvae. LI, L2, etc., first, second, etc. instar larvae. The same capital letter marks control and infected group data originating from one investigation, as follows: A, Schaub (1990a), Tri. infestans; E H , G . A. Schaub (unpublished data): B, Tri. sordida; C , Tri. pallidipennis; D,Tri. spinolai; E, D . maxima; F, R. prolixus; G , R. robustus; H, R. neglectus. Total mortality rate.

290

G. A. SCHAUB

of adult Tri. infestans infected with B. triatomae, larval development and mortality rate were investigated in groups maintained together with uninfected bugs or those infected with B. triatomae (Schaub, 1980; Schaub and Jensen, 1985). In comparison to the parent generation, pathological effects were slightly reduced in the groups which had the possibility of coprophagic infection, but this reduction coincide with reduced infection rates. Infection in vitro of the fourth generation showed that the sensitivity to B. triatomae was not reduced. C.

HOMOXENOUS TRYPANOSOMATIDS IN HYMENOPTERA AND DIPTERA

Only three other species of homoxenous trypanosomatids are known to affect the mortality rate of immature insect hosts. In a pioneer study, H . swainei was found to increase the mortality rate of the jack-pine sawfly (Hymenoptera) only slightly in the late larval instars (up to 20%), and this only in larvae infected during the first two instars. In double infections with a virus, the two parasites did not act synergistically, and when the virus developed successfully, the flagellate was killed (Smirnoff and Lipa, 1970). (Warburg and Ostrovska (1987) also described a detrimental effect of virus infections on the development of Leish. major in the sandfly.) H . swainei overwinters in the cocooned host and, in the initial study, in spring the period of emergence and the number of emerged adults did not differ between infected and uninfected populations (Smirnoff and Lipa, 1970). In a later investigation H . swainei strongly affected the emergence rate: whereas 67.5% of uninfected adults emerged from cocoons (about two-thirds being males) only 20% of infected pupae (half of them males) survived. In an uninfected “wild” population 55% of adults emerged (Smirnoff, 1974). In eye gnats (Diptera) infected with H . muscarum, developmental times of larvae and pupae did not appear to be affected, but under normal rearing conditions at 27°C only 38% of the adults emerged, compared to 69% in uninfected populations (Bailey and Brooks, 1972b). At higher temperatures the developmental times of uninfected and infected populations decreased, as did most mortality rates. At lower temperatures the opposite effect occurred (Bailey and Brooks, 1972b). (This correlation was also obvious in Tri. infestans infected with B. triatomae.) Similarly to H . swainei, H . muscarum seemed to be more virulent if young larvae were infected (Bailey and Brooks, 1972a). Since mortality of infected gnats seems to be caused by the bacterial septicaemia, and since studies of this flagellate were undertaken with laboratory colonies, the bacterial fauna and the importance of parasitization in “wild” populations remain to be investigated. More striking effects are known for B. caliroa, which seemed to be responsible for the collapse of outbreaks of a fruit-tree pest, the hymenop-

EFFECTS OF TRYPANOSOMATIDS ON INSECTS

29 1

teran Caliroa cerasi (Carl, 1976; Lipa et al., 1977). N o laboratory study has been performed with this species, but of 2500 collected larvae, 53% died during rearing, usually in late larval instars, and 92% of these larvae contained heavy flagellate infections in the haemocoele. At another locality a mortality rate of about 40% was associated with the infection. First the colour of the larvae changed (see Section III.E), and then they eventually stopped feeding and died.

V. EFFECTS ON ADULTLIFESPANAND REPRODUCTION RATE The effects on female fecundity can mainly be explained by a parasitogenic reduction of the amount of material needed for egg production. In contrast to helminth-infected intermediate hosts, there is no known example of trypanosomatids increasing the life span of insect hosts by castration or reduction of reproduction. A.

LEISHMANIA AND TR YPANOSOMA

Effects of Leishmania on adult sandflies have been considered only in single, old studies and need confirmation using better rearing conditions. Single observations that sandflies heavily infected with Leish. donovani die within a few days (Smith et al., 1940) were confirmed in an extensive study (Smith et al., 1941): only nine uninfected, but 77 infected, sandflies failed to take up blood and died within the first week. According to Killick-Kendrick (1979) and Molyneux and Killick-Kendrick (1987), two additional studies which reported harmful effects on sandflies are not convincing. In a further investigation of Leish. major and a saurian Leishmania, longevity of two species of sandflies was reduced after simultaneous infection with both parasites (Alekseev et al., 1975; Safyanova and Alexeiev, 1977). These effects were due to infections with that species of Leishmania with which the sandflies are not naturally infected, i.e. the saurian Leishmania affected Phl. papatasi, the natural vector of Leish. major, and vice versa. A comparison of infection rates of parous and gravid Phl. papatasi showed similar infected proportions in both groups; therefore, Leish. major infections did not appear to affect sandfly survival in the field (Yuval, 1991). However, the very rough classification would not indicate slight effects. In phlebotomines presumed to be infected with a bat trypanosome, parasite and insect host seemed to compete for metabolites essential for egg development. Thereby, gonotrophic concordance of blood ingestion and ovarian development was disrupted and longevity must be reduced (Williams, 1976).

292

G . A. SCHAUB

Heavy infections of tsetse flies with trypanosomes are also likely to affect the vital characteristics of this insect. Based on the respiratory rates of trypanosomatids and the energy content of the ingested blood, Bursell (1981) calculated a more than 15% reduction of flight duration for infected flies. These calculations are supported by observations by Ryan (1984) showing significantly higher activity of infected flies and indicating that the nutritional reserves of infected flies may be slightly lower than those of uninfected flies. Results of investigations of the effects of trypanosomes on life span and reproduction rate of tsetse flies are contradictory: G . palpalis and G . morsitans, infected with Tryp. rhodesiense, Tryp. gambiense or Tryp. brucei, showed a tendency for greater longevity than uninfected flies (Duke, 1928; Baker and Robertson, 1957). There was no difference in mean longevity, number of puparia or weight of puparia of G . m. morsitans (ca. 100 or 200 individuals) fed on calves and goats, comparing uninfected flies with those infected with Tryp. vivax, Tryp. congolense or Tryp. brucei, and observed for 63 days (Moloo and Kutuza, 1985). In a later study by this group using Tryp. vivax and G . p . gambiensis, the 88 infected males had a statistically significantly higher mean survival time (82 days) compared with that of uninfected males (71 days), but opposite results (although not statistically significant) were obtained for 100 females (99 and 102 days) (Makumi and Moloo, 1991). The other features investigated, number and weight of puparia, were not affected by the infection. Two reports have described clear effects of Tryp. brucei and/or Tryp. congolense on G . m. morsitans. In both parasite-vector systems, mortality rate was increased within 30 days after infection, and the number of pupae per female within the first 18 days was decreased compared to uninfected flies. Abortion rate, mean weight and viability of pupae were unaffected (Kaaya et al., 1987). However, only 10-12 flies were investigated. The mortality of an initial population of 150 G . m. morsitans, comparing uninfected flies with those infected with Tryp. congolense, was significantly increased in the infected flies from the 10th day after infection. At the end of the observation period ( 1 7 days after infection), 25 infected flies, but only 10 uninfected flies, had died (Nitcheman, 1988). To summarize these investigations, an effect of trypanosome infection on Glossina may be present during the initial phase of infection. The infection of fleas with the rat trypanosome, Tryp. lewisi, caused an initial increase in the mortality rate, corresponding to the time in which the cells of the midgut are invaded (see Section 1II.A.l.c) (Garnham, 1955). This effect did not occur after a second infectious feed and is attributed to the higher sensitivity of newly emerged fleas. This interpretation remains to be verified since it is also possible that all sensitive individuals were killed

EFFECTS OF TRYPANOSOMATIDS ON INSECTS

293

by the first infection. Another often cited example, the pathogenicity of Tryp. melophagium to sheep keds, was later shown to be caused by the experimental conditions (Nelson, 1956, 1981; Hoare, 1972). An infection with a bird trypanosome developing in the haemocoele eventually became so profound that it was lethal to the vector (Macfie and Thompson, 1929); however, this parasite is transmitted not by insects, but by mites, and the suggested pathogenicity needs to be verified by an experimental study. In the experimental vector Aedes aegypti, infections with Tryp. avium reduced the number of eggs produced (Bennett, 1970a). Since this effect was very strong if the birds on which the mosquitoes fed had high parasitaemias, it could be partly due to a reduction in the quality of the blood. Investigations with Tryp. cruzi also led to contradictory results as to whether or not adult triatomines are affected (reviewed by Schaub, 1989b). Two publications mentioned reduced life expectancy of a species of Triatoma and R . prolixus (Carcavallo, 1970; Neves and Peres, 1975), and the latter study also noted a reduced egg-laying period, but essential data were not given (see Schaub, 1989b). In Tri. dimidiata, mean life spans of males and females, as well as the hatching rate of eggs, were apparently unaffected (Zeledon et al., 1970), as were the egg-laying period and mean adult life span of Tri. infestans (Schaub et al., 1985). The reduced egg production of infected Tri. infestans reported by two other authors might have been caused by initial effects of the infection (of adults or fifth instars) or by their having been fed in vitro with defibrinated blood (reviewed by Schaub, 1989b), both indicating a subpathogenic effect of Tryp. cruzi (see Section VI). In our Tryp. cruzi-Tri. infestans system a slight reduction in egg-laying rate during the first weeks, and a slight decrease in the hatching rate, seemed to occur (Schaub et al., 1985). Such studies are complicated by high variability and the effects of blood ingestion and ageing. Daily counting of the number of eggs laid showed that reproduction of infected and uninfected adults and blood ingestion possessed a corresponding periodicity. The effects of ageing were an even greater complication; the periods when no eggs were laid increased with age, and egg weight and hatching rate decreased. Thus, more detailed studies are necessary. However, the slight decreases observed in bugs infected with Tryp. cruzi cannot influence natural populations with good feeding opportunities. Only two investigations compared the adult life span of R. prolixus when uninfected or infected with Tryp. rangeli. No increase of mortality rate in the infected group was observed during the first 3 months after infection of adults (Tobie, 1965); in the investigation by Aiiez et al. (1987), four of 30 infected adults, but none of 10 uninfected bugs, had died by the end of the 3-month observation period.

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In R. prolixus infected with Tryp. rangeli, egg production was reduced by about two-thirds compared to controls; in the infected bugs the percentage of non-viable eggs increased from 5% to 27% (Watkins, 1969). This effect seemed to be due to Tryp. rangeli and not to the intracoelomic inoculation, since bugs infected in the same way with Tryp. cruzi showed no reduction of egg production and only a slight increase in the percentage of non-viable eggs. B.

HOMOXENOUS TRYPANOSOMATIDS

1, Eflects on life span

There are only a few investigations considering the effects of homoxenous trypanosomatids on adult insects. Similarly to Tryp. cruzi, for regularly fed bugs survival time of males and fecundity of females are nearly identical in uninfected water striders and those with variable parasite loads of B. gerridis and/or C . flexonema (Arnqvist and Maki, 1990). However, adult life span was reduced by 9% to 25% in eye gnats infected with H. muscarum if the temperature range permitted activity of the Diptera (Bailey and Brooks, 1972b). According to Lotmar (1946), A. Porter mentioned that H. vespae killed populations of bees in Canada and Natal. Since certain diseases of bees were unknown at that time, and since H. vespae infections of this wellstudied insect have not been reported since, it is more likely that the bees were killed by another pathogen. Also the pathogenicity of H. bombycis, which invades the haemocoele, remains to be reinvestigated since the alleged lethal effect was observed after inoculation into only one butterfly (Levaditi, 1905). More detailed information is available for Tri. infestans infected with B. triatomae, which are greatly affected by the parasite. Individual infected females live up to 20 weeks and males up to 27 weeks, and mean life spans of infected females and males after moulting to the adult stage are 9 and 12 weeks, respectively. However, in uninfected Tri. infestans, the mean life spans are 35 weeks in males and 30 weeks in females (Schaub et al., 1984, 1985; G. A. Schaub, unpublished observations). 2. Eflects on the reproduction rate

Effects on the reproduction rate have been reported in three systems. In an initial study, H. swainei did not seem to affect the reproduction rate of the naturally-infected jack-pine sawfly (Smirnoff and Lipa, 1970). However, in a later study from the same group, dissection of the females and counting of the eggs showed that fecundity of infected flies was reduced by 25%

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(Smirnoff, 1974). According to Jenkins (l964), Ayroza Galvgo and Coutinho (1941) reported that Anopheles infected with B. pessoai (syn. Lept. pessoai) showed a pathogenic action on ovaries and gut. As with parallel studies with Tryp. cruzi, evaluation of the reproductive data of Tri. infestans infected with B. triatomae is complicated by the effects of feeding and ageing, but the effects of B. triatomae are considerable. In infected groups or pairs, the number of eggs laid per day, egg weight, hatching rate and weight of the first instar larvae are always reduced compared to controls (Schaub et al., 1984, 1985; G. A. Schaub, unpublished observations), resulting in a reduction of the reproductive rate by 95%. Recently detailed studies were made of the effects of C. bombi on bumble bees (Shykoff and Schmid-Hempel, 1991a,b,c). Spring queens of Bombus terrestris, but not of B. lucorum, that failed to found nests in the laboratory had less developed ovaries than did uninfected queens (Shykoff and SchmidHempel, 1991b). Some infected queens were able to found nests, but early in the colony cycle the oviposition rate of the infected queens was reduced (Shykoff and Schmid-Hempel, 1991~).In the laboratory, colony productivity of these small colonies did not differ from that of larger colonies of uninfected queens if enough food was offered. However, effects in the field might well occur. Thus, overwintering of the parasite could be complicated, since the parasite seems to overwinter only in infected queens and only large field colonies produce queens. Interestingly, the effect of the parasites on the queen seems to be compensated, since parasites also delay the age-dependent ovarian development in workers. Thereby, worker-laid eggs appear later in infected nests than in uninfected nests. After the worker bumble bees begin egg laying, they reduce the time they invest in foraging and feeding of the queen’s larvae. Therefore, the delayed reproduction of infected workers increases the likelihood of the colony producing more queens (Shykoff and Schmid-Hempel, 1991~). V I.

SYNERGISTIC EFFECTS OF TRYPANOSOMATIDS AND OTHER STRESSORS

In laboratory investigations insects are maintained under optimum conditions. However, natural populations are often subjected to adverse biotic and abiotic stressors. Environmental stress, especially a combination of different stress factors, can cause adverse effects as shown, for example, for mites infected with a weak bacterial pathogen (Lighthart et al., 1988). In infections with those trypanosomatids which are subpathogenic and do not obviously affect the host, the synergistic action of the trypanosomatid and other stressors could result in recognizable effects. In infections with pathogenic trypanosomatids, the intensity of the effects may well be increased.

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So far the effect of the most important abiotic stressors, temperature and relative humidity, on insects infected with trypanosomatids have been investigated only in the B. triatomae-Tri. infestans system (see section IV.B), but the temperature steps used were too large to recognize synergistic effects. Laboratory studies are necessary to clarify whether the significantly lower prevalence of C. bombi in spring queens of bumble bees than in previous summer workers is caused by reduced hibernation success of infected queens or by loss of the parasites during the winter (Shykoff and Schmid-Hempel, I99 1b). Observations by Gorla's group indicate that a synergistic stress of infection and abiotic factors might act on natural populations of Tri. infestans. The percentage of bugs infected with Tryp. cruzi is statistically significantly lower in winter and early spring, with mean daily minimum and maximum environmental temperatures of about 6" and 17"C, respectively, than it is in mid spring and autumn (15" and 27°C) (Giojalas et al., 1990). However, other possibilities, such as temperature dependency of the development of Tryp. cruzi and the age structure of the population, could be excluded only by a detailed study, e.g. using populations in chicken houses (Gorla and Schofield, 1989). Another stress factor for specimens from naturally infected populations could be capture and transport to the laboratory. This was evident with sandflies infected with different trypanosomatids of toads and lizards. The highest rates of infection occurred in flies that did not survive the transport (Ayala, 1973). In other systems synergistic effects of the infection and a second stressor are evident. A.

SENSITIVITY TO INSECTICIDES

The sensitivity of tsetse flies infected with Tryp. brucei to a low dose (50% lethal dose or less) of different insecticides was tested by Golder et al. (1982, 1984). Within 48 h after topical application of endosulfan, about 50% more infected flies than uninfected ones were dead. The increased sensitivity of infected flies was also evident after application of a natural pyrethrum extract, and in both studies males reacted more sensitively than females. In addition, G. m. morsitans infected with Tryp. congolense had reduced resistance to deltamethrine (Nitcheman, 1988). Whereas the results concerning the effect of infection on longevity of flies are contradictory, these insecticide data indicate at least a subpathological effect of the trypanosomes. In our Tryp. cruzi-Tri. infestans system we used different insecticides. The effective dose which killed 50% of the populations did not differ between

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uninfected and infected populations. Also, B. triatomae did not seem to affect the sensitivity of bugs to insecticides. The long-term effect of sublethal doses remains to be investigated (G. A. Schaub and R. Pospischil, unpublished observations). B.

STARVATION RESISTANCE

The effect of short-lasting starvation of R. prolixus naturally infected with Tryp. rangeli has been investigated by Marinkelle (1968). Starvation for 8 weeks increased the mortality rate of regularly fed, laboratory reared, uninfected fourth and fifth instar larvae (normally 10%) only up to l8%, but during a 6-week starvation period 87% of bugs caught in the field died. However, the infection rates detected in dead and surviving larvae were nearly identical and, unexpectedly, very low (7%), in contrast to 46% in the original batch of insects. The low rate detectable in the dead bugs was explained as an artefact due to the delay in determination of the infection after death of the insects, since no motile forms of Tryp. rangeli could be found 1-3 h after death of the bugs (Marinkelle, 1968). Since infection rates and especially the feeding state of bugs caught in the field may vary, determination of starvation resistance of such bugs offers results with only limited value. These difficulties were also evident in a calculation of starvation resistance of Tri. dimidiata naturally infected with Tryp. cruzi (Vargas and Zeledon, 1985). In addition, infected and uninfected groups contained different numbers of bugs of different developmental stages, which vary in their capacity to survive starvation. Therefore, it was uncertain whether the lower mean survival time in the infected group really indicated an adverse effect of the infection on starvation resistance. Using laboratory reared Tri. infestans, which had been infected in the first instar with Tryp. cruzi and given their last feed in the second, third or fourth instar, the mean starvation resistance period was reduced respectively by 3%, 14% and 17% relative to uninfected bugs, the differences between the two latter values being statistically significant by Student’s t test (Schaub and Losch, 1989). The respective data for bugs infected with B. triatomae, 51%, 55% and 32%, demonstrated the strong pathological effect of the homoxenous flagellate. Whereas the most resistant stage, the fourth instar, survived uninfected up to 432 days after the last feed in the third instar (allowing development to the fourth instar), the last bug of this instar infected with Tryp. cruzi died on day 331 and the last bug infected with B. triatomae survived only to day 140. Food remnants were present in the intestines of a higher proportion of dead bugs infected with Tryp. cruzi than in those of uninfected bugs, and in even more larvae infected with B.

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triatomae. Thus, flagellates and bug either seem to compete for essential metabolites whose depletion results in death, or else the flagellates excrete toxic substances (Schaub and Losch, 1989). Similarly to Tryp. cruzi, which does not affect survival time of regularly fed bugs reared under optimum conditions, B. gerridis and/or C .flexonema infections reduced starvation resistance of male water striders (Arnqvist and Maki, 1990). Surviving males showed a statistically significant lower parasite load than those dying of starvation during the first days. Since water striders do not survive such long starvation periods as triatomines (mean survival time of starved males was 5 days), their natural populations are probably more affected than those of triatomines. C.

SENSITIVITY TO ISOLATION AND OVERCROWDING

Maintenance in isolation or in overcrowded conditions can be a stress factor for insects, adversely affecting different life characteristics (Chauvin, 1967; Peters, T. M. and Barbosa, 1977). The occurrence of such effects depends on the natural life style of the species or its developmental stages. In insects which normally live singly the stressor is grouping, and in insects which normally live gregariously, like the triatomines, it can be either isolation or overcrowding (Peters, T. M. and Barbosa, 1977). Whereas it has long been known that crowding can induce outbreaks of insect diseases (e.g. Steinhaus, 1958; further literature cited by Schaub, 1990b), the effects of isolation on insects infected with trypanosomatids have only recently been investigated with triatomines. Reis dos Santos and Lacombe (1985) attributed retarded development of some first instar larvae of Tri. infestans which were infected with Tryp. cruzi to the infection and not to an isolation effect; this effect was not obtained in our Tryp. cruzi-Tri. infestans system (Schaub, 1988d). In a detailed study investigating the importance of group size in Tri. infestans, both uninfected and infected with B. triatomae, the developmental time and mortality of larvae which were maintained in isolation or in groups of 20, 30, 40 and 50 bugs were compared (Schaub, 1990b). In uninfected groups only a minor proportion of isolated older larvae, but no crowded bugs, showed delayed development, demonstrating for the first time an isolation effect on development of uninfected triatomines. The lack of such an effect on development of first instars (Schaub, 1988d) was confirmed in that study. In groups infected with B. triatomae, development of isolated bugs was more retarded than in uninfected bugs. In addition, a synergistic action of overcrowding and infection slightly increased developmental times. At 35-40 weeks after first feeding of first instars of the most crowded groups (50 bugs), only about half of those infected bugs from which adults

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eventually developed had moulted to adults. By that time 20% more bugs had emerged in all the other infected groups (Schaub, 1990b). Mean total mortality rates of about 10% in uninfected groups were unaffected by group size. In most groups of infected bugs the mean mortality rates were about 50%, but in the most crowded groups, consisting of 50 larvae, a higher mean mortality rate of 75% (a statistically significant difference) was observed. This indicates a subpathological overcrowding stress, increased by the synergistic action of the flagellate (Schaub, 1990b). In natural populations, crowding of Tri. infestans reduces blood intake (Schofield, 1982), but isolation is likely to be a less important stress factor for triatomines. The more sensitive reaction of infected bugs to these stressors implies that other stress factors may well also act synergistically with B. triatomae and perhaps also with Tryp. cruzi (see Section VIII).

VII. MECHANISMS OF PATHOGENICITY The mechanism of the pathological effects seems to be clear when the parasites multiply intracellularly in the affected organ, e.g. the intestine. Presumably the altered behaviour of infected blood sucking insects can be attributed to effects on salivary glands, blood flow rate, or receptor function. In the two systems in which many effects are known in detail, R . prolixus infected with Tryp. rangeli and Tri. infestans infected with B. triatomae, we are far from understanding the physiological bases of the similar complex sickness syndrome in the insect. Gomez (1967) suggested that it is not invasion of the haemocoele which causes the pathogenicity of Tryp. rangeli, but an effect in the intestine. In her investigation, larval mortality rate was much higher than that observed by Grewal (1957) (see Section IV.A), but only 2% of her fifth instar larvae which did not moult to the adult stage possessed parasites in the haemocoele, compared to 100% (total infection rate 54%) in the study by Grewal (1957). However, direct inoculation into the haemocoele also harmed the bugs (Grewal, 1957, 1969; Watkins, 1969), and Grewal (1957) emphasized that mortality “is probably not connected with the penetration of the gut wall”. The discussion of Aiiez (1984) indicated that invasion of the haemocoele and continuous development in all tissues were the important pathological mechanisms. Accordingly, the high mortality rate in the first, second and fifth instars could be caused by either early or late invasion of the haemocoele by Tryp. rangeli (Aiiez, 1984). Watkins (1969, 1971a,b) has already referred to two phenomena which are similar to some effects observed in R . prolixus infected with Tryp. rangeli. In larvae infected with this trypanosomatid, and also in those with blocked spiracles, peristalsis of the gut is absent, the rectum is much distended,

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defaecation and feeding stop, and haemolymph volume increases. On the other hand, some of these effects were also evident in aposymbiotic bugs of different species of triatomines if they were fed on humans, rabbits or guinea-pigs; larval developmental times and mortality rates were increased, mainly in the last two instars, bugs also often died during or soon after ecdysis, moulting deformities were common, the cuticle was pale, excretion and digestion were disturbed and some larvae probed but did not ingest blood (summarized by Gumpert and Schwartz, 1962; Auden, 1974). Those few aposymbiotic Rhodnius that became adults produced no eggs (Brecher and Wigglesworth, 1944). Therefore, Watkins (1969, 1971a) suggested an effect of Tryp. rangeli on the gut-colonizing symbionts which are thought to produce B-group vitamins. She observed that cross-sections of midguts contained far fewer symbionts than those of uninfected bugs. However, her tests with smears made from intestinal contents on agar plates (Watkins, 1969) were not totally convincing. Culture material of Tryp. rangeli inhibited the growth of the intestinal bacteria originating from uninfected bugs. In addition, smears made from contents of uninfected intestines yielded a good growth of symbiont-like bacteria, visible at 48 h and heavy after 78 h. With material from infected bugs, growth was first visible on the third day and then ceased. However, this also happened when using intestinal contents from uninfected bugs that showed deformities similar to those of infected bugs. Therefore, independently of infections with Tryp. rangeli, intestinal bacteria may not be viable in sick bugs. Since the sickness syndrome of bugs infected with B. triatomae also resembles the effects in aposymbiotic bugs, we tried to ascertain the number of bacteria in uninfected and infected Tri. infestans, but preliminary tests showed great variation in the number of bacteria in bugs of both groups ( G . A. Schaub and K . Fischer, unpublished observations). However, other experiments support the interpretation that B. triatomae might interfere with the symbionts or their supply of vitamins or other metabolites. Supplementation of blood with B-group vitamins supported the initial, but not late, development of B. triatomae in the small intestine of young, but not old, Tri. infestans (Jensen and Schaub, 1991). Since this supplementation also greatly reduced the pathogenicity, competition of the bug and the flagellates for the vitamins/metabolites or other effects on the symbionts may be the mechanism of the pathogenicity of B. rriatomae (Jensen, 1987). The fact that B. triatomae and Tryp. rangeli affect different groups of bugs might then be due to differences in the sensitivity of the symbionts. In both systems the interactions of parasites and bug symbionts need detailed investigation. This aspect has only once been studied, in bugs infected with Tryp. cruzi (Muhlpfordt, 1959).

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So far we cannot decide what is the sequence of steps in the pathological process. It is possible that anoxia inhibits the growth of symbionts, thereby reducing the vitamin supply. However, it seems more likely that a reduced vitamin/metabolite supply causes reduced growth of the tracheoles, resulting in anoxia. In both triatomine systems these questions could be answered soon, perhaps also providing explanations applicable to other trypanosomatidinsect systems. VIII.

CONCLUSIONS

Although many trypanosomes reach high population densities in the insect host, strong pathological effects are observed in only a few systems (Table 4). Usually the interaction of trypanosomes and insects seems to be balanced, and no ill effects are obvious. However, under natural conditions insects are often exposed to adverse conditions-factors which are rarely considered in laboratory studies-and the parasite might then act synergistically with other stressors (Schaub, 1989b) (see Section VI). Theoretical models indicate that synergistic action with other stressors might be important in regulation of the host population (Anderson, R. M., 1979; Anderson, R. M. and May, 1981). According to the intensity of the effects on the insect host, trypanosomatids can be classified into three groups. Trypanosomatids of the first group reach low population densities in the insect and will not affect the host even under adverse conditions. Since tests under adverse conditions have not been performed with most trypanosomatid-insect systems, the majority of trypanosomatids must be classified into this group. The second group is subpathogenic, but harms the insect if other stress factors act synergistically. This group consists of most Leishmania species and other trypanosomatids of sandflies, the salivarian trypanosomes developing in tsetse flies, Tryp. cruzi, B. gerridislc. jlexonema, C . bombi, Lept. pyrrhocoris and perhaps Lept. pyraustae and B. pessoai. Most of the effects discussed in the present review can be attributed to this group. So far only a few trypanosomatids are known to affect the insect even under optimum conditions, and these can be classified into the third group. They are Tryp. lewisi, Tryp. rangeli, H . swainei, H . muscarum, B. caliroa and B. triatomae. A better grouping will be achieved after more investigations of natural populations like those of Arnqvist and Maki (1990) and Shykoff and Schmid-Hempel(199 1a,b,c). Such studies should be accompanied by laboratory studies under exactly defined conditions including infection doses, temperature and relative humidity, and the exclusion of factors such as

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TABLE 4 Summary of effects of trypanosomatids on insectsa Feature assessedb Trypanosomatid

Host insect

Endotrypanum schaudinni Leishmania donovani L. major L. braziliensis L. amazonensis L. aethiopica Trypanosoma sp. (of toads,

Sandfly

lizards)

Trypanosoma sp. (of bats) T. congolense T. brucei T. lewisi T. avium T. corvi T. cruzi T. rangeli Blastocrithidia triatomae B. gerridis B. pessoai B. caliroa Herpetomonas swainei H.muscarum Crithidia bombi C. cimbexi Leptomonas pyrrhocorris L. oncopelti L. pyraustae

Sandfly Sandfly Sandfly Sandfly Sandfly Sandfly Sandfly Tsetse fly Tsetse fly Rat flea Mosquito Tabanid Mosquito Triatomine Triatomine Bed bug Triatomine Water strider Mosquito Hymenoptera Hymenoptera Diptera Bumble bee Hymenoptera Bug Bug

Corn borer

A

B

C

+ + +

+ + + + + +

D

+

E

F

G

H

I

K

+ +

+

+ + f + f +

+

f f +

+ + + + + + + f + f + + + + + + + + + + + + + + + + + + + + +' +' + + + + + + + + + + + + + + + + + + + -c

References are given in the text. B, probing and engorgement; C, intestine; D, Malpighian tubules; E, haemolymph; F, cuticle; G, further organs; H, larval development and mortality rate; 1, adult longevity and fecundity; K, survival, usually subpathogenically affected (synergistic stressors). +, Affected (sometimes only slight indications for the respective effect); not affected; k, contradictory results. ' Double infections with Crithidiujlexonemu. a

bA, fitness;

-.

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concomitant double infections with other pathogens. In these laboratory studies it is very important that parasites and hosts originate from the same locality (discussed by Boker and Schaub, 1984). Investigations of “wild” populations for several years can provide data on the variation of the genetic heterogeneity. Theoretically, in “wild” populations the proportion of susceptible and tolerant individuals should vary and, therefore, also the percentage of affected insects (Anderson, R. M., 1986). Selection phenomena require more time to develop in insects with long developmental times such as the triatomines. Genetic heterogeneity may also be important in insect populations which are normally less heterogeneous, the social insects. Effects at the population level are indicated by the recent field and laboratory studies comparing uninfected bumble bees and those infected with C. bombi (Shykoff, 1991; Shykoff and Schmid-Hempel, 1991a). Differences in susceptibility result in higher parasite transmission rates between related bees than among those which are unrelated. Assuming that all susceptible individuals react sensitively, subpathogenic trypanosomatids also provide a selection pressure on social insects to maintain genetic variability. Another interesting theoretical aspect is offered by the infection rate of natural populations with B. triatomae. Whereas all specimens of the insectivorous bug Zelus leucogrammus were found to be infected by Carvalho and Deane (1974), though no pathological effect was reported, maximum infection rates of “wild” triatomines were 5.4% in Tri. maculata and less than 1 % in Tri. sordida and Panstrongylus megistus (publications summarized by Schaub and Boker, 1986a; Schaub, 1988a). On the one hand, it is possible that B. triatomae is pathogenic only in infections of insect species which are only occasionally infected in nature. On the other hand, it is possible that the high infection rate of Z. leucogrammus is caused by predation of infected triatomines. The low infection rate of triatomines would then be an indicator of virulence, since a characteristic of highly pathogenic species seems to be a very low infection rate in the host population (Anderson, R. M., 1979). However, Anderson, R. M. (1979) also emphasized another characteristic, the low average parasite burden of the host; this is not the case in bugs infected with B. triatomae. Only B. triatomae seems to be a good candidate for biological control of its host. Unlike viruses and bacteria, a fast action cannot be expected since the main effects occur in late larval instars. An application of B. triatomae combines characteristics of both approaches summarized by McLaughlin (1973). In some aspects it can be considered as belonging to McLaughlin’s second group. It is slow acting, safe, and may act as a major factor or synergistically with others. Additionally, only an infrequent application is necessary since the cysts are resistant stages. On the other hand, B. triatomae has some of the advantages of a microbial insecticide. It can be applied by

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methods commonly available, production costs are low, and it can be stored easily. Since the effects of B. triatomae vary in groups infected with the same dose and maintained under identical conditions, the intensity of the effects may be caused in part by secondary infections. A weakening of the insects has to be considered (see Section III.A.2.c). Thus, there might have been an increase in either the sensitivity to B. triatomae infection or the susceptibility to other pathogens which cannot develop in uninfected bugs, or can do so only to a harmless level. Under natural conditions even a weakening of the insect host would probably be sufficient to permit an adjustment of the insect population at a lower equilibrium level. The correlation of infection rate and mortality rate indicates that the use of B. triatomae as a biological control agent against triatomines is possible only if high infection rates can be achieved. Since large numbers of cysts can be readily obtained from experimentally infected bugs or from cultures in vitro, it is feasible to spray the resting places of the bugs with a suspension of cysts (Schaub and Jensen, 1990; Schaub et al., 1990a). A further advantage of the use of B. triatomae is the suppression of the development of Tryp. cruzi in double infections (G. A. Schaub and M. Mehl, unpublished observations). However, initial exploratory field tests are required to establish whether high infection rates can be achieved and whether B. triatomae will be the first trypanosomatid used for biological or integrated control of insects. ACKNOWLEDGEMENTS

I thank Professors H. Hecker and D. Molyneux for providing electron micrographs and Professors A. N. Alekseev, N. A. Ratcliffe and Y. Schlein and Drs G. Arnqvist and J. A. Shykoff for providing unpublished results, papers in press or valuable information for this review. I am much indebted to Dr J. R. Baker for revision of the English version of the manuscript, and I am deeply grateful for the funding of my investigations from the UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical Diseases and the Deutsche Forschungsgemeinschaft. REFERENCES Adler, S. and Ber, M. (1941). The transmission of Leishmania rropica by the bite of Phlebotomus papatasii. Indian Journal of Medical Research 29, 803-809.

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