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Responses of coelomocytes from Lumbricus terrestris to native and non-native eukaryotic parasites

Posted on: Tuesday, 2 March 2004, 06:00 CST

Summary

Eukaryotic parasites residing in earthworms such as Lumbricus terrestris, have been studied for many years. However, little research has been published on the host-parasite relationship, particularly the immune response of L terrestris and the effect of the parasite on the host. The majority of individuals belonging to the species Lumbricus terrestris are parasitized by a sporozoan of the genus Monocystis, the spores of which can be found in huge numbers in the worms' seminal vesicles. This preliminary study of the Lumbricus terrestris-Monocystis relationship attempts to determine whether a cellular reaction by the earthworm's coelomocytes (immune cells) occurs both in vivo and in vitro. Light and electron microscopic observations indicate that there is no apparent interaction between coelomocytes and parasites in the worms' seminal vesicles. However, in vitro incubations of the Monocystis spores together with coelomocytes produced an encapsulation response: a rapid attachment to and aggregation of coelomocytes around parasites, followed by a complete degranulation of the coelomocytes. The degree of degranulation was monitored by measuring acid phosphatase levels, an enzyme released during degranulation. These were subsequently compared to incubations with equal numbers of other eukaryotic parasites with thick cyst walls (Giardia and Cryptosporidium species), but which do not normally infect the worm. Acid phosphatase levels were significantly higher in Monocystis incubations compared to those of G. lamblia and Cryptosporidium, indicating a greater cellular response to the endogenous parasite. Interestingly, Giardia lamblia cysts actually inhibited degranulation compared to that of control (spontaneous degranulation) coelomocytes, a phenomenon possibly due to the nature of the cyst wall. These experiments show that whereas an immune response against the predominant spore stage of Monocystis does not occur within the seminal vesicles of the earthworm, the hosts' coelomocytes are capable of responding to these spores in vitro, and do so to a greater degree than to non-native parasites.

Key words: Lumbricus terrestris, Monocystis species, encapsulation, coelomocyte, host-parasite relationship, degranulation

Introduction

Lumbricus terrestris, along with many other earthworm species, is host to apicomplexan parasites belonging to the genus Monocystis. These parasites have a complex life cycle, purportedly beginning with the ingestion of spores containing infective sporozoites. After digestion of the spore wall and hatching in the gizzard, the sporozoite exits the worm's intestine, enters the circulatory system and hearts, and then penetrates the seminal vesicle wall. In the seminal vesicle it develops into a trophozoite form, living in and feeding on developing sperm cells. It can then differentiate into gametocytes, develops into gametes which then fuse, and the developing zygote will divide and secrete a spore wall, thus forming the numerous spores seen in the majority of L. terrestris. Despite the parasite's apparent "takeover" in the seminal vesicle, little is known of the effect of the parasite on the earthworm (harmful, beneficial or benign?), or of the earthworm's response to the parasite. In one of the few publications on this subject, Pizl (1985) reported that the application of the herbicide zeazin 50 to the earthworm's soil resulted in a significant increase in the numbers of Monocystis. This finding could imply that the earthworm's defense system was adversely affected and thus does play a role in keeping down the parasite numbers. In a review on cellular defense mechanisms in annelids, Bilej (1994) stated that "...an explanation of host-parasite relationships is still lacking."

Annelid response to parasites is characterized by "encapsulation", whereby coelomocytes (immune defense cells found in the coelomic cavity) attach to and aggregate around the parasite (reviewed by Cooper & Roch 1994; Bilej 1994). This capsule or "granuloma" increases in size, assumes a brown color (now called a "brown body"), moves toward the posterior end of the worm and is released through autotomy (Keilin 1925; Valembois et al. 1992). However, it is unknown whether such a reaction to Monocystis species occurs within L. terrestris' highly parasitized seminal vesicles or whether coelomocytes are capable of recognizing the parasites as foreign, and responding by attachment. Also, if such a reaction does occur, is the response to Monocystis heightened or diminished compared to responses to other, non-native parasites? The objective of this study was to determine answers to these questions, in order to lay the ground work for more detailed study of the L. terrestris - Monocystis relationship. In this study, light and electron micrographs of heavily parasitized seminal vesicles were observed for presence of, and response by coelomocytes. In addition, in vitro incubations of Monocystis spores and coelomocytes were performed, and response determined by degranulation assays. These responses were then compared to those against other (non-native) parasites.

Materials and Methods

Maintenance of earthworms

Specimens of Lumbricus terrestris were obtained from Ward's Biology, #87W4661, and kept in a ventilated styrofoam box containing moistened peat moss at 4 C. They were fed Ward's "Magic Worm Food" (#21W2103).

Light microscopic studies of seminal vesicles

10 earthworms were killed by placing them in 50% ethanol for 3 min, dissected and seminal vesicles removed. Squash preparations were made of each and stained with Wright's Stain (Fisher Healthcare "Hemaquick") for 30 sec, followed by 2-one min rinses in distilled water. Smears of preparations were observed for presence and type of coelomocytes and proximity to Monocystis spores. Results were reported as "number of coelomocytes per 1000x field"; a minimum of 50 fields were observed for each worm.

Collection of coelomocytes for culturing experiments

The method of Eyambe et al. (1991) was used: Each earthworm was rinsed in 4 C saline, placed on a moistened paper towel and the posterior portion was massaged to expel contents of the lower gut. Each worm was then placed in 3 ml. extrusion medium (150 mM NaCl, 50 mM guaicol glycerol ether, 8.6 mM EDTA, 9.8 mM ethanol) for 3 min at room temperature. The extrusion medium containing the extruded coelomocytes was transferred to a conical centrifuge tube and washed 3 times with Lumbricus buffered salt solution (LBSS) (71.5mM NaCl, 4.8mM KCl, UmM MgSO^sub 4^, 0.4 mM KH^sub 2^PO^sub 4^, 0.4 mM Na^sub 2^HPO^sub 4^, 4.2 mM NaHCO^sub 3^, pH 7.0) for 10min at 150 x g, and then resuspended in 1.0ml LBSS.

Obtaining Monocystis spores

Seminal vesicles were removed, homogenized in 1ml LBSS, using a glass homogenizer, washed 3 times in LBSS as above, and then spun in LBSS 2 min at 2000 rpm. This resulted in 2 layers: upper-layer sperm debris and lower-layer Monocystis spores. The lowerlayer was then further concentrated using the Sheather's Sugar Flotation method (500g sucrose, 320ml H2O, 6.5 g phenol) by centrifuging at 500g for 10 min and then removing the upper layer containing spores (Arrowood & Sterling 1987).

In vitro incubations (cultures)

Coelomocytes (collected as above) were adjusted in culture medium (RPMI with 10% fetal calf serum, 100 U ml^sup -1^ penicillin, 100 pg streptomycin) to a count of 5 10^sup 5^ ml^sup -1^ (Cossarizza et al. 1996). Monocystis spores were resuspended in the same medium and adjusted to a count of 2.5 10^sup 5^ ml^sup -1^. (Comparative assays were always done on coelomocytes from either the same worm, or from the same pool of coelomocytes). 100 l of parasite suspension was added to 100 l of coelomocyte suspension in 96 well flat bottom microtiter plates, and rotated slowly on a plate rotator. Control wells contained 100 l of coelomocyte suspension with 100 l of culture fluid only, or 100 l of parasite suspension with culture fluid only. After the incubation period (which varied from 30 min to 3h, depending on the assay), the contents of each well were transferred to a 1.5 ml microfuge tube, centrifuged at 10,000 rpm for one min, supernatant was removed and assayed for acid phosphatase, or the pellet retained for electron microscopy.

Sensitization of worms with parasites

Three worms each were injected posterior to the clitellum into the coelom, using a 26 G - tuberculin syringe (B-D), with 0.2cc of either: LESS (control), 1 10^sup 6^ Monocystis spores or 1 10^sup 6^ Giardia cysts (Waterborne, LA), and kept in separate containers for 24 h The coelomocytes were then extruded and used for in vitro incubations.

Acid phosphatase assay

A variation of the method by Honsi and Stenersen (2000) was used. In 96 well plates 50 l of supernatant from cultures were incubated with 100 l of acid phosphatase substrate (Sigma: equal quantities of p-nitrophenyl phosphate, disodium [40 mg per 10mL] and citrate buffer solution [90 mM citrate and 10 mM chloride, pH 4.8]) for 25 min at 37 C. The reaction was terminated by adding 100 l of stop solution (0.1N NaOH). Absorbance was read on spectrophotometer at 405 nm. Acid phosphatase assays were reported as the difference in absorbance between the coelomocyte control incubations and the coelomocyte-parasite incubations.

Transmission electron microscopy

Pellets fromthe cultures were washed with 0.1M phosphate buffer, centrifuged, the supernatant removed and fixed with 2.5 % glutaraldehyde (prepared in the same buffer) for 1 hour. The pellet was washed 3 with buffer for 15min each and post-fixed in 1% osmium tetroxide for 1 h, and then was washed as above. Pellets were dehydrated in 25%, 50%, 75%, 95 % and 100 % ethanol for 15 min each, and then rinsed twice with propylene oxide for 15 min each. After propylene oxide was removed, a 1:1 propylene oxide:resin (Embed 812 resin from Electron Microscopy Sciences) was added for 1h followed by a 1:3 propylene oxide resin solution overnight. The diluted resin was replaced by pure resin for 2-3 h and then embedded in fresh resin in Beem capsule at 60 C for 2d. ~80nm sections were taken with a diamond knife, stained with uranyl acetate and lead citrate and viewed on a Hitachi H-600 transmission electron microscope at 75 KV.

Killing cysts/spores with formalin

Monocystis spores and Giardia cysts were treated with 10% formalin for 48 hours in order to kill them and prevent possible secretory activity. Prior to use in assays, treated spores and cysts were washed 3 in LBSS.

Results

Light microscopic observations of seminal vesicles

Wright's stained smears of dissected seminal vesicles showed large numbers of Monocystis spores, an occasional Monocystis trophozoite and very few coelomocytes (~ 1 or less per 1000 field were seen). The coelomocytes were either acidophils or basophils, and did not appear to adhere to any of the parasites. There were no signs of encapsulation within the seminal vesicles (data not shown).

In vitro cultures of coelomocytes with parasites

Coelomocytes incubated with Monocystis spores showed evidence of granuloina formation within 30 minutes, and coelomocytes appeared to be completely degranulated by 120 minutes (Fig.1). As described earlier, the coelomocytes adhered to the spores, aggregated into a granuloma (often many were caught up into one large granuloma), and the coelomocytes degranulated. In order to assess granulomas quantitatively, the amount of acid phosphatase released from the granules was measured. Following a 2 h co-incubation of extruded worm coelomocytes with Monocystis spores, there was a substantial release of acid phosphatase compared to spontaneous degranulation in control coelomocyte incubations (controls contained an equal number of coelomocytes from the same worm, incubated for the same time period but without the addition of a parasite). When incubations (using coelomocytes from the same worm) were performed using Cryptosporidium spores or Giardia cysts, there was not only a decrease in the amount of acid phosphatase produced, but in the case of the Giardia cysts, acid phosphatase levels were substantially lower than the spontaneous degranulation in the control coelomocytes (Fig. 2).

Effects of in vivo sensitization with parasites

Worms injected intracoelomically with parasites 24 h prior to extruding coelomocytes were then used for in vitro incubations. This was done in order to determine if an in vivo encounter with the parasite would affect the degree of response by these sensitized coelomocytes to the parasites. Coelomocytes from worms injected with Monocystis spores showed even higher levels of acid phosphatase production when incubated with Monocystis, compared to non- sensitized worms. In fact, injection ("sensitization") with Monocytsis spores even enhanced the in vitro response of coelomocytes to the Giardia cysts (Fig. 3)! Accordingly, in vivo sensitization with Giardia cysts slightly diminished the in vitro coelomocyte response both to Monocystis spores and Giardia.

Fig.1. a-c Degranulation of coelomocytes around Monocystis a. 60 min, b. 90 min, c. 120 min (Bar = 25 m)

Fig. 2. Acid phosphatase levels after 2 h in vitro incubation of coelomocytes + parasites, expressed as the difference between control coelomocytes (due to add phosphatase from spontaneous degranulation) and coelomocytes incubated with parasites:

1. Monocystissp., 2. Cryptosporidium sp.,

2. Giardia sp. (n = 3, +/- std. dev.)

Transmission electron microscopy of in vitro granulomas

Fig. 4b illustrates the interaction between the coelomocytes and Monocystis spores (the spores' internal sporozoites showed only partial preservation, probably due to incomplete penetration of the spore wall by the fixatives). The coelomocytes appear to be completely degranulated and show membrane-spore wall interaction. In contrast, there appears to be neither interaction between the coelomocytes and Giardia cysts walls, nor even close proximity, despite the fact that these cells were pelleted through centrifugation (Fig. 4a).

Fig. 3. Add phosphatase levels after 3 h incubation of parasites with pre-sensitized coelomocytes (coelomocytes extruded from worms injected with parasites 24 h previous to assay): 1. Monocystis + coelomocytes presensitized with Monocystis, 2. Giardia + coelomocytes pre-sensitized with Giardia, 3. Monocystis + coelomocytes pre-sensitized with Giardia, 4. Giardia + coelomocytes presensitized with Monocystis

Fig. 4. Electron micrographs of coelomocytes + parasites after 3 h incubation: a. Giardia cyst b. Monocystis spore (12,000x)

Fig. 5. Acid phosphatase levels after in vitro incubation of coelomocytes + parasites, expressed as the difference between control coelomocytes and coelomocytes incubated with parasites: 1. 1h incubation, 2. 2h incubation (n = 3, +/- std. dev.)

In vitro cultures of live vs. dead parasites

In an effort to determine whether the inhibitory effect of the Giardia cysts was possibly due to a soluble substance secreted by the parasite rather than a cell-cell interaction, in vitro cultures were performed using both live and dead (formalinized) parasites. Acid phosphatase levels from the supernatants from these cultures indicated that the inhibitory effect was actually greater on the formalinized cultures, and therefore it is unlikely that secretions from live cysts play a role in the inhibition of the coelomocyte response (Fig. 5).

Discussion

Microscopic examinations of seminal vesicles indicated that there was no coelomocyte response to the parasites within the seminal vesicles. The presence of a few non-adherent coelomocytes could be due either to cells that were present on the exterior portion of the seminal vesicles (in contact with coelomic fluid), or to response- inhibitory factors within the seminal vesicles. However, in vitro cultures of the parasites with host coelomocytes showed that coelomocytes do respond to Monocystis spores, and therefore the lack of response within the seminal vesicles was not because the Monocystis is non-immunogenic (incapable of stimulating a coelomocyte response). Previous studies on the polychaete Nereis diversicolor and one of its coccidian parasites indicated that only the motile stages of the parasite were susceptible to immune attack, and that stages which formed a cyst wall were not susceptible (Porchet-Hennere et al. 1987). This does not appear to be the case in the current study. Rather, it is more likely that the Monocystis have exploited a site in the worm that is hidden from penetration by immune cells, or if cells do enter, they are inhibited by factors present in the seminal vesicles. It is well known that certain sites in vertebrates are "immune privileged", due either to lack of vascular accessibility or to lack of MHC expression (Fiszer & Kurpisz 1998).

Not only were the coelomocytes capable of responding to Monocystis, but the degree of response (as measured by levels of acid phosphatase released through degranulation) was significantly greater than spontaneous degranulation of control coelomocytes. The acid phosphatase levels were also substantially higher than in cultures using a thick-walled organism that does not parasitize L. terrestris, namely cysts from Giardia lamblia. Even though the earthworm is capable of only innate immune responses and therefore lacks immune memory, these results might reflect the fact that all coelomocytes in this study came from worms that had at least some degree of Monocystis infection, and that in fact some degree of pre- sensitization could be occurring. (Unfortunately, neither we nor apparently any other group have been able to obtain sufficient numbers of worms that do not contain Monocystis, which could be used as controls.) In fact, in the experiments in which we injected either the Monocystis or Giardia into the coelomic cavity 24 hrs prior to performing the in vitro assays, coelomic encounter to the Monocystis did appear to enhance the responses of coelomocytes both to the Monocystis and the Giardia.

A very interesting and unanticipated finding was that in vitro cultures using Giardia cysts exhibited not only a decreased response as compared to the Monocystis cultures, but that degranulation by the coelomocytes, as measured by acid phosphatase levels, was actually less than spontaneous degranulation by control coelomocytes. This indicates that not only are these cysts non- immunogenic, but they appear to be actually inhibiting the coelomocyte response. Also, in experiments in which worms were injected with Giardia cysts 24 hrs. prior to the in vitro assays, this intracoelomic sensitization appeared to result in lower in vitro levels of acid phosphatase when tested with both the Giardia and the Monocystis, as compared to assays using coelomocytes from worms that had been previously injected with the Monocystis spores. The possibility thus exists that either the cyst wall itself has some inhibitory characteristic, or the cyst is secreting some type of suppressant. Immune suppressive parasite surfaces and secretory products have been intensively studied in parasites of vertebrate hosts (review by Zambrano-Villa et al. 2002). Though this has not been as widely researched in invertebrates, Webb & Luckhart (1996) extracted a suppressive substance from the eggs of \the parasite Campoletis sonorensis, and then induced an encapsulation response to these eggs by its host, Heliothis virescens. Data shown here indicated that the inhibition is probably not due to any secreted substances, since formalinized cysts also inhibited degranulation.

In summary, in vitro cultures showed that the L. terrestris' coelomocytes are capable of recognizing and vigorously responding to the spore stage of its ubiquitous parasites belonging to the genus Monocystis. The coelomocytes aggregate and attach to the spores, and then rapidly degranulate, releasing the enzymes contained in their granules. This response appears to be significantly greater than in vitro reactions to Giardia cysts, which actually inhibited coelomocyte degranulation. However, such a response to Monocystis does not occur within the seminal vesicles, allowing the predominant spore stage of this parasite to remain unharmed.

Acknowledgements. The authors wish to thank Margaret Kasschau (University of the Sciences in Philadelphia) for advice and for the use of her laboratory, and Sally Shrom (Villanova University) for assistance with the electron microscopy.

References

Arrowood, M. J., Sterling C. R. (1987) Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic percoll gradients. Journal of Parasitology 73, 314- 319.

Bilej, M. (1994) Cellular defense mechanisms. In: Vetvicka, V., Sima, P., Cooper E. L., Bilej, M., Roch, P. (eds) Immunology of Annelids. CRC Press, Inc., pp. 167-200.

Cooper, E. L., Roch, P. (1994) Immunolgical profile of annelids: transplantation immunity. In: Vetvicka, V., Sima, P., Cooper E. L., Bilej, M., Roch, P. (eds) Immunology of Annelids. CRC Press, Inc., pp. 210-243.

Cossarizza, A., Cooper, E. L., Suzuki, M. M., Salvioli, S., Capri, M., Gri, G., Quaglino, D., Franceschi, C. (1996) Earthworm Leukocytes that are not phagocytic and cross-react with several human epitopes can kill human tumor cell lines. Experimental Cell Research 224, 174-182.

Eyambe, G. S., Goven, A. J., Fitzpatrick, L. C., Venables, B. J., Cooper, E. L. (1991) A noninvasive technique for sequential collection of earthworm (Lumbricus terrestris) leukocytes during subchronic immunotoxicity studies. Laboratory Animals 25, 61-67.

Fiszer, D., Kurpisz, M. (1998) Major histocompatibility complex expression on human, male germ cells: a review. American Journal of Reproductive Immunology 40(3), 172-176.

Honsi, T. G., Stenersen, J. (2000) Activity and localisation of the lysosomal marker enzymes acid phosphatase, N-acetyl-[beta]-D- glucosaminidase, and [beta]-galactosidase in the earthworms Eisenia fetida and E. veneta. Comparative Biochemistry and Physiology Part B 125, 429-437.

Keilin, N. D. (1925) Parasitic autotomy of the host: a mode of liberation of coelomic parasites from the body of the earthworm. Parasitology 17, 170-172.

Pizl, V. (1985) The effect of the herbicide Zeazin 50 on the earthworm infection by monocystid Gregarines. Pedobiologica 28, 399- 402.

Porchet-Hennere, E., M'Beri, M., Chalnaut, A., Porchet, M. (1987) Ultrastructural study of the encapsulation response of the polychaete annelid Nereis diversicolor. Cell and Tissue Research 248, 463-471.

Valembois, P., Lassegues, M., Roch, P. (1992) Formation of brown bodies in the coelomic cavity of the earthworm Eisenia fetida andrei and attendant changes in shape and adhesive capacity of constituitive cells. Developmental and Comparative Immunology 16, 95- 101.

Webb, B. A., Luckhart, S. (1996) Factors mediating short- and long-term immune suppression in a parasitized insect. Journal of Insect Physiology 42, 33-40.

Zambrano-Villa, S., Rosales-Borjas, D., Carrero, J. C., Ortiz- Ortiz, L. (2002) How protozoan parasites evade the immune response. Trends in Parasitology 18, 272-278.

Margaret Reinhart1,2* and Norman Dollahon2

1 University of the Sciences in Philadelphia, 600 S. 43rd Street, Philadelphia PA 19104, USA

2 Villanova University, Lancaster Avenue, Villanova, PA 19085, USA

Submitted September 6, 2002 * Accepted May 8, 2003

* E-mail corresponding author: m.reinha@usip.edu

Copyright Urban & Fischer Verlag 2003

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