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Echinostoma paraensei: Hemocytes of Biomphalaria glabrata as targets of echinostome mediated interference with host snail resistance to Schistosoma mansoni
EXPERIMENTALPARASITOLOGY
62, 149-154(1986)
Echinostoma paraensei: Targets of Echinostome Resistance
Hemocytes of Biomphalaria glabrata as Mediated interference with Host Snail to Schistosoma mansoni
ERIC S. LOKER,~ CHRISTOPHER J. BAYNE, AND MARY A. YUI Department of Zoology, Oregon State University, Cowallis, Oregon 97331, U.S.A. (Accepted for publication 2 January 1986) LOKER, E. S., BAYNE. C. J., AND YUI, M. A. 1986. Echinostomaparaensei: Hemocytes of Biornphalaria glabrata as targets of echinostome mediated interference with host snail resistance to Schistosoma mansoni. Experimental Parasitology 62, 149- 154. Earlier in vivo work by Lie et a/. (1977) indicated that the innate resistance of the lOR2 strain of Biompha[aria glabrata to PRl Schistosoma mansoni could be interfered with if the snails were infected previously with another trematode, Echinostoma paraensei. We have studied this interference phenomenon using in vitro methods in an attempt to understand its mechanistic basis. Hemolymph, derived from lOR2 snails infected with E. paraensei for 14-28 days, killed 25% of S. mansoni sporocysts in vitro, significantly less (P < 0.001) than the 90% killing rate observed with hemolymph from uninfected, control lOR2 snails. Hemolymph from the infected lOR2 snails and from schistosome susceptible M line snails did not differ significantly (P > 0.1) in their relative inability to kill S. mansoni sporocysts in vitro. The defect in sporocyst killing exhibited by echinostome infected lOR2 snails was traced to the cellular, rather than the humoral, component of the hemolymph. Preparations containing uninfected lOR2 snail hemolymph and echinostome daughter rediae exhibited significantly less (P < 0.001) killing of S. mansoni sporocysts than did controls containing only lOR2 hemolymph and S. mansoni sporocysts. Our results suggest that echinostome larvae release factors that interfere with the ability of B. glabrata hemocytes to kill S. mansoni sporocysts. 0 1986 Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Schistosoma mansoni; Echinostoma paraensei; Trematodes; Biomphalaria glabrata; Snail; Immunoparasitology; Hemolymph; Hemocytes; Interference; Fluorescein isothiocyanate (FITC); Cytotoxicity, cell mediated (CMC); Medium F base (MFB).
result of echinostome induced interference, host resistance to other trematode species As a means to ensure their survival, par- is also diminished; the innate resistance of asites commonly exert suppressive effects the 10R2 strain of B. glabrata to PRI upon the immune systems of their hosts. In Schistosoma mansoni is reduced by prior the trematode-mollusc model, Echino- echinostome infection (Lie et al. 1977). stoma paraensei-Biomphalaria glabrata, a Echinostome infected snails respond to phenomenon referred to as interference by several other insults, implying their inits discoverers (Lie and Heyneman 1976) ternal defense systems are otherwise intact exists, as a consequence of which normal (Lie et al. 1981). age dependent resistance to the parasite Understanding the mechanisms which fails to develop in hosts infected during sustain the host-parasite balance is extheir juvenile phase of susceptibility. As a traordinarily hard to achieve in most in vivo models due to the dynamism of the complex metabolic interplay. Understanding ’ Present address: Biology Department, University of New Mexico, Albuquerque, New Mexico 87131, can sometimes be gained by the use of in vitro models. Using such a model, we have USA. 149 0014-4894/86$3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
150
LOKER, BAYNE,
found that echinostome mediated interference with the natural resistance of B. glabrata for S. mansoni is a consequence of the echinostome inducing a physiological defect in the host hemocyte. The effect can be elicited by echinostomes in the culture system at the time when snail hemocytes encounter schistosomes, but is not mediated by plasma taken from infected snails. MATERIALS
AND METHODS
AND YUI
were held on a microscope slide at 26 C, in a moist chamber, for a minimum of 24 hr. The number of living and dead S. mansoni sporocysts in contact with hemocytes was recorded after l-4 hr and 24-hr coincubation. During scoring, the identity of the preparation was withheld from the observer. Large opaque aggregates, in which sporocysts could not be clearly discerned, and the few sporocysts not bound by hemocytes, were disregarded in these experiments (see below). For replicates 3-6 and for negative control preparations using M line hemolymph, a refined protocol was followed that facilitated detection of sporocysts and assessment of their condition. This procedure involved vitally prestaining the sporocysts with FITC and incorporating eosin Y into the culture fluid (medium or snail plasma), as described by Boswell and Bayne (1985). The FITC enhances the accuracy with which an observer can determine the presence of a sporocyst in a large cell ball, and the eosin Y, which enters sporocysts as they die, permits scoring (by a switch of filters on the microscope) of their viability. To determine whether interference is due to alteration of the cytotoxic capacity of the hemocytes alone, or whether it was due to a soluble factor in the plasma during the course of the experiments, sporocysts were exposed to heterologous combinations of hemocytes and plasma from E. paraensei infected and uninfected lOR2 snails. The first such preparation was made by removing the plasma after cell aggregations had formed (within the first hour of coincubation), washing the aggregations with tissue culture medium, and replacing the plasma in a heterologous fashion. To avoid the potential for significant adsorption of plasma factors to sporocysts during the 1-hr incubation, a second such preparation was made using a Ficoll cushion to separate hemocytes and plasma components, as described by Bayne et al. (1980b). These experiments were run simultaneously with replicates 4 and 5 described above. The killing of sporocysts during the course of the 24-hr experiments was determined using the formula
The pulmonate snail Biomphalaria glabrata (M line and lOR2 strains) and the PRI strain of Schistosoma mansoni were maintained as described by Stibbs et al. (1979). S. mansoni was propagated in outbred SwissWebster mice and M line snails. Echinostoma paraensei was supplied to us in infected B. glabrata by Dr. K. J. Lie (University of California, San Francisco, CA, USA); it was passaged in Syrian hamsters and M line B. glabrata according to methods specified by Dr. K. J. Lie and Dr. K. Jeong (personal communication), essentially as described by Lie and Basch (1967). Sporocysts of S. mansoni were transformed from miracidia in Complete Medium F (Stibbs et al. 1979). Snail hemolymph was obtained by cardiac puncture of adult individuals (lo-12 mm diameter) after they had soaked for 20-30 min in 2% penicillin/streptomycin/ fungizone (Microbiological Associates) in artificial spring water (Ulmer 1970), and after the shell was swabbed with 70% alcohol and dried as described previously (Bayne et al. 1980a; Loker et al. 1984). Hemolymph for experimental preparations was derived from lOR2 snails harboring prepatent 14-28 day infections of E. paraensei, evidenced by sporocysts in the heart or rediae in the digestive gland. Control preparations used hemolymph from unexposed lOR2 snails. For replicates 1 and 2, 25-50 S. mansoni sporocysts and 1SOp,l of the appropriate hemolymph were added to agarose cytotoxicity tubes prepared as described by Bayne et al. (1980a). Hemocytes were al(% sporocysts alive at start) lowed to settle onto the flat agarose surface and onto (% sporocysts alive at end) sporocysts for 1 hr, excess plasma was removed, and % sporocysts alive at start the remaining contents of the tube were transferred to a 50 ~1 flat capillary tube (Microslides, 0.3 mm path length, Vitro Dynamics, Rockaway, NJ, USA). This The mean percentage of sporocysts killed in experiprotocol favored the formation of several small aggre- mental and control preparations was compared using gates of sporocysts and hemocytes rather than a single Student’s t test. M line B. glabrata were exposed to lo-20 miracidia large aggregate as described in earlier studies (Bayne of E. paraensei; 40-50 days later, daughter rediae et al. 198Oa,b; Loker and Bayne 1982). Use of microwere dissected from the digestive gland and ovotestis slides also permitted repeated examination of individual hemocyte/sporocyst aggregates. Most such ag- lof infected snails. Rediae were rinsed five times in gregates were sufficiently small as to allow direct as- ‘,MFB, our basic culture medium but lacking mammalian serum and antibiotics. Daughter rediae remain acsessment of sporocyst viability, as judged by motility, flame cell activity, and general appearance. The tubes tive in MFB for several days. To each experimental
Echinostoma paraensei: INTERFERENCE IN VITRO tube was added 25-50 5’. mansoni primary sporocysts, IO-30 E. paruensei daughter rediae, and 150 ~1 of hemolymph from uninfected lOR2 or M line B. glabrata. Control preparations lacked rediae but were otherwise set up and observed in the same manner. After 1 hr, sporocyst/hemocyte aggregates and rediae were transferred to microslides. For these experiments, viability was assessed by direct observation of sporocysts, without the use of FITC or eosin Y.
RESULTS
The cytotoxic capacity of hemolymph (hemocytes and plasma) from lOR2 snails for Schistosoma mansoni primary sporocysts was depressed if the snails were infected with Echinostoma paraensei (Table I). Two types of controls were included. Positive controls were sporocysts incubated with hemocytes from uninfected lOR2 Biomphalaria glabrata. Negative controls were sporocysts incubated with hemolymph from naive M line snails, in which sporocyst viability is maintained (Bayne et al. 1980b; Loker and Bayne 1982). These results indicate that echinostome induced interference with the resistance of 10R2 snails to S. mansoni is a consequence of altered hemolymph responsiveness of infected snails. Hemolymph from schistosome susceptible M line snails and from echinostome infected lOR2 snails were equally ineffective in killing S. mansoni sporocysts (P > 0.1). Experiments using heterologous combinations of infected and uninfected lOR2 hemolymph components (Table I) suggested that hemocytes, rather than plasma factors, from infected snails were affected by the echinostomes. Whereas it is clear that interference is due to altered hemocyte competence and that plasma from infected snails does not contain (sufficient) humoral factor(s) capable of interfering with CMC during our 24-hr assays, it is also to be expected that, in vivo, the echinostome secretes factors which have the observed effect on the hemocytes. We found evidence supporting this assertion (Table II). When E. paraensei
151
rediae, dissected from M line snails, were present in the culture during the 24-hr CMC assay, the cytotoxic capacity of 10R2 hemocytes was reduced. DISCUSSION
The initial evidence regarding the ability of echinostome trematodes to interfere with the immune responses of their molluscan hosts was provided by the in vivo studies of Lie, Heyneman, and colleagues (reviewed by Lie 1982). For example, Lie et al. (1977) observed that juvenile Biomphalaria glabrata lOR2 snails, which are normally resistant to Schistosoma mansoni (Richards and Merritt 1972), if first infected with Echinostoma paraensei, become susceptible to S. mansoni. Since 10R2 resistance is normally evidenced by a cellular attack on schistosome sporocysts (Lie et al. 1980; Loker et al. 1982), it was suspected that interference would be mediated via an effect on host hemocytes. Our experiments, in addition to providing an in vitro model of the interference effects, have shown this to be the case. In one of our experiments, daughter rediae, some of which released actively swimming echinostome cercariae, were placed adjacent to smaller schistosome primary sporocysts. Echinostome rediae and cercariae did not visibly damage schistosome sporocysts nor did they prevent hemocytes from adhering to them. Hemocytes were not observed to adhere to rediae . In vivo, echinostome rediae prey upon schistosome sporocysts (Lim and Heyneman 1972) and this has been seen in vitro (Basch and DiConza 1975). The smaller numbers of sporocysts we found in experimental relative to control preparations (Table II) was probably not a consequence of redial predation however; the large, relatively immobile daughter rediae were never seen to ingest sporocysts. In our control lOR2 preparations, the appearance of hemocyte capsules changed
152
LOKER, BAYNE,
AND YUI
TABLE I Exposure of PRl Schistosoma munsoni Sporocysts to Hemolymph Components from lOR2 and M Line Snails Snail strain
Experiment No.
10R2
1 2 3 4 5 6
M line
1 2 3 4 5
Experimentals n
Controls
% killed
21 20.5 43 14.3 8 25.0 21 4.8 26 67.0 37 16.6 X = 24.7 F 21.8
n
% killed
27 51.9 20 100.0 11 100.0 23 100.0 8 100.0 x = 90.4 2 21.5 3 33.0 23 16.0 33 33.0 31 16.1 19 10.5 X = 21.7 I 10.5
Infected lOR2 cells + uninfected lOR2 plasma 4 5
30 21
0.0 28.2 j;: = 14.1
Uninfected lOR2 cells + infected lOR2 plasma 4 5
100.0 92.0
12 39 X = 96.0
Note. Experimental lOR2 hemolymph was derived from snails infected with Echinostoma paraensei (age of infection 14-28 days). Control 10R2 and M line hemolymph was derived from unexposed snails of comparable size. Preparations containing heterologous combinations of infected and uninfected lOR2 hemolymph components (see text) were run concurrently with the controls. The number of sporocysts examined (n) is listed for each replicate as is the percentage killed during the experiment. For each treatment, the mean percentage of sporocysts killed (+ SD) is indicated. A significantly higher percentage of sporocysts was killed in control than in experimental lOR2 preparations (P < 0.001). Experimental lOR2 and M line controls did not differ significantly in this regard (P 1 0.1).
with time. Early in the coincubation period, hemocytes and sporocysts were in intimate contact, with much or all of the sporocyst surface covered with hemocytes. Frequently, by 24 hr, hemocytes retained contact with either end of the dead sporocyst, but no longer enveloped its surface. How is interference established? It is reasonable to suggest that echinostome emitted products, targeted at hemocytes,
are responsible (Lie 1982), a suggestion supported by our data. In addition, we have found that patently infected B. glabrata carry in their plasma a number of large molecular weight antigens which are crossreactive with rabbit antiparasite #antibodies (unpublished results); putative S. munsoni antigens detected using polyclonal, antiparasite antisera have been reported on hemocytes of infected snails
1.53
Echinostoma paraensei: INTERFERENCE IN VITRO TABLE11 Exposure of PRl Schistosoma mansoni Primary Sporocysts to Hemolymph of Uninfected lOR2 or M Line Biomphalaria glabrata
Snail strain
Replicate No.
Number rediae present
lOR2
1 2 3 4 5
27 18 10 I1 8
M line
1
23
Experimentals (Sporocysts + rediae) n
Controls (Sporocysts only)
% killed
15 57.0 9 55.6 13 38.5 14 42.9 6 50.0 X = 48.9 rt 8.2 29 0.0
n
% killed
14 85.7 29 82.8 21 85.7 8 62.5 31 77.4 X = 78.8 ? 9.7 22 13.6
Note. Experimental preparations also contained daughter rediae of Echinostomn paraensei; controls lacked rediae. Listed for both experimentals and controls is the number of sporocysts examined per replicate (n) and the percentage of these sporocysts killed during the course of the experiment. The mean percentage of sporocysts killed (i SD) is also indicated. For the lOR2 strain, the percentage of sporocysts killed in control preparations was significantly higher than in experimentals (P < 0.001). M line preparations served as negative controls.
(Abdul-Salam and Michelson 1983). These findings further support the notion that echinostome rediae secrete factors which interfere directly with CMC in our in vitro assays. Although the precise molecular nature of the interference mechanism is unknown, it is apparently selective, in that the ability of echinostome infected Biomphalaria glabrata snails to encapsulate latex spheres, nematodes, and miracidial plates of Schistosoma mansoni is not affected (Lie et al. 1981). Clearly, a total abrogation of host responsiveness to foreignness would be deleterious to the echinostome as it would lead to premature host death (Lie et al. 1981). This selectivity also suggests that interference is not simply a manifestation of nonspecific physiological deterioration resulting from infection. Our work demonstrates that parasite strategies of immunosuppression, which are better known in the context of vertebrate immunology, are also utilized effectively in invertebrate hosts of important human pathogens. The presumed molecular interactions between interference factors and hemocytes raises fascinating
problems, the elucidation of which will surely enhance our general understanding of parasite evasive strategies and of nonself recognition in molluscs. ACKNOWLEDGMENTS Our studies would not have been possible without the guidance and materials provided by the pioneering work of Dr. Kian Joe Lie and of Dr. Charles S. Richards. We wish to dedicate this paper to them, to mark the occasion of their retirements. We are grateful to Dr. Lie for sending us infected snails; to Lindsley Bennett and Berkeley Bayne for preparation of sporocysts; to Carl Boswell for help in the laboratory and for critically reviewing the manuscript; and to the U.S. National Institutes of Health for Grant AI-16137. C.J.B. utilized the facilities of the Department of Experimental Animal Morphology and Cell Biology, Wageningen University, The Netherlands, for the preparation of this manuscript. REFERENCES ABDUL-SALAM, J. M., AND MICHELSON, E. H. 1983. Schistosoma mansoni: Immunofluorescent detection of its antigen reacting with Biomphalaria glabrata amoebocytes. Experimental Parasitology 55, 132-137. BASCH, P. F., AND DICONZA, J. J. 1975. Predation by echinostome rediae upon schistosome sporocysts in vitro. Journal of Parasitology 61, 1044-1047. BAYNE, C. J., BUCKLEY, P. M., AND DEWAN, P. C. 1980a. Macrophagelike hemocytes of resistant
154
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BAYNE,
Biomphalaria glabrata are cytotoxic for sporocysts of Schistosoma mansoni in vitro. Journal of Parasitology 66, 413-419.
BAYNE, C. J., BUCKLEY, P. M., AND DEWAN, P. C. 1980b. Schistosoma mansoni: Cytotoxicity of hemocytes from susceptible snail hosts for sporocysts in plasma from resistant Biomphalaria glabrata. Experimental
Parasitology
50, 409-416.
BOSWELL, C. A., AND BAYNE, C. J. 1985. Schistosoma mansoni: Lectin-dependent cytotoxicity of hemocytes from susceptible host snails, Biomphalaria
glabrata.
Experimental
Parasitology
60,
133-138. LIE, K J. 1982. Survival of Schistosoma mansoni and other trematode larvae in the snail Biomphalaria glabrata. A discussion of the interference theory. Tropical and Geographic Medicine 34, 11 l- 122. LIE, K. .I., AND BASCH, PE 1967. The life history of Echinostoma paraensei sp. n. (Trematoda: Echinostomatidae). Journal ofParasitology 53, 1192-1199. LIE, K. J., AND HEYNEMAN, D. 1976. Studies on resistance in snails. 6. Escape of Echinostoma lindoense sporocysts from encapsulation in the snail heart and subsequent loss of the host’s ability to resist infection by the same parasite. Jourrial of Parasitology
62, 298-302.
LIE, K. J., HEYNEMAN, D., AND RICHARDS, C. S. 1977. Studies on resistance in snails: Interference by nonirradiated echinostome larvae with natural resistance to Schistosoma mansoni in Biomphalaria glabrata.
Journal
of Invertebrate
Pathology
29,
118-125: LIE, K. J., JEONG, K. H., AND HEYNEMAN, D. 1980. Tissue reactions induced by Schistosoma mansoni in Biomphalaria glabrata. Annals of Tropical Medicine and Parasitology 74, 157-166. LIE, K. J., JEONG, K. H., AND HEYNEMAN,
D. 1981.
Selective interference with granulocyte function in-
AND YUI
duced by Echinostoma larvae in Biomphalaria Journal of Parasitology
paraensei glabrata 61, 790-796.
(Trematoda) (Mollusca).
LIM, H. K., AND HEYNEMAN, D. 1972. Intramolluscan inter-trematode antagonism: Review of factors influencing the host-parasite system and its possible role in biological control. Advances in Parasitology 10, 191-268. LOKER, E. S., AND BAYNE, C. J. 1982. In vitro encounters between Schistosoma mansoni primary sporocysts and hemolymph components of susceptible and resistant strains of Biomphalaria glabrata. American Journal of Tropical giene 31, 999- 1005.
Medicine
and Hy-
LOKER, E. S., BAYNE, C. J., BUCKLEY, P. M., AND KRUSE, K. T. 1982. Ultrastructure of encapsulation of Schistosoma mansoni mother sporocysts by hemocytes of juveniles of the lo-R2 strain of Biomphalaria
glabrata.
Journal
of Parasitology
68,
84-94. LOKER, E. S., YUI, M. A., AND BAYNE, C. J. 1984. Schistosoma mansoni: Agglutination of sporocysts, and formation of gels on miracidia transforming in plasma of Biomphalaria glabrata. Experimental Parasitology
58, 56-62.
RICHARDS,C. S., AND MERRITT, J. W., JR. 1972. Genetic factors in the susceptibility of juvenile Biomphalaria glabrata to Schistosoma mansoni infection. American Journal of Tropical Medicine and Hygiene
21, 425-434.
STIBBS, H. H., OWCZARZAK,A., BAYNE, C. J., AND DEWAN, P. 1979. Schistosome sporocyst-killing cells isolated from Biomphalaria glabrata. Journal of Znvertebrate
Pathology
33, 1.59-170.
ULMER, M. J. 1970. Notes for the rearing of snails in the laboratory. In “Experiments and Techniques in Parasitology” (A. J. MacInnis and M. Voge, eds.), pp. 143-144. Freeman, San Francisco.
62, 149-154(1986)
Echinostoma paraensei: Targets of Echinostome Resistance
Hemocytes of Biomphalaria glabrata as Mediated interference with Host Snail to Schistosoma mansoni
ERIC S. LOKER,~ CHRISTOPHER J. BAYNE, AND MARY A. YUI Department of Zoology, Oregon State University, Cowallis, Oregon 97331, U.S.A. (Accepted for publication 2 January 1986) LOKER, E. S., BAYNE. C. J., AND YUI, M. A. 1986. Echinostomaparaensei: Hemocytes of Biornphalaria glabrata as targets of echinostome mediated interference with host snail resistance to Schistosoma mansoni. Experimental Parasitology 62, 149- 154. Earlier in vivo work by Lie et a/. (1977) indicated that the innate resistance of the lOR2 strain of Biompha[aria glabrata to PRl Schistosoma mansoni could be interfered with if the snails were infected previously with another trematode, Echinostoma paraensei. We have studied this interference phenomenon using in vitro methods in an attempt to understand its mechanistic basis. Hemolymph, derived from lOR2 snails infected with E. paraensei for 14-28 days, killed 25% of S. mansoni sporocysts in vitro, significantly less (P < 0.001) than the 90% killing rate observed with hemolymph from uninfected, control lOR2 snails. Hemolymph from the infected lOR2 snails and from schistosome susceptible M line snails did not differ significantly (P > 0.1) in their relative inability to kill S. mansoni sporocysts in vitro. The defect in sporocyst killing exhibited by echinostome infected lOR2 snails was traced to the cellular, rather than the humoral, component of the hemolymph. Preparations containing uninfected lOR2 snail hemolymph and echinostome daughter rediae exhibited significantly less (P < 0.001) killing of S. mansoni sporocysts than did controls containing only lOR2 hemolymph and S. mansoni sporocysts. Our results suggest that echinostome larvae release factors that interfere with the ability of B. glabrata hemocytes to kill S. mansoni sporocysts. 0 1986 Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Schistosoma mansoni; Echinostoma paraensei; Trematodes; Biomphalaria glabrata; Snail; Immunoparasitology; Hemolymph; Hemocytes; Interference; Fluorescein isothiocyanate (FITC); Cytotoxicity, cell mediated (CMC); Medium F base (MFB).
result of echinostome induced interference, host resistance to other trematode species As a means to ensure their survival, par- is also diminished; the innate resistance of asites commonly exert suppressive effects the 10R2 strain of B. glabrata to PRI upon the immune systems of their hosts. In Schistosoma mansoni is reduced by prior the trematode-mollusc model, Echino- echinostome infection (Lie et al. 1977). stoma paraensei-Biomphalaria glabrata, a Echinostome infected snails respond to phenomenon referred to as interference by several other insults, implying their inits discoverers (Lie and Heyneman 1976) ternal defense systems are otherwise intact exists, as a consequence of which normal (Lie et al. 1981). age dependent resistance to the parasite Understanding the mechanisms which fails to develop in hosts infected during sustain the host-parasite balance is extheir juvenile phase of susceptibility. As a traordinarily hard to achieve in most in vivo models due to the dynamism of the complex metabolic interplay. Understanding ’ Present address: Biology Department, University of New Mexico, Albuquerque, New Mexico 87131, can sometimes be gained by the use of in vitro models. Using such a model, we have USA. 149 0014-4894/86$3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
150
LOKER, BAYNE,
found that echinostome mediated interference with the natural resistance of B. glabrata for S. mansoni is a consequence of the echinostome inducing a physiological defect in the host hemocyte. The effect can be elicited by echinostomes in the culture system at the time when snail hemocytes encounter schistosomes, but is not mediated by plasma taken from infected snails. MATERIALS
AND METHODS
AND YUI
were held on a microscope slide at 26 C, in a moist chamber, for a minimum of 24 hr. The number of living and dead S. mansoni sporocysts in contact with hemocytes was recorded after l-4 hr and 24-hr coincubation. During scoring, the identity of the preparation was withheld from the observer. Large opaque aggregates, in which sporocysts could not be clearly discerned, and the few sporocysts not bound by hemocytes, were disregarded in these experiments (see below). For replicates 3-6 and for negative control preparations using M line hemolymph, a refined protocol was followed that facilitated detection of sporocysts and assessment of their condition. This procedure involved vitally prestaining the sporocysts with FITC and incorporating eosin Y into the culture fluid (medium or snail plasma), as described by Boswell and Bayne (1985). The FITC enhances the accuracy with which an observer can determine the presence of a sporocyst in a large cell ball, and the eosin Y, which enters sporocysts as they die, permits scoring (by a switch of filters on the microscope) of their viability. To determine whether interference is due to alteration of the cytotoxic capacity of the hemocytes alone, or whether it was due to a soluble factor in the plasma during the course of the experiments, sporocysts were exposed to heterologous combinations of hemocytes and plasma from E. paraensei infected and uninfected lOR2 snails. The first such preparation was made by removing the plasma after cell aggregations had formed (within the first hour of coincubation), washing the aggregations with tissue culture medium, and replacing the plasma in a heterologous fashion. To avoid the potential for significant adsorption of plasma factors to sporocysts during the 1-hr incubation, a second such preparation was made using a Ficoll cushion to separate hemocytes and plasma components, as described by Bayne et al. (1980b). These experiments were run simultaneously with replicates 4 and 5 described above. The killing of sporocysts during the course of the 24-hr experiments was determined using the formula
The pulmonate snail Biomphalaria glabrata (M line and lOR2 strains) and the PRI strain of Schistosoma mansoni were maintained as described by Stibbs et al. (1979). S. mansoni was propagated in outbred SwissWebster mice and M line snails. Echinostoma paraensei was supplied to us in infected B. glabrata by Dr. K. J. Lie (University of California, San Francisco, CA, USA); it was passaged in Syrian hamsters and M line B. glabrata according to methods specified by Dr. K. J. Lie and Dr. K. Jeong (personal communication), essentially as described by Lie and Basch (1967). Sporocysts of S. mansoni were transformed from miracidia in Complete Medium F (Stibbs et al. 1979). Snail hemolymph was obtained by cardiac puncture of adult individuals (lo-12 mm diameter) after they had soaked for 20-30 min in 2% penicillin/streptomycin/ fungizone (Microbiological Associates) in artificial spring water (Ulmer 1970), and after the shell was swabbed with 70% alcohol and dried as described previously (Bayne et al. 1980a; Loker et al. 1984). Hemolymph for experimental preparations was derived from lOR2 snails harboring prepatent 14-28 day infections of E. paraensei, evidenced by sporocysts in the heart or rediae in the digestive gland. Control preparations used hemolymph from unexposed lOR2 snails. For replicates 1 and 2, 25-50 S. mansoni sporocysts and 1SOp,l of the appropriate hemolymph were added to agarose cytotoxicity tubes prepared as described by Bayne et al. (1980a). Hemocytes were al(% sporocysts alive at start) lowed to settle onto the flat agarose surface and onto (% sporocysts alive at end) sporocysts for 1 hr, excess plasma was removed, and % sporocysts alive at start the remaining contents of the tube were transferred to a 50 ~1 flat capillary tube (Microslides, 0.3 mm path length, Vitro Dynamics, Rockaway, NJ, USA). This The mean percentage of sporocysts killed in experiprotocol favored the formation of several small aggre- mental and control preparations was compared using gates of sporocysts and hemocytes rather than a single Student’s t test. M line B. glabrata were exposed to lo-20 miracidia large aggregate as described in earlier studies (Bayne of E. paraensei; 40-50 days later, daughter rediae et al. 198Oa,b; Loker and Bayne 1982). Use of microwere dissected from the digestive gland and ovotestis slides also permitted repeated examination of individual hemocyte/sporocyst aggregates. Most such ag- lof infected snails. Rediae were rinsed five times in gregates were sufficiently small as to allow direct as- ‘,MFB, our basic culture medium but lacking mammalian serum and antibiotics. Daughter rediae remain acsessment of sporocyst viability, as judged by motility, flame cell activity, and general appearance. The tubes tive in MFB for several days. To each experimental
Echinostoma paraensei: INTERFERENCE IN VITRO tube was added 25-50 5’. mansoni primary sporocysts, IO-30 E. paruensei daughter rediae, and 150 ~1 of hemolymph from uninfected lOR2 or M line B. glabrata. Control preparations lacked rediae but were otherwise set up and observed in the same manner. After 1 hr, sporocyst/hemocyte aggregates and rediae were transferred to microslides. For these experiments, viability was assessed by direct observation of sporocysts, without the use of FITC or eosin Y.
RESULTS
The cytotoxic capacity of hemolymph (hemocytes and plasma) from lOR2 snails for Schistosoma mansoni primary sporocysts was depressed if the snails were infected with Echinostoma paraensei (Table I). Two types of controls were included. Positive controls were sporocysts incubated with hemocytes from uninfected lOR2 Biomphalaria glabrata. Negative controls were sporocysts incubated with hemolymph from naive M line snails, in which sporocyst viability is maintained (Bayne et al. 1980b; Loker and Bayne 1982). These results indicate that echinostome induced interference with the resistance of 10R2 snails to S. mansoni is a consequence of altered hemolymph responsiveness of infected snails. Hemolymph from schistosome susceptible M line snails and from echinostome infected lOR2 snails were equally ineffective in killing S. mansoni sporocysts (P > 0.1). Experiments using heterologous combinations of infected and uninfected lOR2 hemolymph components (Table I) suggested that hemocytes, rather than plasma factors, from infected snails were affected by the echinostomes. Whereas it is clear that interference is due to altered hemocyte competence and that plasma from infected snails does not contain (sufficient) humoral factor(s) capable of interfering with CMC during our 24-hr assays, it is also to be expected that, in vivo, the echinostome secretes factors which have the observed effect on the hemocytes. We found evidence supporting this assertion (Table II). When E. paraensei
151
rediae, dissected from M line snails, were present in the culture during the 24-hr CMC assay, the cytotoxic capacity of 10R2 hemocytes was reduced. DISCUSSION
The initial evidence regarding the ability of echinostome trematodes to interfere with the immune responses of their molluscan hosts was provided by the in vivo studies of Lie, Heyneman, and colleagues (reviewed by Lie 1982). For example, Lie et al. (1977) observed that juvenile Biomphalaria glabrata lOR2 snails, which are normally resistant to Schistosoma mansoni (Richards and Merritt 1972), if first infected with Echinostoma paraensei, become susceptible to S. mansoni. Since 10R2 resistance is normally evidenced by a cellular attack on schistosome sporocysts (Lie et al. 1980; Loker et al. 1982), it was suspected that interference would be mediated via an effect on host hemocytes. Our experiments, in addition to providing an in vitro model of the interference effects, have shown this to be the case. In one of our experiments, daughter rediae, some of which released actively swimming echinostome cercariae, were placed adjacent to smaller schistosome primary sporocysts. Echinostome rediae and cercariae did not visibly damage schistosome sporocysts nor did they prevent hemocytes from adhering to them. Hemocytes were not observed to adhere to rediae . In vivo, echinostome rediae prey upon schistosome sporocysts (Lim and Heyneman 1972) and this has been seen in vitro (Basch and DiConza 1975). The smaller numbers of sporocysts we found in experimental relative to control preparations (Table II) was probably not a consequence of redial predation however; the large, relatively immobile daughter rediae were never seen to ingest sporocysts. In our control lOR2 preparations, the appearance of hemocyte capsules changed
152
LOKER, BAYNE,
AND YUI
TABLE I Exposure of PRl Schistosoma munsoni Sporocysts to Hemolymph Components from lOR2 and M Line Snails Snail strain
Experiment No.
10R2
1 2 3 4 5 6
M line
1 2 3 4 5
Experimentals n
Controls
% killed
21 20.5 43 14.3 8 25.0 21 4.8 26 67.0 37 16.6 X = 24.7 F 21.8
n
% killed
27 51.9 20 100.0 11 100.0 23 100.0 8 100.0 x = 90.4 2 21.5 3 33.0 23 16.0 33 33.0 31 16.1 19 10.5 X = 21.7 I 10.5
Infected lOR2 cells + uninfected lOR2 plasma 4 5
30 21
0.0 28.2 j;: = 14.1
Uninfected lOR2 cells + infected lOR2 plasma 4 5
100.0 92.0
12 39 X = 96.0
Note. Experimental lOR2 hemolymph was derived from snails infected with Echinostoma paraensei (age of infection 14-28 days). Control 10R2 and M line hemolymph was derived from unexposed snails of comparable size. Preparations containing heterologous combinations of infected and uninfected lOR2 hemolymph components (see text) were run concurrently with the controls. The number of sporocysts examined (n) is listed for each replicate as is the percentage killed during the experiment. For each treatment, the mean percentage of sporocysts killed (+ SD) is indicated. A significantly higher percentage of sporocysts was killed in control than in experimental lOR2 preparations (P < 0.001). Experimental lOR2 and M line controls did not differ significantly in this regard (P 1 0.1).
with time. Early in the coincubation period, hemocytes and sporocysts were in intimate contact, with much or all of the sporocyst surface covered with hemocytes. Frequently, by 24 hr, hemocytes retained contact with either end of the dead sporocyst, but no longer enveloped its surface. How is interference established? It is reasonable to suggest that echinostome emitted products, targeted at hemocytes,
are responsible (Lie 1982), a suggestion supported by our data. In addition, we have found that patently infected B. glabrata carry in their plasma a number of large molecular weight antigens which are crossreactive with rabbit antiparasite #antibodies (unpublished results); putative S. munsoni antigens detected using polyclonal, antiparasite antisera have been reported on hemocytes of infected snails
1.53
Echinostoma paraensei: INTERFERENCE IN VITRO TABLE11 Exposure of PRl Schistosoma mansoni Primary Sporocysts to Hemolymph of Uninfected lOR2 or M Line Biomphalaria glabrata
Snail strain
Replicate No.
Number rediae present
lOR2
1 2 3 4 5
27 18 10 I1 8
M line
1
23
Experimentals (Sporocysts + rediae) n
Controls (Sporocysts only)
% killed
15 57.0 9 55.6 13 38.5 14 42.9 6 50.0 X = 48.9 rt 8.2 29 0.0
n
% killed
14 85.7 29 82.8 21 85.7 8 62.5 31 77.4 X = 78.8 ? 9.7 22 13.6
Note. Experimental preparations also contained daughter rediae of Echinostomn paraensei; controls lacked rediae. Listed for both experimentals and controls is the number of sporocysts examined per replicate (n) and the percentage of these sporocysts killed during the course of the experiment. The mean percentage of sporocysts killed (i SD) is also indicated. For the lOR2 strain, the percentage of sporocysts killed in control preparations was significantly higher than in experimentals (P < 0.001). M line preparations served as negative controls.
(Abdul-Salam and Michelson 1983). These findings further support the notion that echinostome rediae secrete factors which interfere directly with CMC in our in vitro assays. Although the precise molecular nature of the interference mechanism is unknown, it is apparently selective, in that the ability of echinostome infected Biomphalaria glabrata snails to encapsulate latex spheres, nematodes, and miracidial plates of Schistosoma mansoni is not affected (Lie et al. 1981). Clearly, a total abrogation of host responsiveness to foreignness would be deleterious to the echinostome as it would lead to premature host death (Lie et al. 1981). This selectivity also suggests that interference is not simply a manifestation of nonspecific physiological deterioration resulting from infection. Our work demonstrates that parasite strategies of immunosuppression, which are better known in the context of vertebrate immunology, are also utilized effectively in invertebrate hosts of important human pathogens. The presumed molecular interactions between interference factors and hemocytes raises fascinating
problems, the elucidation of which will surely enhance our general understanding of parasite evasive strategies and of nonself recognition in molluscs. ACKNOWLEDGMENTS Our studies would not have been possible without the guidance and materials provided by the pioneering work of Dr. Kian Joe Lie and of Dr. Charles S. Richards. We wish to dedicate this paper to them, to mark the occasion of their retirements. We are grateful to Dr. Lie for sending us infected snails; to Lindsley Bennett and Berkeley Bayne for preparation of sporocysts; to Carl Boswell for help in the laboratory and for critically reviewing the manuscript; and to the U.S. National Institutes of Health for Grant AI-16137. C.J.B. utilized the facilities of the Department of Experimental Animal Morphology and Cell Biology, Wageningen University, The Netherlands, for the preparation of this manuscript. REFERENCES ABDUL-SALAM, J. M., AND MICHELSON, E. H. 1983. Schistosoma mansoni: Immunofluorescent detection of its antigen reacting with Biomphalaria glabrata amoebocytes. Experimental Parasitology 55, 132-137. BASCH, P. F., AND DICONZA, J. J. 1975. Predation by echinostome rediae upon schistosome sporocysts in vitro. Journal of Parasitology 61, 1044-1047. BAYNE, C. J., BUCKLEY, P. M., AND DEWAN, P. C. 1980a. Macrophagelike hemocytes of resistant
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