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Formation of brown bodies in the coelomic cavity of the earthworm Eisenia fetida andrei and attendant changes in shape and adhesive capacity of constitutive cells
Developmental and Comparative Immunology,Vol. 16, pp. 95-101, 1992 Printed in the USA. All rights reserved.
0145-305XJ92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
FORMATION OF BROWN BODIES IN THE COELOMIC CAVITY OF THE EARTHWORM Eisenia fetida andrei AND ATTENDANT CHANGES IN SHAPE AND ADHESIVE CAPACITY OF CONSTITUTIVE CELLS Pierre Valembois, Maguy Lassegues, and Philippe Roch Department de Physiologie des Invert6br~s, URA CNRS 1138, Universite de Bordeaux 1, 33405 Talence, France
(Submitted November 1990; Accepted June 1991)
[]Abstract--The formation of brown bodies in the coelomic cavity may result from an aggregation of coelomocytes around offending foreign cells such as bacteria, gregarines, incompatible graft fragments, and altered self structures such as setae or necrotic muscle cells. The initial nodule rapidly increases in volume by aggregation of new coelomocytes and various waste particles. When a brown body has reached a diameter of 1-2 nun, its external cells flatten and lose their adhesiveness toward free coelomocytes or waste particles and its pigment rapidly darkens. Brown bodies play an important role in homeostasis. []Keywords - - Invertebrates; Earthworm defense; Encapsulation; Brown body; Coelomic cells; Capsule formation; Bacteria aggregation; Coelomocyte adhesion.
parasite in a few hours (2). In oligochaetes, the structure of these capsules, also called brown bodies, was first described nearly a century ago (3,4). Brown body formation requires generally several months and finally produces a convex disk-shaped mass of 1-2 mm in diameter. This mass, which darkens only during the last weeks of its maturation, migrates to the hindmost segments of the body cavity, where it is eliminated by autotomy (5). This study presents both light and scanning e l e c t r o n m i c r o s c o p i c (SEM) investigations of: (i) the mechanism of brown body formation; (ii) the adhesive activity exhibited by coelomocytes during brown body formation.
Introduction
Materials and Methods
Numerous invertebrates neutralise nonself or altered self material by elaborating, in their body cavities, multicellular nodules that often accumulate melanin as reviewed by Ratcliffe et al. (1). This mechanism of segregating waste material has mainly been documented in arthropods, in which it constitutes an efficient and rapid process of defense. This allows, for instance, the encapsulation of a
Earthworms and Distribution of Brown Bodies
Earthworms, Eisenia fetida andrei (E. f. andrei) (6), were collected from farm manure or derived from our laboratory colonies. Mature or immature brown bodies were removed by dissection from the posterior segments. In populations obtained from laboratory breedings, only one earthworm of five exhibited visible brown bodies in the posterior region of Address correspondence to Pierre Valemits body cavity, whereas one of two were bois, Drpartement de Physiologie des Inverfound in farm manure populations. trbrrs de l'Universit6 de Bordeaux 1, Institut de Biologie Animale, Avenue des Facultrs, These proportions were established after examining 650 laboratory worms and 400 33405 Talence, Crdex, France. 95
96
worms from farm manure collected in May 1990.
Harvesting and Treatment of Brown Bodies for Microscopy Studies Primordial brown bodies, in initial stages of formation, were collected from: (i) naive earthworms (n = 100); (ii) worms injected with LDs0 doses of Bacillus megaterium (n = 50); (iii) worms injected with equivalent doses of Aeromonas hydrophila (n = 50) (7). Injected segments were opened dorsally with scissors 12-96 h after injections and cellular contents of the coelomic cavity deposited on glass slides for direct observation under phase contrast microscopy (four of every five of the earthworms injected) or on 18-mm round glass coverslips (one of every five of the earthworms) for SEM observation after fixat i o n a n d c r i t i c a l p o i n t d r y i n g as previously described (8). Mature brown bodies for examination by SEM were fixed and dehydrated similarly. Observations were made using a JEM 840 A equipped with a goniometer specimen stage.
Relationships between Brown Bodies and Surrounding Tissues To elucidate the relationships between brown bodies and neighboring tissues, such as the coelomic epithelia, frozen transverse sections were made from pieces arising from 25 earthworms fixed 5 rain in 2% glutaraldehyde. Serial sections (10 ~m) were then cut using a Leitz freezing microtome, stained several minutes with a supravital dye such as Nile blue sulphate, toluidine blue, or neutral red, and rapidly observed.
Results Gross Appearance of Brown Bodies During collection of brown bodies from the coelomic cavity, we observed
P. Valembois eta/.
that the colour changed with size and localisation. In the anterior region of the body, multiceUular aggregates were generally yellowish and smaller than 0.5 mm in diameter. In the intermediary region (posteriorly to the clitellum), brown bodies increased in size and were red to brown-red. In the last five posterior segments, the size was large (I-2 mm) and the pigmentation distinctly brown. The term "brown b o d y " is therefore only correct when referring to large structures found in the posterior segments. Using the terms " n o d u l e " or " a g g r e g a t e " seems more suitable as generic terms. Except in the very hindmost segments, where brown bodies were completely free, nodules strongly adhered to the coelomic wall and required the use of scissors to be isolated. Histological observations of transverse sections of worms at different levels of the body agreed with the macroscopic observations. Nodules smaller than 1 mm in diameter exhibited strands of cellular material that bound them to the coelomic epithelium (Fig. 1). As the nodules became larger, they were bound to the coelomic epithelium by cellular processes, becoming more and more narrow before completely disappearing (Figs. 2 and 3). We never observed in the nodules the existence of an inner noncellular zone after using low concentration of aldehyde fixations not exceeding 1 h (9).
Phase Contrast Microscopy Examination of 120 small (150-250 p.m) nodules collected from 42 naive worms revealed the simultaneous presence of coelomocytes and foreign particles such as nematodes (less than 1% of the nodules observed), agglutinated bacteria (29%), and gregarines (7%). Most nodules (85%) contained tissue wastes (e.g., necrotic muscle cells) and especially setae (Table 1). These percentages
Brown body formation in earthworms
97
Figure 1. Transverse frozen section at 10 p.m through an immature nodule (toluidine blue stain). The nodule is closely bound to the coelomic layer coating the body wall (bw), the septa (s), and the intestinal wall (iw). Figure 2, Frozen section at 10 ~m through a nodule at a later step of maturation (Nile blue sulphate stain). When compared to Fig. 1, the nodule has increased in volume by fixing and aggregating coelomocytes and waste particles such as setae (arrow). The simultaneous presence in the same nodule of two distinct masses of agglutinated bacteria (encircled areas) suggests the possibility of growth of nodules by coalescence of initial aggregates. Figure 3. Frozen section at 10 p.m through a brown body in stage of maturation (toluidine blue stain). The mature brown body is completely free inside the coelomic cavity (compare with micrography of Fig. 1).
were probably underestimated with regard to the presence of bacteria and mainly gregarines, which were difficult to detect because of their small size and weak tendency to be agglutinated. When larger, most nodules simultaneously contained several categories of offending particles (bacteria, gregarines, setae, etc.) (Fig. 2).
SEM of Small Nodules When coelomic fluid was harvested 6-12 h after bacterial injection, we found numerous clusters (about 10 in each earthworm) of 10-20 coelomocytes aggregated together with agglutinated bacteria (Fig. 4). Similar results, in smaller numbers (one cluster as a mean in each
Table 1. Nature and number of foreign organisms or fragments of senescent tissue present in small nodules (diameter 150-250 ixm).* Offending Particles
Number of observations
Nematodes
Bacteria
Gregarines
Altered Self Structures
1
35
8
102
* One hundred twenty brown bodies arising from the coelomic cavity of 42 earthworms were observed in phase contrast microscopy. One hundred worms were dissected to collect their brown bodies, but 58 animals had no aggregate or only brown bodies of dimension greater than 250 p.m. In some cases, two or three types of foreign (or degenerative) particles were found in the same nodule.
3
CO
Brown body formation in earthworms
earthworm), were also observed in coelomic fluid cellular content from naive worms. Coelomocytes forming these clusters were of two types: (i) elongated morula-shaped cells identified as chloragocytes (8); (ii) round or oval 5- to 15Ixm leucocytes whose surfaces were smooth or bristled with short pseudopods. As also illustrated by Figure 4, contact surfaces between cells in clusters at 6-12 h were restricted to spots or narrow areas. Most clusters harvested from worms dissected 48 h after injecting bacteria were composed of 50-100 coelomocytes (Fig. 5). The contact surfaces between constitutive coelomocytes were generally more extensive than in clusters harvested at 6-12 h. The increase of reciprocal adhesive surfaces is accompanied by a flattening of the cells whose shape becomes homogeneous, making difficult the identification of nodular cells as chloragocytes or leucocytes.
SEM of Large Nodules When nodules of different sizes were compared by SEM at low magnification,
99
their external surface exhibited different features (Fig. 6). The surface of large brown bodies appeared smooth and homogeneously dark because of a uniform repartition of the gold palladium coat. In contrast, the surface of intermediary nodules was irregularly bristled with bright material independently of their orientation with respect to the electron beam. This bright aspect may have been caused by the phenomenon of "charging" as a consequence of an irregular cell surface difficult to coat homogeneously with gold palladium. At higher magnifications, the entire external surfaces of large brown bodies, as well as the smooth surfaced areas of intermediary nodules, were composed of a layer of flattened cells (Fig. 7). The limits between these cells became less and less visible when the surfaces of dark areas increased and were difficult to detect on the surfaces of large brown bodies (Fig. 8). At the surface of bright areas of intermediary nodules, at high magnification, we observed well-structured leucocytes or chloragocytes bound by elongated cytoplasmic processes to flattened underlying cells. In these areas, isolated
Figure 4. SEM micrograph of an initial nodule formed by the aggregation of about 20 leucocytes and chloragocytes (Chl) around a mass of agglutinated bacteria (delimited by arrows). These bacteria (Aeromonas hydrophi/a) were injected to the coelomic cavity 24 h before harvesting and fixing the coelomic fluid's cellular content. Note that coelomocytes forming the initial aggregate only adhere to each other by a limited part of their cell surface. Figure 5. SEM micrograph of a nodule harvested 48 h after injecting bacteria. Cells forming the nodule are more numerous and the contact surface between neighboring cells more extensive than at 24 h. Agglutinated to the flattened cells one can observe some of the inducing bacteria, Bacillus megaterium (arrows). Figure 6. SEM micrograph of isolated brown bodies at low magnification. On the left, there is a relatively young nodule bristled with bright material. The brown body, on the right, at a later stage of growth, is uniformly opaque except for a small bright area. Higher magnifications of the areas enclosed in the rectangles numbered 7, 8, and 9 are given in the following corresponding figures. Figure 7. At medium or high magnification, there are in bright areas spreading over the major part of the surface of the growing nodule, well-structured leucocytes or chloragocytes (Chl) bound to each other and to underlying cells by cytoplasmic processes (arrows). Also, there are individual microorganisms such as gregarine cysts (g) fixed at the surface of the nodule. Rgure 8. SEM micrograph of a dark area at the surface of an immature nodule. In dark areas whose surface increases as the nodule becomes older, external cells are closely joined and form a sort of flat pavement. Each individual cell presents an irregular and sinuous outline (arrows). Figure 9. Mature brown body observed in SEM at the end of its growth. Its surface is almost uniformly opaque except for limited areas that have maintained their ability to fix coelomocytes or waste particles such as setae (arrows).
100
gregarines, bacteria (isolated or in small clusters), and setae were commonly observed attached to the surfaces of the nodules (Fig. 9). Discussion In most cases, the formation of a brown body is initiated by a contact between free coelomocytes and foreign particles or altered self tissue fragments. It seems that the presence of a minimal volume of offending particles was required to initiate the formation of a brown body (isolated bacteria or gregarines had no initiating capacity). In fact, in contrast with what often happens in arthropods (2), nonself material introduced into the earthworm is not immediately encapsulated but begins to undergo a degradation process mediated by various humoral factors. For instance, bacteria observed in the initial aggregate were previously agglutinated and altered by humoral factors such as the protein system of 40-45 KDa (10,11) or lysozyme (12). Incompatible grafted tissues, which can also provoke the formation of brown bodies (13-15), are initially attacked by leucocytes and partially lysed by lysosomal hydrolases. Coelomocytes, in contact with altered self or nonself material, modify their adhesive activity. Present as free cells in the coelomic fluid, they change into adhesive cells mediating contact with neighboring cells and with altered particles. The problem of factors responsible for this change in adhesive activity will be investigated in light of information found in arthropods and especially in the crustacea, in which molecular determination of adhesion during encapsulation has been investigated extensively (16,17). The growth of the initial nodule results from a high adhesive capacity of external cells (18) occurring simultaneously with a flattening of underlying cells. Waste and pathogenic bacteria ad-
P. Valembois eta/.
hering to the nodule during this period include not only altered voluminous clusters but also undamaged microscopic cells such as isolated bacteria or wellstructured cysts of gregarines. Another mechanism for growth may likely be due to the coalescence of elementary nodules as suggested by the observation of nodules in which several distinct masses of agglutinated bacteria were present simultaneously (Fig. 2). Two different reasons may explain the attachment of the growing nodule to the peritoneal wall: (i) either free coelomocytes fixing at the surface of the nodule also manifest adhesive activity against the coelomic epithelial cells; or (ii) cells of the coelomic epithelium acquire adhesive abilities and membrane activity allowing them to bind to external cells of the nodule by cytoplasmic processes. Examination by light microscopy seems to favor the first hypothesis. Cells that adhere well to their neighbors or to the underlying coelomocytes flatten and progressively lose their ability to fix free coelomocytes and waste particles. From this difference in adhesive ability manifested by different cells, according to their shape or their state of senescence, it may be inferred that, besides external humoral factors, cellular membrane features also interact as mediators of adhesive activity. The end of the stage of growth is marked by a progressive loss of adhesiveness of the nodule's surface toward coelomic epithelial cells, free coelomocytes, and waste particles present in the coelomic fluid. Thus, this stage, when compared to the others, appears as a period of r e d u c e d e x c h a n g e b e t w e e n brown bodies and other tissues of the earthworm. N e v e r t h e l e s s , some exchange of information still occurs since the presence of mature brown bodies apparently induces the autotomy of the hindmost segments. Various observations support the hypothesis of a significant enzymatic activ-
Brown body formation in earthworms
101
ity of brown bodies during the stage of maturation. The darkening of the pigmentation is completed during this stage. The aggregation to the nodule of wellstructured coelomocytes, known to be rich in hydrolases (19-22) and in oxidative enzymes (23,24), occurs until a late period. In spite of their essentially descriptive
character, our data reveal a significant activity of cells involved in formation of brown bodies and particularly an important adhesive activity. This activity and also the initiation role played by offending material in the formation of brown bodies suggest that these structures actively participate in the immune defense of earthworms.
References 1. Ratcliffe, N. A.; Rowley, A. E; Fitzgerald, S. W.; Rhodes, C. P. Invertebrate immunity: basic concepts and recent advances. Int. Rev. Cytol. 97:183-349; 1985. 2. G6tz, E Encapsulation in arthropods. In: Breh61in, M., ed. Immunity in invertebrates. Bedim Springer Verlag; 1986:153-170. 3. Metchnikoff, E. Lemons sur la pathologic compar~e de l'inflammation. Paris: G. Masson; 1892:78-86. 4. Cu~not, L. Etudes physiologiques sur les oligoch~tes. Arch. Biol. 15:79-124; 1898. 5. Keilin, N. D. Parasitic autotomy of the host as a mode of liberation of coelomic parasites from the body of the earthworm. Parasitology 17:170-172; 1925. 6. Bouch6, M. B. Lombriciens de France. Ecologie et syst6matique. Ann. Zool. INRA Paris Publ. 77-82; 1972. 7. Lass~gues, M. ; Roch, Ph.; Vaiembois, P. Antibacterial activity of Eisenia fetida andrei coelomic fluid: Evidence, induction and animal protection. J. Invertebr. Pathol. 53:1-6; 1989. 8. Valembois, P.; Lass~gues, M. ; Roch, Ph.; Vaillier, J. Scanning electron microscopic study of the involvement of coelomic cells in earthworm antibacterial defense. Cell. Tissue Res. 240:479-484; 1985. 9. Poinar, G. O.; Hess, R. T. Immune response in the earthworm Aporrectodea trapezoides (Annelida), against Rhabditis pellio (Nematoda). Comp. Pathobiol. 3:69-84; 1977. 10. Roch, Ph.; Lass~gues, M.; Valembois, P.; Vaillier, J. Amino-acid composition and relationships of five earthworm defense proteins. Comp. Biochem. Physiol. 85B:747-751; 1986. 11. Roch, Ph.; Lass~gues, M.; Valembois, P. Antibacterial activity ofEiseniafetida andrei coelomic fluid: 3--Relationship with the polymorphism of hemolysins. Dev. Comp. Immunol. 15:27-32; 1991. 12. Lassaile, E; Lass~.gues, M.; Roch, Ph. Protein analysis of earthworm coelomic fluid: 4 - - E v idence, activity, induction and purification of Eisenia fetida andrei lysozyme (Annelidae). Comp. Biochem. Physiol. 91B:187-192; 1989.
13. Vaiembois, P. Evolution de la musculature d'un x6nogreffon de paroi du corps chez un lombricien. J. Microscopic 11:339-352; 1971. 14. Hostetter, R. K.; Cooper, E. L. Earthworm coelomocyte immunity. In: Cooper, E. L., ed. Invertebrate immunology; Contemporary topics in immunobiology. New York: Plenum Press; 1974:91-107. 15. Dales, R. E The basis of graft rejection in the earthworm Lumbricus terrestris and Eisenia foetida. J. Invertebr. Pathol. 32:264-277; 1978. 16. S6derh~ill, K. Fungal cell wail 13-1-3-glucans induce clotting and phenoloxidase attachment to foreign surfaces of crayfish hemocyte lysate. Dev. Comp. Immunol. 5:565-573; 1981. 17. Johansson, W.; S6derh~ill, K. Isolation and purification of a cell adhesion factor from crayfish blood cells. J. Cell Biol. 106:1795-1803; 1988. 18. Cameron, G. R. Inflammation in earthworms. J. Pathol. Bacteriol. 35-933-972; 1932. 19. Semal-Van Gansen, E Les cellules chloragog6nes des lombriciens. Bull. Biol. France Belgique 90:335-358; 1956. 20. Fischer, E. Histochemische untersuschunger tiber die metabolische aktivitfit des chloragosomen yon Lumbricus terrestris. Acta Histochem. 46:1-9; 1973. 21. Varute, A. T.; More, N. K. Lysosomal acid hydrolases in the chioragogen cells of earthworms. Comp. Biochem. Physiol. 45A:607635; 1973. 22. Stein, E. A.; Cooper, E. L. Cytochemical observations of coelomocytes from the earthworm Lumbricus terrestris. Histochem. J. 10:657-678; 1978. 23. Fischer, E. DOPA peroxidase activity in the chloragogen cells of the earthworm Lumbricus terrestris L. Acta Histochem. 63:210-233; 1978. 24. Valembois, P.; Seymour, J.; Roch, Ph. Evidence and cellular localization of an oxidative activity in the coelomic fluid of the earthworm Eisenia fetida andrei. J. Invertebr. Pathoi. 57:177-183; 1991.
0145-305XJ92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
FORMATION OF BROWN BODIES IN THE COELOMIC CAVITY OF THE EARTHWORM Eisenia fetida andrei AND ATTENDANT CHANGES IN SHAPE AND ADHESIVE CAPACITY OF CONSTITUTIVE CELLS Pierre Valembois, Maguy Lassegues, and Philippe Roch Department de Physiologie des Invert6br~s, URA CNRS 1138, Universite de Bordeaux 1, 33405 Talence, France
(Submitted November 1990; Accepted June 1991)
[]Abstract--The formation of brown bodies in the coelomic cavity may result from an aggregation of coelomocytes around offending foreign cells such as bacteria, gregarines, incompatible graft fragments, and altered self structures such as setae or necrotic muscle cells. The initial nodule rapidly increases in volume by aggregation of new coelomocytes and various waste particles. When a brown body has reached a diameter of 1-2 nun, its external cells flatten and lose their adhesiveness toward free coelomocytes or waste particles and its pigment rapidly darkens. Brown bodies play an important role in homeostasis. []Keywords - - Invertebrates; Earthworm defense; Encapsulation; Brown body; Coelomic cells; Capsule formation; Bacteria aggregation; Coelomocyte adhesion.
parasite in a few hours (2). In oligochaetes, the structure of these capsules, also called brown bodies, was first described nearly a century ago (3,4). Brown body formation requires generally several months and finally produces a convex disk-shaped mass of 1-2 mm in diameter. This mass, which darkens only during the last weeks of its maturation, migrates to the hindmost segments of the body cavity, where it is eliminated by autotomy (5). This study presents both light and scanning e l e c t r o n m i c r o s c o p i c (SEM) investigations of: (i) the mechanism of brown body formation; (ii) the adhesive activity exhibited by coelomocytes during brown body formation.
Introduction
Materials and Methods
Numerous invertebrates neutralise nonself or altered self material by elaborating, in their body cavities, multicellular nodules that often accumulate melanin as reviewed by Ratcliffe et al. (1). This mechanism of segregating waste material has mainly been documented in arthropods, in which it constitutes an efficient and rapid process of defense. This allows, for instance, the encapsulation of a
Earthworms and Distribution of Brown Bodies
Earthworms, Eisenia fetida andrei (E. f. andrei) (6), were collected from farm manure or derived from our laboratory colonies. Mature or immature brown bodies were removed by dissection from the posterior segments. In populations obtained from laboratory breedings, only one earthworm of five exhibited visible brown bodies in the posterior region of Address correspondence to Pierre Valemits body cavity, whereas one of two were bois, Drpartement de Physiologie des Inverfound in farm manure populations. trbrrs de l'Universit6 de Bordeaux 1, Institut de Biologie Animale, Avenue des Facultrs, These proportions were established after examining 650 laboratory worms and 400 33405 Talence, Crdex, France. 95
96
worms from farm manure collected in May 1990.
Harvesting and Treatment of Brown Bodies for Microscopy Studies Primordial brown bodies, in initial stages of formation, were collected from: (i) naive earthworms (n = 100); (ii) worms injected with LDs0 doses of Bacillus megaterium (n = 50); (iii) worms injected with equivalent doses of Aeromonas hydrophila (n = 50) (7). Injected segments were opened dorsally with scissors 12-96 h after injections and cellular contents of the coelomic cavity deposited on glass slides for direct observation under phase contrast microscopy (four of every five of the earthworms injected) or on 18-mm round glass coverslips (one of every five of the earthworms) for SEM observation after fixat i o n a n d c r i t i c a l p o i n t d r y i n g as previously described (8). Mature brown bodies for examination by SEM were fixed and dehydrated similarly. Observations were made using a JEM 840 A equipped with a goniometer specimen stage.
Relationships between Brown Bodies and Surrounding Tissues To elucidate the relationships between brown bodies and neighboring tissues, such as the coelomic epithelia, frozen transverse sections were made from pieces arising from 25 earthworms fixed 5 rain in 2% glutaraldehyde. Serial sections (10 ~m) were then cut using a Leitz freezing microtome, stained several minutes with a supravital dye such as Nile blue sulphate, toluidine blue, or neutral red, and rapidly observed.
Results Gross Appearance of Brown Bodies During collection of brown bodies from the coelomic cavity, we observed
P. Valembois eta/.
that the colour changed with size and localisation. In the anterior region of the body, multiceUular aggregates were generally yellowish and smaller than 0.5 mm in diameter. In the intermediary region (posteriorly to the clitellum), brown bodies increased in size and were red to brown-red. In the last five posterior segments, the size was large (I-2 mm) and the pigmentation distinctly brown. The term "brown b o d y " is therefore only correct when referring to large structures found in the posterior segments. Using the terms " n o d u l e " or " a g g r e g a t e " seems more suitable as generic terms. Except in the very hindmost segments, where brown bodies were completely free, nodules strongly adhered to the coelomic wall and required the use of scissors to be isolated. Histological observations of transverse sections of worms at different levels of the body agreed with the macroscopic observations. Nodules smaller than 1 mm in diameter exhibited strands of cellular material that bound them to the coelomic epithelium (Fig. 1). As the nodules became larger, they were bound to the coelomic epithelium by cellular processes, becoming more and more narrow before completely disappearing (Figs. 2 and 3). We never observed in the nodules the existence of an inner noncellular zone after using low concentration of aldehyde fixations not exceeding 1 h (9).
Phase Contrast Microscopy Examination of 120 small (150-250 p.m) nodules collected from 42 naive worms revealed the simultaneous presence of coelomocytes and foreign particles such as nematodes (less than 1% of the nodules observed), agglutinated bacteria (29%), and gregarines (7%). Most nodules (85%) contained tissue wastes (e.g., necrotic muscle cells) and especially setae (Table 1). These percentages
Brown body formation in earthworms
97
Figure 1. Transverse frozen section at 10 p.m through an immature nodule (toluidine blue stain). The nodule is closely bound to the coelomic layer coating the body wall (bw), the septa (s), and the intestinal wall (iw). Figure 2, Frozen section at 10 ~m through a nodule at a later step of maturation (Nile blue sulphate stain). When compared to Fig. 1, the nodule has increased in volume by fixing and aggregating coelomocytes and waste particles such as setae (arrow). The simultaneous presence in the same nodule of two distinct masses of agglutinated bacteria (encircled areas) suggests the possibility of growth of nodules by coalescence of initial aggregates. Figure 3. Frozen section at 10 p.m through a brown body in stage of maturation (toluidine blue stain). The mature brown body is completely free inside the coelomic cavity (compare with micrography of Fig. 1).
were probably underestimated with regard to the presence of bacteria and mainly gregarines, which were difficult to detect because of their small size and weak tendency to be agglutinated. When larger, most nodules simultaneously contained several categories of offending particles (bacteria, gregarines, setae, etc.) (Fig. 2).
SEM of Small Nodules When coelomic fluid was harvested 6-12 h after bacterial injection, we found numerous clusters (about 10 in each earthworm) of 10-20 coelomocytes aggregated together with agglutinated bacteria (Fig. 4). Similar results, in smaller numbers (one cluster as a mean in each
Table 1. Nature and number of foreign organisms or fragments of senescent tissue present in small nodules (diameter 150-250 ixm).* Offending Particles
Number of observations
Nematodes
Bacteria
Gregarines
Altered Self Structures
1
35
8
102
* One hundred twenty brown bodies arising from the coelomic cavity of 42 earthworms were observed in phase contrast microscopy. One hundred worms were dissected to collect their brown bodies, but 58 animals had no aggregate or only brown bodies of dimension greater than 250 p.m. In some cases, two or three types of foreign (or degenerative) particles were found in the same nodule.
3
CO
Brown body formation in earthworms
earthworm), were also observed in coelomic fluid cellular content from naive worms. Coelomocytes forming these clusters were of two types: (i) elongated morula-shaped cells identified as chloragocytes (8); (ii) round or oval 5- to 15Ixm leucocytes whose surfaces were smooth or bristled with short pseudopods. As also illustrated by Figure 4, contact surfaces between cells in clusters at 6-12 h were restricted to spots or narrow areas. Most clusters harvested from worms dissected 48 h after injecting bacteria were composed of 50-100 coelomocytes (Fig. 5). The contact surfaces between constitutive coelomocytes were generally more extensive than in clusters harvested at 6-12 h. The increase of reciprocal adhesive surfaces is accompanied by a flattening of the cells whose shape becomes homogeneous, making difficult the identification of nodular cells as chloragocytes or leucocytes.
SEM of Large Nodules When nodules of different sizes were compared by SEM at low magnification,
99
their external surface exhibited different features (Fig. 6). The surface of large brown bodies appeared smooth and homogeneously dark because of a uniform repartition of the gold palladium coat. In contrast, the surface of intermediary nodules was irregularly bristled with bright material independently of their orientation with respect to the electron beam. This bright aspect may have been caused by the phenomenon of "charging" as a consequence of an irregular cell surface difficult to coat homogeneously with gold palladium. At higher magnifications, the entire external surfaces of large brown bodies, as well as the smooth surfaced areas of intermediary nodules, were composed of a layer of flattened cells (Fig. 7). The limits between these cells became less and less visible when the surfaces of dark areas increased and were difficult to detect on the surfaces of large brown bodies (Fig. 8). At the surface of bright areas of intermediary nodules, at high magnification, we observed well-structured leucocytes or chloragocytes bound by elongated cytoplasmic processes to flattened underlying cells. In these areas, isolated
Figure 4. SEM micrograph of an initial nodule formed by the aggregation of about 20 leucocytes and chloragocytes (Chl) around a mass of agglutinated bacteria (delimited by arrows). These bacteria (Aeromonas hydrophi/a) were injected to the coelomic cavity 24 h before harvesting and fixing the coelomic fluid's cellular content. Note that coelomocytes forming the initial aggregate only adhere to each other by a limited part of their cell surface. Figure 5. SEM micrograph of a nodule harvested 48 h after injecting bacteria. Cells forming the nodule are more numerous and the contact surface between neighboring cells more extensive than at 24 h. Agglutinated to the flattened cells one can observe some of the inducing bacteria, Bacillus megaterium (arrows). Figure 6. SEM micrograph of isolated brown bodies at low magnification. On the left, there is a relatively young nodule bristled with bright material. The brown body, on the right, at a later stage of growth, is uniformly opaque except for a small bright area. Higher magnifications of the areas enclosed in the rectangles numbered 7, 8, and 9 are given in the following corresponding figures. Figure 7. At medium or high magnification, there are in bright areas spreading over the major part of the surface of the growing nodule, well-structured leucocytes or chloragocytes (Chl) bound to each other and to underlying cells by cytoplasmic processes (arrows). Also, there are individual microorganisms such as gregarine cysts (g) fixed at the surface of the nodule. Rgure 8. SEM micrograph of a dark area at the surface of an immature nodule. In dark areas whose surface increases as the nodule becomes older, external cells are closely joined and form a sort of flat pavement. Each individual cell presents an irregular and sinuous outline (arrows). Figure 9. Mature brown body observed in SEM at the end of its growth. Its surface is almost uniformly opaque except for limited areas that have maintained their ability to fix coelomocytes or waste particles such as setae (arrows).
100
gregarines, bacteria (isolated or in small clusters), and setae were commonly observed attached to the surfaces of the nodules (Fig. 9). Discussion In most cases, the formation of a brown body is initiated by a contact between free coelomocytes and foreign particles or altered self tissue fragments. It seems that the presence of a minimal volume of offending particles was required to initiate the formation of a brown body (isolated bacteria or gregarines had no initiating capacity). In fact, in contrast with what often happens in arthropods (2), nonself material introduced into the earthworm is not immediately encapsulated but begins to undergo a degradation process mediated by various humoral factors. For instance, bacteria observed in the initial aggregate were previously agglutinated and altered by humoral factors such as the protein system of 40-45 KDa (10,11) or lysozyme (12). Incompatible grafted tissues, which can also provoke the formation of brown bodies (13-15), are initially attacked by leucocytes and partially lysed by lysosomal hydrolases. Coelomocytes, in contact with altered self or nonself material, modify their adhesive activity. Present as free cells in the coelomic fluid, they change into adhesive cells mediating contact with neighboring cells and with altered particles. The problem of factors responsible for this change in adhesive activity will be investigated in light of information found in arthropods and especially in the crustacea, in which molecular determination of adhesion during encapsulation has been investigated extensively (16,17). The growth of the initial nodule results from a high adhesive capacity of external cells (18) occurring simultaneously with a flattening of underlying cells. Waste and pathogenic bacteria ad-
P. Valembois eta/.
hering to the nodule during this period include not only altered voluminous clusters but also undamaged microscopic cells such as isolated bacteria or wellstructured cysts of gregarines. Another mechanism for growth may likely be due to the coalescence of elementary nodules as suggested by the observation of nodules in which several distinct masses of agglutinated bacteria were present simultaneously (Fig. 2). Two different reasons may explain the attachment of the growing nodule to the peritoneal wall: (i) either free coelomocytes fixing at the surface of the nodule also manifest adhesive activity against the coelomic epithelial cells; or (ii) cells of the coelomic epithelium acquire adhesive abilities and membrane activity allowing them to bind to external cells of the nodule by cytoplasmic processes. Examination by light microscopy seems to favor the first hypothesis. Cells that adhere well to their neighbors or to the underlying coelomocytes flatten and progressively lose their ability to fix free coelomocytes and waste particles. From this difference in adhesive ability manifested by different cells, according to their shape or their state of senescence, it may be inferred that, besides external humoral factors, cellular membrane features also interact as mediators of adhesive activity. The end of the stage of growth is marked by a progressive loss of adhesiveness of the nodule's surface toward coelomic epithelial cells, free coelomocytes, and waste particles present in the coelomic fluid. Thus, this stage, when compared to the others, appears as a period of r e d u c e d e x c h a n g e b e t w e e n brown bodies and other tissues of the earthworm. N e v e r t h e l e s s , some exchange of information still occurs since the presence of mature brown bodies apparently induces the autotomy of the hindmost segments. Various observations support the hypothesis of a significant enzymatic activ-
Brown body formation in earthworms
101
ity of brown bodies during the stage of maturation. The darkening of the pigmentation is completed during this stage. The aggregation to the nodule of wellstructured coelomocytes, known to be rich in hydrolases (19-22) and in oxidative enzymes (23,24), occurs until a late period. In spite of their essentially descriptive
character, our data reveal a significant activity of cells involved in formation of brown bodies and particularly an important adhesive activity. This activity and also the initiation role played by offending material in the formation of brown bodies suggest that these structures actively participate in the immune defense of earthworms.
References 1. Ratcliffe, N. A.; Rowley, A. E; Fitzgerald, S. W.; Rhodes, C. P. Invertebrate immunity: basic concepts and recent advances. Int. Rev. Cytol. 97:183-349; 1985. 2. G6tz, E Encapsulation in arthropods. In: Breh61in, M., ed. Immunity in invertebrates. Bedim Springer Verlag; 1986:153-170. 3. Metchnikoff, E. Lemons sur la pathologic compar~e de l'inflammation. Paris: G. Masson; 1892:78-86. 4. Cu~not, L. Etudes physiologiques sur les oligoch~tes. Arch. Biol. 15:79-124; 1898. 5. Keilin, N. D. Parasitic autotomy of the host as a mode of liberation of coelomic parasites from the body of the earthworm. Parasitology 17:170-172; 1925. 6. Bouch6, M. B. Lombriciens de France. Ecologie et syst6matique. Ann. Zool. INRA Paris Publ. 77-82; 1972. 7. Lass~gues, M. ; Roch, Ph.; Vaiembois, P. Antibacterial activity of Eisenia fetida andrei coelomic fluid: Evidence, induction and animal protection. J. Invertebr. Pathol. 53:1-6; 1989. 8. Valembois, P.; Lass~gues, M. ; Roch, Ph.; Vaillier, J. Scanning electron microscopic study of the involvement of coelomic cells in earthworm antibacterial defense. Cell. Tissue Res. 240:479-484; 1985. 9. Poinar, G. O.; Hess, R. T. Immune response in the earthworm Aporrectodea trapezoides (Annelida), against Rhabditis pellio (Nematoda). Comp. Pathobiol. 3:69-84; 1977. 10. Roch, Ph.; Lass~gues, M.; Valembois, P.; Vaillier, J. Amino-acid composition and relationships of five earthworm defense proteins. Comp. Biochem. Physiol. 85B:747-751; 1986. 11. Roch, Ph.; Lass~gues, M.; Valembois, P. Antibacterial activity ofEiseniafetida andrei coelomic fluid: 3--Relationship with the polymorphism of hemolysins. Dev. Comp. Immunol. 15:27-32; 1991. 12. Lassaile, E; Lass~.gues, M.; Roch, Ph. Protein analysis of earthworm coelomic fluid: 4 - - E v idence, activity, induction and purification of Eisenia fetida andrei lysozyme (Annelidae). Comp. Biochem. Physiol. 91B:187-192; 1989.
13. Vaiembois, P. Evolution de la musculature d'un x6nogreffon de paroi du corps chez un lombricien. J. Microscopic 11:339-352; 1971. 14. Hostetter, R. K.; Cooper, E. L. Earthworm coelomocyte immunity. In: Cooper, E. L., ed. Invertebrate immunology; Contemporary topics in immunobiology. New York: Plenum Press; 1974:91-107. 15. Dales, R. E The basis of graft rejection in the earthworm Lumbricus terrestris and Eisenia foetida. J. Invertebr. Pathol. 32:264-277; 1978. 16. S6derh~ill, K. Fungal cell wail 13-1-3-glucans induce clotting and phenoloxidase attachment to foreign surfaces of crayfish hemocyte lysate. Dev. Comp. Immunol. 5:565-573; 1981. 17. Johansson, W.; S6derh~ill, K. Isolation and purification of a cell adhesion factor from crayfish blood cells. J. Cell Biol. 106:1795-1803; 1988. 18. Cameron, G. R. Inflammation in earthworms. J. Pathol. Bacteriol. 35-933-972; 1932. 19. Semal-Van Gansen, E Les cellules chloragog6nes des lombriciens. Bull. Biol. France Belgique 90:335-358; 1956. 20. Fischer, E. Histochemische untersuschunger tiber die metabolische aktivitfit des chloragosomen yon Lumbricus terrestris. Acta Histochem. 46:1-9; 1973. 21. Varute, A. T.; More, N. K. Lysosomal acid hydrolases in the chioragogen cells of earthworms. Comp. Biochem. Physiol. 45A:607635; 1973. 22. Stein, E. A.; Cooper, E. L. Cytochemical observations of coelomocytes from the earthworm Lumbricus terrestris. Histochem. J. 10:657-678; 1978. 23. Fischer, E. DOPA peroxidase activity in the chloragogen cells of the earthworm Lumbricus terrestris L. Acta Histochem. 63:210-233; 1978. 24. Valembois, P.; Seymour, J.; Roch, Ph. Evidence and cellular localization of an oxidative activity in the coelomic fluid of the earthworm Eisenia fetida andrei. J. Invertebr. Pathoi. 57:177-183; 1991.