immune defense and biological responses induced by toxics ...€¦ · annelida react to physical...

Immune defense and biological responses inducedby toxics in Annelida1

André Dhainaut and Patrick Scaps

Abstract: The phylum Annelida comprises primitive coelomates that possess specially developed cellular immunityagainst pathogens. Active phagocytosis by coelomocytes occurs in the struggle against bacteria in Polychaeta andOligochaeta. Encapsulation plays an important role in defense against parasites, and experimental studies have demon-strated that cooperation between different coelomocyte populations occurs in this process. Spontaneous cytotoxicity ofcoelomocytes against xenogenic or allogenic cells is analogous with that of vertebrate natural killer cells. Graft rejec-tion is a model for studying the activity of these cells. Accelerated rejection following multiple transplantation revealsthat the cellular immune defense system has a short-term memory. In humoral immunity, agglutinins aggregate foreignmaterial and their level is enhanced by antigens; in Annelida, however, no specificity analogous to vertebrate antibodieshas been revealed, except for weak specificity of some antigen-binding proteins. Hemolytic substances have been de-tected, particularly in Oligochaeta, where a fetidin possesses bactericidal activity. Lysozyme and some antibacterial pro-teins also occur in Polychaeta. Annelida react to physical and chemical insults by various processes. These responsesare mainly due to synthesis of stress-induced proteins, inhibition of enzyme activity, and modulation (inhibition orstimulation) of the activity of enzymes involved in the detoxification of xenobiotics. Moreover, these responses fre-quently differ from those of vertebrates, particularly in terms of the nature of inducers. In other respects, these re-sponses are extremely variable in Annelida, even in closely related species.Reviews / Synthèses 253

Résumé: Les annélides sont des coelomates primitifs dont l’immunité cellulaire contre les agents pathogènes est parti-culièrement développée. La phagocytose intense opérée par les coelomocytes assure la lutte contre les bactéries, aussibien chez les polychètes que chez les oligochètes. L’encapsulation intervient davantage dans la lutte contre les parasi-tes. Des expériences ont mis en lumière la coopération de diverses populations de coelomocytes dans ce processus. Lacytotoxicité spontanée des coelomocytes contre les cellules xénogènes et allogènes n’est pas sans rappeler celle des cel-lules lymphocytes tueurs des vertébrés. Le rejet de greffe peut servir de modèle dans l’étude de l’activité de ces cellu-les. À la suite de greffes consécutives, le temps de rejet devient de plus en plus court, ce qui reflète la mémoire àcourt terme des cellules du système immunitaire. Dans l’immunité humorale, les agglutinines agglomèrent des particu-les étrangères et leur teneur est stimulée par ces antigènes. Cependant, chez les annélides, il ne semble pas y avoir despécificité comparable à celle des anticorps de vertébrés, sauf peut-être une faible spécificité dans le cas de certainesprotéines qui se lient aux antigènes. Des substances hémolytiques ont été détectées, particulièrement chez des oligochè-tes où a également été trouvée une fétidine à propriétés bactéricides. Du lysozyme et quelques protéines antibactérien-nes ont également été rencontrés chez les polychètes. Les annélides réagissent de diverses façons aux assauts physiqueset chimiques de leur environnement, par la synthèse des protéines due au stress, par l’inhibition de l’activité enzyma-tique et par la modulation (inhibition ou stimulation) des enzymes responsables du processus de détoxication des xéno-biotiques. Ces réponses sont souvent différentes de celles observées chez des vertébrés, particulièrement en ce quiconcerne la nature des agents inducteurs. À d’autres égards, les réactions sont extrêmement variables au sein du phy-lum des annélides, même chez des espèces très apparentées.

Can. J. Zool.79: 233–253 (2001) © 2001 NRC Canada

233

DOI: 10.1139/cjz-79-2-233

Received October 21, 2000. Accepted September 29, 2000. Published on the NRC Research Press Web site on February 9, 2001.

A. Dhainaut. Laboratoire d’Endocrinologie des Annélides, Unité Propre de la Recherche Scientifique Assocée A 8017 CentreNational de la Recherche Scientifique, F-59655 Villeneuve d’Ascq CEDEX, France.P. Scaps.Laboratoire Ecosystèmes Littoraux et Côtiers, Unité Propre de la Recherche Scientifique Assocée A 8013, CentreNational de la Recherche Scientifique, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d’Ascq CEDEX,France.

1This review is one of a series dealing with aspects of the biology of the phylum Annelida. This series is one of several virtualsymposia on the biology of neglected groups that will be published in the Journal from time to time.

Biology of neglected groups: Annelida /Biologie des groupes négligés : Annelida

J:\cjz\cjz79\cjz-02\Z00-196.vpFriday, February 02, 2001 9:36:26 AM

Color profile: DisabledComposite Default screen

1. Introduction 2342. Immunity in Annelida 234

2.1. Introduction 2342.2. Cell immunity 235

2.2.1. Phagocytosis 2352.2.2. Encapsulation response 235

2.2.2.1. Encapsulation and parasitism 2362.2.2.2. Experimental encapsulation 236

2.2.3. Grafts 2362.2.4. Cytotoxic cells 237

2.3. Humoral immunity 2372.3.1. Agglutinins and hemolysins 238

2.3.1.1. Agglutinins 2382.3.1.2. Antigen-binding proteins 2382.3.1.3. Hemolysins (directed against

erythrocytes) 2382.3.1.4. Hemolytic and antibacterial proteins 239

2.3.2. Other antibacterial molecules 2392.3.2.1. Lysozyme 2392.3.2.2. Bactericidal factors 2392.3.2.3. Antibacterial protein 2402.3.2.4. Enkelytin 240

2.4. Influence of toxics on the immune response:utilization as biomarkers 240

2.4.1. Cellular response 2402.4.2. Humoral response 240

3. Physical and chemical insults 2403.1. Introduction 2403.2. Inhibition of activity: cholinesterase model 241

3.2.1. Characterization and functions 2413.2.1.1. Acetylcholinesterases 2413.2.1.2. Propionylcholinesterases 241

3.2.2. Inhibition of ChE activity byorganophosphate andcarbamate pesticides 241

3.2.3. Effects of exposure to heavy metals 2423.3. Stress-induced proteins 242

3.3.1. Heat-shock proteins 2423.3.1.1. Characteristics and functions 2423.3.1.2. Induction of HSPs 242

3.3.2. Metal-binding proteins 2423.3.2.1. Metallothioneins 242

3.3.2.1.1. Structure and properties 2423.3.2.1.2. Inducibility ofL. rubellusMT 243

3.3.2.2. Metalloproteins 2433.3.2.2.1. Metallothionein-like proteins 2433.3.2.2.2. Non-metallothionein-like proteins 244

3.3.2.3. Non-metallothionein cysteine-richprotein 244

3.4. Enzymes involved in the detoxification ofxenobiotics 244

3.4.1. The mixed-function oxidase enzyme system 2453.4.1.1. Phase 1 cytochrome P-450

mediated MFO 2453.4.1.1.1. Isolation and characterization of

the cytochrome P-450dependentmono-oxygenase system 246

3.4.1.2. Phase 2 conjugation enzyme:glutathioneS-transferases 246

3.4.1.2.1. Isolation and characterization ofglutathioneS-transferases 246

3.4.1.2.2. Inducibility of GSTs 247

3.4.2. Oxidative stress and antioxidant defenses 2474. Conclusion 2475. References 248

1. Introduction

During the course of evolution, Annelida have developeddefense strategies against various pathogens. These patho-gens are, firstly, bacteria living in water or soil that are in-gested during feeding or introduced into the body followinginjury. Parasites, particularly larval forms, which representthe dissemination phase, are an other important group ofpotentially pathogenic agents. Reaction against these livingpathogens constitutes immune defense.

Physical and chemical insults occurred in the past (volca-nic eruptions, exposure to metals through erosion of naturalveins, etc.), but they have increased greatly as a result of hu-man activities (release of industrial heavy metals and emis-sion of various pollutants). Defense mechanisms induced bythe presence of these toxic materials are less specific thanimmune reactions and induce various responses: some de-press physiological activities, while others enhance the syn-thesis of molecules required for detoxification.

Only a limited number of species in the three classes ofAnnelida have been investigated in this respect: in particular,except for neuroimmunity (see Salzet 2001), little is knownabout defense mechanisms in Achaeta.

2. Immunity in Annelida

2.1. Introduction

Immunity can be defined as the natural defenses of the or-ganism (self) against external invasions (non-self) from theenvironment. Polychaeta, which are essentially marine worms,are aggressed by bacteria living in seawater and sedimentand also by numerous parasites. Most Oligochaeta are incontact with bacteria in the soil. During evolution, adapta-tion has occurred through increasing efficiency of both celland humoral immunity.

For a comprehensive survey of immune processes, it isnecessary to review the anatomy of Polychaeta andOligochaeta.These worms are characterized by the presenceof two compartments containing free cells: (i) the blood sys-tem with hemocytes; this compartment does not seem to beactively involved in immunity; and (ii ) the coelom, whichcontains several populations of cells.

An extensive review of older papers describing the fea-tures of hemocytes and coelomocytes in many families ofPolychaeta, determined by means of light microscopy, waspublished by Dales and Dixon (1981). Various terms havebeen used by the authors of these older papers: amoebocyte,lymphocyte-like, etc. For consistency in terminology, only“granulocyte” and “eleocyte” (a term introduced by Romieu1923) will be used in this review.

In the family Nereididae, “granulocyte” was introduced byBaskin (1974) for cells containing many electron-dense gran-ules. InNereis(Hediste) diversicolor, three types of granulo-cytes have been described, first from their morphology stud-ied by electron microscopy (EM) (Dhainaut 1984a; Dhainautand Porchet-Henneré 1988) and then with the aid of mono-

© 2001 NRC Canada

234 Can. J. Zool. Vol. 79, 2001



clonal antibodies (Porchet-Henneré et al. 1987a; Porchet-Henneré 1990). Type I granulocytes, termed G1 cells(observedalso in Arenicolidae by Dales and Dixon 1981),are large, fusiform cells that contain two types of cyto-plasmic granules and bundles of microfilaments that extendthroughout the cell. Type II granulocytes (G2 cells) havefrequently been observed in Polychaeta. They were firstmentioned as granular amoebocytes occurring in several fam-ilies and described, using EM, in Nereididae (Baskin 1974;Dhainaut 1984a), Terebellidae (Eckelberger 1976), andArenicolidae (Dales and Dixon 1981). G2 granulocytes haveno microfilaments and their cytoplasmic granules are morenumerous than in type I cells. Abundant phagocytic vacuolesare distributed in the cortical cytoplasm. In vitro, G2 granu-locytes produce hyaline veil-like pseudopodia, and adhere tothe substrate by means of radially distributed actin fibers.Type III granulocytes (G3) are small cells. They have a highnucleoplasmic ratio and contain a few rounded granules.They do not adhere to glass slides. The eleocytes are charac-terized by their abundant nutritive inclusions. These cellsshow close analogy with the chloragogen-cell lineage ofOligochaeta.

Despite the advent of ultrastructural studies, the classifica-tion of coelomocytes in Oligochaeta remains confused(Jamieson1981). Valembois (1971) provided a precise clas-sification based on both morphology and origin of the coelo-mocytes inEisenia fetida,but it is only partially compatiblewith the description of coelomocytes fromLumbricus terrestrisby Linthicum et al. (1977b). Those authors recognize threetypes (their apparent equivalent in Valembois’ system is givenin parentheses): (1) lymphocytic coelomocyte types I and II(lymphocytoid cells, splancnopleural lineage); (2) granulocyticcoelomocyte type I (macrophage, splanchnopleural lineage)and type II (late lymphocytoid); (3) inclusion-containing coelo-mocyte (possibly vacuolar leucocyte).

Chloragocytes (chloragogen cells) are specialized peritonealcells of the intestine and dorsal blood vessel, which detachfrom the peritoneum and differentiate into eleocytes, whichremain free in the coelomic fluid (CF). The chloragocytesand eleocytes have been said to resemble the vertebrate liverin function: the metabolism and storage of glycogen andlipids and excretion (see Jamieson 1981).

All these coelomic cells are implicated to various degreesin the process of cell immunity and, through their secretions,in humoral immunity.

2.2. Cell immunity

2.2.1.Phagocytosis

This process, one of the oldest defense mechanisms in theAnimal Kingdom, is usually linked with the induction of anoxidative burst of metabolism. Production of highly reactiveoxygen radicals represents an effective way of destroyingengulfed microorganisms.

In Polychaeta, active pagocytosis occurs in the struggleagainst bacteria. Experimentally, this process has often beenexamined by injecting small particles such as ferritin, indiaink, or thorium dioxide, which are taken up by endocytosisand stored in the vacuoles of the coelomic cells. This uptakemay be followed by partial degranulation of the cells. InTerebellidae and Arenicolidae, Braunbeck and Dales (1984)

observed that larger particles such as vertebrate erythro-cytes are also taken up by G2 granulocytes by means ofphagocytosis. Thephagocytic cells are then carried to theheart bodies and the chloragogen tissues.

An important function of granulocytes is the clearanceof bacteria from the coelom. In Arenicolidae, those ofMicroccussp. appeared to be engulfed by phagocytic cells,which revealed strong phosphatase activity (Dales and Dixon1981). In the same family, Fitzgerald and Ratcliffe (1982)noted a reduction in the number of free circulating coelomo-cytes and the appearance of cellular aggregates after inocu-lation with Gram-positive and Gram-negative bacteria. InNereididae, whenVibrio alginolyticus, previously labelledby incorporation of tritiated thymidine, were injected in thecoelomic cavity (A. Dhainaut, unpublished data), the radio-activity of the CF dropped quickly. Simultaneously, the num-ber of circulating granulocytes decreased, whereas the num-ber of eleocytes did not change. EM observations revealedthat bacteria were phagocytosed by granulocytes and that G2granulocytes were especially active in antibacterial defense(Dhainaut and Porchet-Henneré 1988).

The role of the eleocytes is rather enigmatic. These cellscontain abundant storage material (glycogen and fat drop-lets). In female Nereididae, macromolecules synthesized bythe eleocytes play an important role in vitellogenesis. Eleocytesappear to be the site of production of a complex molecule,vitellogenin, the precursor of the oocyte yolk glycoprotein(Dhainaut 1984b; Fischer and Dhainaut 1985; Baert andSlomianny 1987).

Immediately prior to spawning, the muscle cells ofNereididae undergo histolysis during metamorphosis to theswimming epitokous form (Dehorne and Defretin 1933). EMinvestigations inNereis pelagicaandN. diversicolor(Dhainaut1966, 1984a) andNereis longissima(Baskin 1974) revealedthat the eleocytes are specialized for phagocytosis of musclefragments (sarcolytes). Eleocytes restrict their phagocytosisto the sarcolytes, which are degenerating “self” components.They never contribute to phagocytosis of the “non-self,” suchas bacteria.

In Oligochaeta, all coelomocyte types inL. terrestris, withthe exception of chloragogen cells, produce pseudopodia andare capable of phagocytosis (Stein et al. 1977). In this spe-cies, phagocytotic activity of leucocytes is enhanced by thevertebrate opsonins IgG and complement C3b fragment (Steinand Cooper 1981; Laulan et al. 1988). InE. foetida, Dalesand Kalac (1992) followed phagocytosis of three species ofbacteria and confirmed the role of the coelomocytes: thebacteria multiply in vitro in CF devoid of coelomocytes.However, repeated vaccination did not affect the rate ofphagocytosis. The processes of both bacterial clearance(Valembois et al. 1993) and generation of reactive oxygenspecies (Valembois and Lassegues 1995) have been reportedin E. fetida.

2.2.2.Encapsulation response

A defense reaction that neutralizes foreign bodies that aretoo large for phagocytosis is found in all invertebrates (seethe review in Ratcliffe et al. 1985). In the polychaeteN. diversicolor, brown tumors containing degeneratingoocytes or chaetae were described some time ago (Thomas

© 2001 NRC Canada

Reviews / Synthèses 235



1930), and the formation of capsules can be experimentallyinduced (Dales 1983). In the oligochaeteE. fetida, formationof brown bodies in the coelomic cavity may result from ag-gregation of coelomocytes around offending foreign cellssuch as bacteria, gregarines, incompatible graft fragments, ormodified “self structures” (Valembois et al. 1992). Lipofuscinand melanin have been detected in these brown bodies(Valembois et al. 1994).

2.2.2.1.Encapsulation and parasitism

In Polychaeta, encapsulation plays an important role indefense against parasites. As early as 1904, Brasil (cited byDales and Dixon 1981) described in some detail the reactionof Pectinaria korenicells to the gregarineUrospora lagidis,concluding that the amoebocytes encapsulated the sporozoitesonly when the parasites were developing. In cirratulids para-sitized by Gonospora longissima, the amoebocytes wereable to destroy the living trophozoites (Caullery and Mesnil1898, cited by Dales and Dixon 1981).

More recently, the development of a coelomic coccidian,Coelotropha durchoni, in N. diversicolor(Vivier and Porchet-Henneré 1964; Porchet-Henneré and Dugimont 1992) con-stituted an interesting model for analyzing a host–parasiterelationship. The coccidian develops successfully in its hostbecause of its ability to circumvent the host’s internal de-fense system. First, it avoids phagocytes by penetrating othercells, principally eleocytes and muscle cells, where it under-goes a phase of intracellular development. After it becomesextracellular, a thick coat protects it from attack by granulo-cytes. This coat then ruptures to permit fertilization, then athick cystic envelope forms to protect the fertilized zygotes.Parasites that have lost their coat and remain unfertilized aresurrounded with granulocytes and destroyed by encapsula-tion. A hormonal correlation exists between the biologicalcycles of the parasite and its host (Porchet-Henneré 1969).Under natural conditions, infection by the coelomic coccidianstage occurs in mature worms, which at this stage undergo astrong decrease in their neuroendocrine activity. Experimentally,when the brain of the worm is removed, the intracellularstages ofC. durchonialso undergo considerable growth andthe construction of the coat is enhanced.

2.2.2.2.Experimental encapsulation

The encapsulation process has been experimentally stud-ied in N. diversicolor, using implanted latex beads as micro-carriers (Porchet-Henneré et al. 1987b). Cooperation betweendifferent coelomocyte populations in their responses has beeninvestigated by transmission electron microscopy (TEM) andscanning electron microscopy (SEM) and using monoclonalantibodies (Porchet-Henneré 1990).

In the minutes following implantation of the foreign body(latex bead), the first cells to come into contact with it wereG3 cells, which are reactive to the monoclonal antibodyCC13. Almost immediately after fixation to the surface ofthe bead, these cells underwent lysis. The nucleus, cellorganites, and dense granules were extruded and all formeda coating around the implant.

So G3 cells are involved in the first step in recognizingthe “non-self” character of the implant. This process proba-bly implies the existence of membrane receptors. This pro-cess is poorly understood in Polychaeta. Lectins, with their

specific sugar-binding properties, have been used to analyzethe glycoconjugates composing the surface membrane of bloodcells from a variety of invertebrate species (for a review seeYoshino 1986), and the capacity of the host to carry out en-capsulation could be related to certain cell types. Using thismethod, M’Beri et al. (1988) studied the nature of membranereceptors in coelomocyte subpopulations inN. diversicolorand showed that G3 cells can be distinguished from othergranulocytes by their affinity for a specific lectin-bindingfucose residue.

Several hours after implantation, numerous granulocytes,mainly G2 cells, are recruited to the latex bead, where theybecome flattened and stacked to form concentric sheetsaround the foreign body. G2 cells are linked by numerousdesmosomes. Degranulation occurs in the inner sheets ofcoelomocytes and gives rise to a large amount of electron-dense fluid, which runs into the intercellular spaces andflows onto the surface of the implant.

The material produced by the G2 cells during the immunereaction of encapsulation is brown. Cytochemistry has estab-lished its melanic nature. Moreover, the G2 cells are capableof producing the enzyme phenoloxidase, which triggers mel-anin biosynthesis (Porchet-Henneré and Vernet 1992). Theenzyme was detected in vacuoles and the Golgi apparatus ofthe granulocytes, and was activated by the presence of theforeign bodies. The G2 cells may therefore be compared to amelanocyte in which melanin is not stored as in mammalcells, but immediately extruded following synthesis in theform of a thick fluid. In arthropods, it has been found that inaddition to their function in encapsulation, intermediates in themelanin pathway are cytotoxic and fungistatic and partici-pate in hemolymph coagulation (Johansson and Soderhall1989). The same features may be postulated forN. diversicolor;degranulation of G2 cells has been shown to kill parasites(Porchet-Henneré et al. 1987a). The earthwormE. fetidashowed evidence of lipofuscin and melanin production in thebrown bodies, which also resulted from aggregation ofcoelomocytes (Valembois et al. 1994).

Encapsulation is a complex process that implies coopera-tion between the different granulocyte populations (eleocytesare never involved). Very little is known about the factors in-volved in cell recognition in Polychaeta. Interleukin-like 1(IL-1) has been detected in the brain and eleocytes ofN. diversicolor by immunnocytochemisty (Paemen et al.1992). Preliminary experiments in this species suggest thatIL-1 could induce chemotaxis and stimulate the granulocytes(E. Porchet-Henneré, unpublished data).

2.2.3.Grafts

There have been few studies of graft processing inPolychaeta, and observations are restricted to the familyNereididae. Grafts of various organs (parapods, body wall,etc.) have been exchanged between individuals of the sameNereis species (allograft) or between species (xenograft)(Boilly-Marer 1979). Development of the grafts and of thehost has been followed for 1 or 2 years until normalreproduction (and death) of the grafted worms occurred.Autografts are always accepted. For both allografts andxenografts, the frequency of rejection is greatest about 2 weeksafter transplantation. However, important differences areobserved according to the origin of the grafts. When

© 2001 NRC Canada

236 Can. J. Zool. Vol. 79, 2001



© 2001 NRC Canada


N. pelagica is used as the host, the rate of rejection ofallografts is moderate: from 6% (body-wall graft) to 24%(parapod graft). In the case of xenografts, two types ofresponses can be distinguished: (1) no compatible inter-specific associations; grafts fromN. diversicolor, Perinereiscultifera, andPlatynereis massiliensiswere rejected (100%);and (2) semicompatible interspecific associations; somegrafts of body-wall tissue fromN. longissima(25%) orNereisirrorata (34%) were not rejected.

Tolerance of xenografts is very unusual in the AnimalKingdom, although durable interspecific associations form-ing chimerae have been constructed for nemertines (Bierneand Langlet 1974; Langlet and Bierne 1977). In Nereididae,tolerance could be related to some antigenic identity be-tweenN. pelagicaand the two semicompatible species, iden-tity that could be associated with their close phylogeneticrelationship. Unfortunately, the taxon Nereididae is mono-phyletic (Fauchald and Rouse 1997) and little is knownabout the relationships between species.

Graft development has been extensively investigated inOligochaeta. InL. terrestris, rejection of xenografts byE. fetidahas been carefully studied by Linthicum et al. (1977b), usingEM. Two major events occurred: a generalized inflammatoryresponse and an immunospecific response. The generalizedinflammatory response (within 1–3 days) appears to be re-lated primarily to wound-healing. In sham-operated controls,autografts, and xenografts, granulocytic coelomocytes mi-grate to the graft site and participate in closing the wound.In the immunospecific response (days 11–13), lymphocyticgranulocytes, which are present in xenografts, engage in ac-tive phagocytosis of muscle fibers. Intervention by this celltype is not observed in autografts and sham-operated controls.Adaptive coelomocyte transfer experiments confirm that graftrejection has a immunospecific component mediated by “ac-tivated” coelomocytes (Valembois 1971). Valembois (1974)presents a somewhat different interpretation of the results ofxenograft experiments employingE. fetida as a host andAllolobophora caliginosaas a xenograft. The first-set graftis destroyed, apparently as a result of induced autolysis in-volving synthesis and release of lysosomal enzymes by thetransplanted tissues. Destruction of the second-set graft resultsfrom coelomocytic invasion, as with the first-set xenograftof L. terrestris.

Second-set xenografts fromE. fetida transplanted ontoL. terrestris hosts undergo accelerated rejection if they aregrafted 1 week after the first-set xenografts. In contrast,second-set xenografts transplanted at longer intervals afterfirst-set grafts undergo no accelerated rejection. The essentialvariable in these experiment has been the timing of second-set transplantation. Accelerated rejection of repeated xenograftsis apparently one measure of the short-term “memory” of thecellular immune defense system and probably results fromthe intense responses of coelomocytes activated by a firsttransplant that are still present at the time of second-setgrafting (Cooper 1971, 1980). Thus, the annelids studieddemonstrate recognition of self involving an efficient cellu-lar response in the form of attacking and destroying non-selftissue.

2.2.4.Cytotoxic cells

The study of graft rejection has emphasized the role of

cell cytotoxicity. The natural cytotoxic activity of leucocyteswas first demonstrated by Boiledieu and Valembois (1977) inSipunculoidea, a group related to Annelida. Then spontaneouscytotoxicity was investigated in vitro in both Polychaeta andOligochaeta.

Evidence of the presence of cytotoxic cells inN. diversicolorwas reported by Porchet-Henneré et al. (1992). Severalpopulations of coelomic cells were mixed in vitro withArenicola sp. coelomocytes or vertebrate erythrocytes (hu-man or mouse). After 20 min incubation, G3 granulocytes(revealed by the antibody CC10) bind very tightly to the for-eign cells. SEM examination showed that many filopodia ofG3 cells extend over and stick to the erythrocytes. TEMrevealedthat the positions of the Golgi apparatus and centro-some in G3 cells were rearranged, and they were directed to-wards the target as soon as they became bound to it. Thenthe G3 granulocytes released electron-dense granules byexocytosis onto the foreign cells, which finally underwentlysis. Porelike structures were observed on the membrane oflysed cells after they came into contact with coelomic cells,suggesting that a pore-forming protein (perforine-like?) mightbe operating in theNereissystem.

In Oligochaeta, spontaneous allogenic cytotoxicity of thecoelomocytes ofE. fetida (Valembois et al. 1980) andL. terrestris (Suzuki and Cooper, 1995a) has been investi-gated in vitro using different assays (trypan blue, lactatedehydrogenase, and51Cr release). Suzuki and Cooper (1995a)investigated xenogenic reactions against human tumor cells.TEM and SEM studies showed that close contact of the tar-get cell withLumbricuscells (granular coelomocytes) is fol-lowed by lysis. The results confirm that effector cell / targetcell contact is essential for cytotoxicity to occur.

These cytological events present analogies with the cytotoxicactivity that occurs in vertebrate natural killer (NK) cells.They support the idea, postulated by Franceschi et al. (1991),that a primitive NK cell (analogous to vertebrate NK cells)developed early in phylogeny.

As in the NK-cell system, target specificity is exceedinglybroad, since xenogeneic, allogeneic, and even syngenicerythrocytes are killed under appropriate conditions. In thepolychaeteGlycera sp., Decker et al. (1981) demonstratedthat cytoxicity of coelomocytes is independent of prior antigenexposure or the addition of exogenous antibody or lectin.The specificity of these cytotoxic cells may be directed atcell-surface glycoproteins on the target cell surface, since avariety of defined mono- and di-saccharides can block cellkilling. Suzuki and Cooper (1995b) demonstrated that modi-fication of surface antigen can affect the level of cell-mediatedcytotoxicity. They observed no significant spontaneous cyto-toxicity against autogeneic target coelomocytes haptened with2,4,6,-trinitrobenzene sulfonic acid, but coelomocytes effectedsignificant spontaneous cytoxicity against a haptened allo-genic target.

2.3. Humoral immunity

Invertebrates lack the immune system of vertebrates, whichis characterized by the production of specific antibodies.Therefore, the protective mechanisms of invertebrate immu-nity, in addition to cell immunity, are lysis and agglutinationenhanced by components from the CF. Research studies inthis field are numerous, especially in Oligochaeta, but are



difficult to classify for two reasons: firstly, cytolytic activitywith agglutinins, hemolysins, and other antibacterial mole-cules often occurs simultaneously in the CF; secondly, undernatural conditions, these factors react against foreign particles(bacteria, fungi, parasites). In the laboratory, experimentalchallenge often involves the use of vertebrate erythrocytes,and comparisons with natural infections are often impossible.

2.3.1.Agglutinins and hemolysins

Hemagglutinins and hemolysins are both present, and ex-hibit various activity levels, in the CF of Polychaeta andOligochaeta. Sheep and rabbit erythrocytes are both lysedand agglutinated inGlycera dibranchiata(Anderson 1980),Neoamphitrite figulus, and Arenicola marina(Dales 1982).Hemolysis and hemagglutination occur simultaneously in theCF and cocoon albumen ofE. fetida(Valembois et al. 1984).Cross-reactivity of hemolytic and hemagglutinating proteinshas been demonstrated in the CF of several earthworm species.The ability of the hemolytic and hemagglutinating factorsin the CF to bind specifically to rabbit erythrocyte mem-branes was used by Mohrig et al. (1996) to purify these com-pounds and prepare them for the production of polyclonalantisera. The antibody raised against the CF ofA. caliginosais able to inhibit the hemolytic activity ofE. fetida andA. caliginosa and also the hemagglutination capacities ofL. terrestris, Lumbricus rubellus, andDendrodrillus rubidus.Fetuin andα-1-acid glycoprotein were demonstrated to bestrong inhibitors of both hemolytic and agglutinating activi-ties in all species investigated.

2.3.1.1.Agglutinins

Agglutinins participate in defense mechanisms of inverte-brates by aiding phagocytosis. Agglutinin production is in-duced by antigens. These may function to aggregate foreignmaterial and may serve as opsonins. In Polychaeta, resultsconcerning the nature of these molecules have been obtainedprincipally in Nereis virens(Russel et al. 1983; Lai et al.1989). The CF of this worm contains three classes ofhemagglutinins: (1) four low molecular mass glycoproteinsthat are heat-sensitive, (2) a mixture of lipid agglutinins thatare heat-insensitve, and (3) a high molecular mass lipid-associated proteoglycan that is insensitive to divalent cat-ions. In Oligochaeta, the CF ofL. terrestriscontains low lev-els of naturally occurring agglutinins for both erythrocytes(Stein et al. 1982) and several strains of bacteria (Stein et al.1986). However, if worms are injected intracoelomically withrabbit erythrocytes (Stein et al. 1982) or some strains of bac-teria (Stein et al. 1986), agglutinin levels increase signifi-cantly within 24 h (up to 7-fold). Agglutinins are inhibitedby a number of glycoproteins and polysaccharides. The onlysimple sugar to show specific inhibition is a monosaccharidefound in bacteria (2-keto-3-deoxyoctonate) (Stein and Coo-per 1983). Separation of the CF ofL. terrestrisby gel chro-matography revealed a single agglutinin, with a molecularmass of 30 kDa, which was unaffected by heating at either60 or 100°C and was composed primarily of glycolipids(Stein et al. 1989). The agglutinins from antigenically chal-lenged CF were more complex; three were unaffected byheat and are considered lipids and two were sensitive to heatand are considered proteins (Stein et al. 1990). These last re-sults seem to indicate that the increase in agglutinin levels in

the CF after induction is not simply the release of stored ag-glutinins, but indicates a specific process.

The origin of agglutinins inE. fetidahas been investigatedby Valembois et al. (1982). Observations using SEM haveshown that chloragocytes are able to agglutinate vertebrateerythrocytes and pathogenic bacteria such asBacillusmegaterium; however, bacteria known to be nonpathogenicto this worm, such asAcinetobactersp., were not aggluti-nated. In vitro, chloragocytes released their chloragosomesand exhibited stronger agglutination activity against patho-genic bacteria than in situ. According to those authors, undernormal living conditions, chloragocytes probably intervenein antibacterial defense mainly after being extruded from thecoelomic cavity and spreading and degranulating at the sur-face of the tegument.

Experimentally, hemagglutinins are responsible for theformation of spontaneous rosettes of sheep erythrocytes aroundnon-adhering coelomocytes (Toupin and Lamoureux 1976);these are also called secretory rosettes (SRs) (Goven et al.1994).

2.3.1.2.Antigen-binding proteins

Laulan et al. (1985) reported thatL. terrestrissynthesizedspecific substances in response to immunization with a syn-thetic hapten. In response to antigenic stimulation by ahapten (arsenilic acid) coupled with serum human albumin,Tuckova et al. (1988) identified an adaptatively formed 56-kDa-molecule antigen-binding protein (ABP) consisting oftwo disulfide-linked polypeptide chains (31 and 33 kDa),both of which participate in forming the antigen-bindingsite. Investigation of the kinetics of ABP formation revealedthat the response reached a maximum level between 4 and8 days after the first dose and approximately 4 days after thesecond challenge (Tuckova et al. 1991a). The ABP was iso-lated and monoclonal antibodies were raised against thismolecule (Tuckova et al. 1991b). Using Western blotting,Bilej et al. (1995b) investigated both humoral and cellularlevels of the ABP in vitro. The binding was significantlyhigher when the same protein was used for successive stimu-lations in vivo. The degree of specificity of the ABP in-creased after the secondary in vivo challenge, but even so itwas considerably lower than that of vertebrate immuno-globulins. Knowledge of the ABP sequence would be partic-ularly interesting for investigating putative analogies withbinding molecules from invertebrates and vertebrates.

2.3.1.3.Hemolysins (directed against erythrocytes)

In the sabellidSpirographis spallanzani, the CF (Parrinelloand Rindone 1981) and external mucus (Canicatti et al.1992) possess powerful lytic activity against a variety of ver-tebrate erythrocyte types. In this species, the hemolysin is anon-enzymatic, calcium-dependent, zinc-inhibitable factorthat occurs naturally in the CF. This activity is not signifi-cantly reduced by thiols or disulfide covalent modifying re-agents (Canicatti and Roch 1993). This finding differentiatesthis hemolysin from many thiol-activated hemolytic toxinsthat occur in different animal groups. It is a low molecularmass molecule (6–8 kDa) whose characteristics remain un-determined. Apart from sialic acid, none of the saccharidestested were able to prevent hemolysis of rabbit erythrocytes,suggesting that carbohydrates are probably not the receptor

© 2001 NRC Canada

238 Can. J. Zool. Vol. 79, 2001



molecules responsible for binding lytic molecules on theerythrocyte surface.

Eight other polychaete species (Nainereis laevigata, Orbiniacuvieri, Cirriformia tentaculata, Notomastus latericeus,Arenicola claparedii, Petaloproctus terricola, Marphysasanguinea, Nereis sp.) belonging to five different orderswere investigated by Roch et al. (1990). Homogenates fromonly three species (N. laevigata, O. cuvieri, andP. terricola)induced hemolysis in sheep erythrocytes. Important differ-ences between the three hemolytic species in hemolytic titers,calcium- or magnesium-dependence, and time-course responseswere noted. In all polychaete species examined, hemolysinsare thermolabile and divalent-cation-dependent. The resultsconcerning the thermolability of hemolytic polychaete activ-ity are in agreement. In all species examined, and in manyother invertebrates studied, temperatures above 55°C gener-ally completely abrogate hemolytic activity. Thermolabilityis probably one of the common characteristics of all lyticmolecules. In this study, however, supernatants from wholehomogenized worms were used. This procedure does not al-low the origin of the lytic factor (gut, coelomic cells) to beprecisely determined, and the biological activity may be dueto secondary reactions resulting from homogenization.

By contrast, Roch et al. (1981) recorded calcium- andmagnesium-independent hemolytic activity in the CF ofOligochaeta. In the earthwormEisenia fetida andrei, thechelating agent EDTA did not reduce the hemolytic responseof the erythrocytes. Such divergent results indicate large dif-ferences between the hemolytic systems of Polychaeta andOligochaeta. To date, how cations act on the hemolytic mol-ecules of Polychaeta is not very well known. As suggestedby Roch et al. 1990, these ions could act (i) to stabilize thestructure of hemolytic molecules; (ii ) to mediate the interac-tion between hemolysins and the target-cell membrane; or(iii ) to accelerate hemolysin polymerization, resulting in mem-branedamage.

2.3.1.4.Hemolytic and antibacterial proteins

The CF of the lumbricidE. f. andrei exhibits stronghemolytic activity against the erythrocytes of various mam-malian species and bacteria (Milochau et al. 1997). This ac-tivity is mediated by two proteins called fetidins, of apparentmolecular masses 40 and 45 kDa. The 45-kDa fetidin ismonophilic, while the 40-kDa fetidin exists in severalisoforms. In the 40-kDa fetidin, five tryptophans were pre-dicted from the cDNA sequence and their presence was con-firmed by biochemical assay (Lassegues et al. 1997). Therecombinant protein inhibited the growth ofB. megaterium.Besides its hemolytic activity, the protein also exhibitedperoxidase acitivity and a peroxidase motif was identified inresidues 52–62.

In E. fetida, Eue et al. (1998) distinguished three proteins(40, 43, and 46 kDa). The 40-kDa molecule was demon-strated to be a bifunctional protein that can lyse and aggluti-nate erythrocytes. At 56°C, the hemolytic activity of theproteins was inactivated but the agglutination activity of the40-kDa protein was stable. These proteins constitute a majorpeptidic component of CF (20% of total CF proteins) andare released from the chloragocytes (Valembois et al.1982).Fetidins contribute about 75% of the bactericidal activity ofthe CF; the remaining bacterial activity resides in the yellow

pigment (riboflavin) and lysozyme. The presence of divalentcations is not required for hemolytic activity of the fetidins(Milochau et al. 1997). Bilej et al. (1995a) demonstrated thatthe cytolytic protein of the earthworm CF is also capable oflysing different mammalian tumor cells.

Cytotoxic activity of fetidin can be stimulated in vitro byseveral serine proteases. Roch et al. (1998) have character-ized a 14-kDa plant-related serine protease inhibitor in theCF, which acts in the regulation of cytotoxic activity. Be-yond these data, little is known about the regulation ofimmunodefense in Annelida. Kauschke et al. (1997) sug-gested that the increase in proteases in the CF ofL. terrestristhat is seen after an antigenic challenge argues for theinvolvement of serine proteases in the earthworm immunesystem.

2.3.2.Other antibacterial molecules

2.3.2.1.Lysozyme

Lysozyme is an enzyme that cleaves theβ-1–4 bondsbetween N-acetylglucosamine andN-acetylmuramicacid ofGram-positive bacterial cell walls. Lysozyme is often de-tected using its action againstMicroccocus lysodeikticus.

The occurrence of lysozyme inNephthys hombergiihasbeen described by Jolles and Zuili (1960) and Perin andJolles (1972). InN. diversicolor, lysozyme was undetectablein the CF of control worms (M’Beri 1988), but has been de-tected in granulocytes of these worms. In the CF, lysozymeappears about 24 h after the worm has been challengedby bacterial injection. The intensity of the response variesaccording to the bacteria injected;Escherischia coliandPseudomonas fluorescenswere the most potent stimulators.Lysozyme activity was found in the CF of the earthwormE. fetida (Lassalle et al. 1988). Base-line activity was en-hanced 20-fold by one injection of either Gram-positive orGram-negative bacteria or sheep erythrocytes. Maximum ac-tivity was induced 4–5 h after injection. Regulation of thetranscription and translation of lysozyme has been investi-gated by Hirigoyenberry et al. (1990). This humoral defenseprocess requires RNA and de novo protein synthesis in re-sponse to pathogenic bacteria. By contrast, fetidins requireonly the translation of stable RNAs, and their activity peaksoccur later (4 h for lysozyme, 3 days for fetidins) (Milochauet al. 1997).

Lysozyme activity occurs also in several species ofHirudinea: Herpobdella octoculata, Hemopsis sanguisuga,and Hirudo medicinalis(Schubert and Messner 1971).

2.3.2.2.Bactericidal factors

With the exception of lysozyme, few antibacterial factorshave been revealed in polychaetes. Chain and Anderson(1983a) studied the action of a bactericidal factor against theGram-negative bacteriumSerratia marcescensin the CF ofG. dibranchiata. This factor, partially purified, has beenshown to be a glycoprotein (250–450 kDa) containing bounddivalent cations and at least one disulfide bridge (Chain andAnderson 1983b).

Dales and Dixon (1980) found no effective naturalbactericidin in three polychaete species, even after immuni-zation with bacteria.

© 2001 NRC Canada




© 2001 NRC Canada

240 Can. J. Zool. Vol. 79, 2001

2.3.2.3.Antibacterial protein

More recently, an antibacterial protein (MP II) has beendetected in the CF ofN. diversicolorthat inhibits the growthof E. coli and Micrococcus kristinae(Dhainaut et al. 1989).The antibacterial activity is very weak in non-immunizedworms. The bacteria used for immunization were found toexert an influence:E. coli and Pseudomonas aeruginosatriggered the strongest antibacterial reaction.Finally, the re-sponse is much reduced in winter (A. Dhainaut, unpublisheddata).

MP II is a metalloprotein (for the features of this moleculesee the section “ Physical and chemical insults”) that hasa bacteriostatic action. MP II probably acts by competingwith iron, which is essential for the growth of bacteria(sideroprivation)(for a description of the capacity of MP II tobind with metals see the section “Physical and chemical in-sults”). Immunocytochemical studies with antibodies againstMP II, either polyclonal (Dhainaut-Courtois et al. 1987) ormonoclonal (Porchet-Henneré et al. 1987a), have led to thelocalization of MP II in coelomic cells identified as G1granulocytes. Detection of mRNA encoding MP II has beenachieved by in situ hybridization (Salzet-Raveillon et al.1993). Based on a partial amino acid sequence of MP II, twooligonucleotide primers were synthesized and used to gener-ate a cDNA fragment by the polymerase chain reaction(PCR). The specificity of the PCR-synthesized 220-base-pair(bp) fragment was verified by hybrid-arrest translation andsequencing. In situ hybridization performed with this cDNAfragment onN. diversicolorwhole body defined two specificrecognition sites: (1) two types of muscles (perineural andoblique) and (2) a cluster of cells floating in the coelom. Thecluster of cells seems to be the site of genesis of G1 granulo-cytes. The encoding mRNA is high at this site. Surprisingly,when the G1 granulocytes are released in the coelomic cav-ity, mRNA expression in the mature G1 granulocytes disap-pears. By contrast, immunocytochemical techniques reveal ahigh MP II content in these cells.

This last observation raises the problem of the genesis anddifferentiation of the coelomic cells, which remain very poorlystudied in Polychaeta.

2.3.2.4.Enkelytin

Enkelytin is an antibacterial peptide (29 amino acids) thatacts on Gram-negative bacteria. It is liberated by cleavage ofa neuropeptide precursor, proenkephalin A. Initially discov-ered in bovine chromaffin cells (Goumon et al. 1996), thismolecule was then reported in the molluscMytilus sp. (Stefanoand Salzet 1999). Recently, in our laboratory, the release ofenkelytin has been described in the hirudineanTheromyzontessulatumfollowing wounding or stimulation by bacteriallipopolysacharides (F.A. Tasiemski, unpublished data).

2.4. Influence of toxics on the immune response:utilization as biomarkers

Immune reactions are influenced to various degrees byenvironmental factors. The immune response to pollutants(heavy metals or pesticides) in the biotope of the worms canbe quantified and the animals then used as biomarkers to de-tect and quantify toxics. The immune-reactivity test is oftenmore sensitive than other laboratory methods (measurement

of toxicity, lethality, or bioaccumulation). Both cellular andhumoral immune-defense responses under toxic conditionshave been investigated.

2.4.1.Cellular response

The influence of heavy metals on phagocytosis ofcoelomocytes inL. terrestriswas studied in vitro by inges-tion of fluorescent microspheres and flow cytometry (Fugèreet al. 1996) Exposure to any form of mercury, cadmium, orzinc was relatively toxic and affected both cell viability andphagocytosis, whereas lead was relatively well tolerated bycoelomocytes. Those authors have also distinguished differ-ences in phagocytic reactivity between the various popula-tions of coeleomocytes.

Polychlorinated biphenyls (PCBs) also cause both acuteand chronic inhibition of the phagocytic competence ofcoelomocytes (Goven et al. 1994; Ville et al. 1995). PCBscommonly inhibit macophage-related functions, such as woundhealing and elimination of non-pathogenic bacteria (Ville etal. 1995). They also depress the natural allogenic cytotoxicityof earthworm coelomocytes (Suzuki et al. 1995). These re-sults suggest that PCBs can suppress NK-like activity.

Lysosome-membrane stability is affected by chemical andnonchemical factors and may be useful as an integrativebiomarker of mutiple stressors (Weeks and Svendsen 1996).The permeability of the lysosome membrane increases in re-sponse to stress. Retention of neutral red by lysosomes fromcoelomocytes appears to be a simple biomarker of exposureto heavy metals: the higher the body metal concentration,the shorter the neutral red retention time (Svendsen et al.1996).

2.4.2.Humoral response

Unlike cellular immunity, humoral activity is stimulatedby xenobiotics (pesticides, herbicides, etc.). PCBs increasedlysozyme activity in three earthworm species (E. fetida, Eiseniahortensis, and L. terrestris) and enhanced other molecularactivities (antibacterial hemolysis, action of proteases) (Villeet al. 1995). InL. terrestris, however, lysozyme activity isinhibited by copper (Goven et al. 1994).

Coelomocytes synthesize and secrete agglutinins that par-ticipate in the humoral immune response. Inhibition of thehumoral response in the earthwormL. terrestris due tochemical exposure can result in immunodepression, i.e., at-tenuated host resistance to infection. SR tests were done toenumerate agglutinin-producing coelomocytes. SR formationindicates the ability of coelomocytes to produce agglutininfactors in response to challenge by foreign bodies, such asbacterial or fungal infections. These factors serve to aggre-gate particular antigens and act like opsonins to facilitatephagocytosis. SRs decreased under the influence of PCBs.SR immunoassay is sensitive to PCB levels within an orderof magnitude of those reported for a wide variety of wildlifefrom contaminated areas (Goven et al. 1994).

3. Physical and chemical insults

3.1. Introduction

The annelids constitute an important part of the biomassof the seashore, estuaries, fresh water, and terrestrial soils.



They occupy a central position in the trophic network, as amajor food source for fishes, birds, and terrestrial fauna.Therefore, they represent a potentially important source ofpollutants for these predators and in the subsequent foodchain. Moreover, micropollutants can also directly affect theviability of these animals, thus interrupting vital links inbiotopes. The principal pollutants contaminating biotopes areheavy metals and xenobiotics (pesticides, herbicides, etc.).

The biological reactions induced by these toxics are manyand include (i) inhibition of enzyme activity; (ii ) synthesisof stress-induced proteins; (iii ) modulation of the activitiesof enzymes involved in the detoxification of xenobiotics.

3.2. Inhibition of activity: cholinesterase model

3.2.1.Characterization and functions

Cholinesterases (ChEs) were discovered in 1906 (Huntand Taveaux 1906). Among annelids, ChEs have been char-acterized in the marine polychaeteN. diversicolor(Scaps etal. 1996), in two species of terrestrial oligochaete,A. caliginosa(Principato et al. 1978, 1979, 1989; Kaloustian 1981) andE. fetida (Andersen et al. 1978; Stenersen 1980a, 1980b;Scaps et al. 1996), and in the freshwater hirudineanH. medicinalis (Principato et al. 1981, 1983; Talesa et al.1995).

3.2.1.1.Acetylcholinesterases

Acetylcholinesterases (AChEs) (EC 3.1.1.7) preferentiallyhydrolyze acetylcholine as substrate and are very sensitive toeserine inhibition. A surplus of substrate reduces AChE ac-tivity. AChEs are involved in synaptic transmission. Themain function of AChEs is the hydrolysis of acetylcholine,the mediator of cholinergic synapses in the nervous system;so AChEs are essential to the correct transmission of nerveimpulses.

An enzyme sharing the properties of AChE is present inthe soluble fraction of the nereidid polychaeteN. diversicolor.Using 7.5% disc polyacrylamide gel electrophoresis (disc-PAGE), Scaps et al. (1996) observed 6 isoforms of AChEs.In N. diversicolor the AChE is represented by two compo-nents with similar molecular masses (47 and 55 kDa), a thirdcomponent with lower molecular mass (18 kDa), and a veryimportant fraction that does not enter the gel (Scaps et al.1996).

3.2.1.2.Propionylcholinesterases

Augustinsson (1963) used the term propionylcholinesterase(PrChE) to distinguish enzymes with a higher activity to-wards propionylthiocholine iodide (PrTCh) than towardsother esters.

Using 7.5% disc-PAGE, Scaps et al. (1996) observed 2isoforms of ChEs in the oligochaeteE. fetida, while in thesame species Stenersen (1980b) found three different isoforms,but with a polyacrylamide gel concentration of 5%. Previouswork by Stenersen (1980b) revealed the existence in thisspecies of two types of ChEs called E1 and E2 by that au-thor. E1 is a PrChE, whereas E2 is a nonspecific ChE. E1 isrepresented by four molecular-mass forms: about 65, 105,235, and 312 kDa. E2 was not further characterized becauseof its high molecular mass; it remained in the stacking gel.By contrast, Andersen et al. (1978) found only one PrChE in

this species, with a molecular mass of about 108 kDa.Components with molecular masses of 53.8, 106, 235, and310 kDa and a very high molecular mass form that remainedin the stacking gel were also observed by Scaps et al. (1996)and confirm the results obtained by Stenersen (1980b). Anadditional component with a molecular mass of 628 kDawas sometimes observed. According to Stenersen (1980b),the high molecular mass form of ChEs probably results froman assemblage of unstable active monomers with a molecu-lar mass of 65 kDa.

In a preliminary study, Principato et al. (1978) partiallypurified and characterized, using acetylthiocholine iodide(ATCh) assubstrate, the ChEs of the oligochaeteA. caliginosaand called them AChEs. During estivation this speciespresentsadditional AChE isozyme, which coincides withthe activation of collagenase (Kaloustian 1981). In a furtherstudy, Principato et al. (1989) showed that the ChEs ofA. caliginosahydrolyze PrTCh optimally and should corre-spond to PrChEs. This species contains one PrChE of 180 kDathat could be a tetramer of two molecular-mass subunitswith molecular masses of 28 and 62 kDa.

Talesa et al. (1995) purified and characterized two ChEsin the medical leechH. medicinalisthat differ in substrateand inhibitor specificity. Both hydrolysed PrTCh optimally.This substrate preference might permit them to be calledPrChEs. Actually, however, the physiological function ofPrChEs is not clearly defined, although propionylcholine(PrCh) has been found in animal tissues (Augustinsson1963; Winners et al. 1978). Although these enzymes hydro-lyzed PrTCh optimally, those authors used AChEs for bothenzymes to underline their fulfillment of conditions charac-teristic of AChEs (EC 3.1.1.7): they hydrolyzed ATCh sig-nificantly, they were inhibited by eserine at concentrationsusual for AChEs, and they were inhibited by excess ATCh,so it is possible that they act biochemically and physiologicallylike AChEs. A “spontaneously soluble” portion of AChE ac-tivity (SS-AChE) was recovered from hemolymph and fromtissues macerated in low-salt buffer, whereas a detergent-soluble AChE (DS-AChE) was obtained after extraction oftissues in low-salt buffer containing 1% Triton × 100. NativeSS-AChE and DS-AChE have a molecular mass of 66 and130 kDa, respectively, whereas denaturing SDS-PAGE underreducing conditions gave one band at 30 kDa for SS-AChEand 66 kDa for DS-AChE. SS-AChE is a hydrophylicmonomer and DS-AChE an amphiphilic glycolipid-anchoreddimer. This study contrasts with previous reports (Principatoet al. 1981, 1983) of only one PrChE in this species. Never-theless, previous studies were restricted to SS-AChE, withno extraction of tissues; moreover, kinetic parameters deter-mined previously by Principato et al. (1983) are in goodagreement with those obtained for SS-AChE by Talesa et al.(1995).

3.2.2. Inhibition of ChE activity by organophosphate andcarbamate pesticides

Organophosphate and carbamate pesticides inhibit the ac-tivity of AChEs at nerve endings. When the enzymes areblocked, they can no longer participate in the hydrolysis ofacetylcholine. Thus, the neurotransmitter accumulates, its ac-tion is enhanced, and it finally causes death.

© 2001 NRC Canada




Inhibitory effects on AChE activity of the ragworm,N. diversicolor, were determined by Scaps et al. (1997a) at aconcentration of 10–6 M for three OP compounds (mala-thion, ethyl parathion, and phosalone) and a carbamate pesti-cide (carbaryl) during a toxicity test performed in thelaboratory with individuals maintained in U-shaped glasstubes (mimicking natural burrows). Only short-term effectswere measured, therefore no cumulative effect was found.Percent inhibition of AChE activity reached a maximumbetween 2 (carbaryl) and 7 (organophosphate compounds)days after exposure and then remained stable.

Stenersen et al. (1992) tested the toxicity of ChE-inhibitingpesticides to earthworms of the genusEisenia(E. f. andrei,E. fetida, andEisenia veneta) using a standard paper-contacttest performed by the Organization for Economic Coopera-tion and Development (OECD 1984) for ecotoxicologicaltesting of industrial chemicals. In vitro inhibition experimentswith carbaryl and paraoxon (diethyl 4-nitrophenyl phosphate)indicated that both species have at least two ChEs with dif-ferent bimolecular inhibition constants. InE. f. andrei andE. fetida, carbaryl discriminated completely between differ-ent ChEs because one was completely resistant (E2) and theother two (E1a and E1b) were very sensitive, but the bimo-lecular constants were sufficiently different to discriminatebetween them. In these two species, the low lethality of car-baryl can be explained by the presence of E2. Two ChEscould be distinguished inE. venetaby carbaryl. However,this species lacks the carbaryl-resistant enzyme and is there-fore killed by minute amounts of this toxicant.Eisenia venetais probably more similar toL. terrestris and other earth-worms with regard to reactions towards cholinesterase-inhibiting pesticides.

In E. f. andrei, parathion inhibits ChE activity in vivo butnot in vitro, whereas parathion must be oxidized to paraoxonbefore being able to inhibit ChE (Stenersen 1979). In vivo,an oxidative and a hydrolytic step must occur involving thecytochrome P-450 dependent mono-oxygenase system (seethe section “Mixed-function oxidase enzyme system” be-low).

3.2.3.Effects of exposure to heavy metals

Scaps et al. (1997b) showed that ChE activity in the earth-worm E. fetidawas not inhibited when individuals were ex-posed for 8 weeks to artificial soil containing either 8 or80 ppm of cadmium or 100 or 2000 ppm of lead. The resultsdiffer from those reported by Labrot et al. (1996) for aclosely related species of earthworm,E. f. andrei. In thisspecies, significant decreases in ChE activity (up to 60%)were noted after exposure to both lead and uranium in vivoand in vitro. However, it should be noted that those authorsused dishes containing moistened filter paper and aerateddaily for in vivo exposure as described in the OECD (1984)standard test.

3.3. Stress-induced proteins

3.3.1.Heat-shock proteins

3.3.1.1.Characteristics and functions

In response to various stressors such as elevated tempera-ture, UV light, xenobiotics, and heavy metals, organisms in-

crease synthesis of the so-called stress proteins. As the ma-jority of these proteins are heat-inducible they were at firsttermed heat-shock proteins (HSPs).

3.3.1.2.Induction of HSPs

In annelids, induction of HSPs was studied in only onespecies of estuarian polychaete, the ragwormN. diversicolor.At the present time, no studies ofthis nature have been per-formed with oligochaetes or achaetes.

When metameres of theN. diversicolorwere exposed invitro to heat shock or cadmium, they reacted by synthesizingstress proteins (Ruffin et al. 1994). In the case of cadmiumexposure, the induction process takes longer and a prelimi-nary 24-h exposure of whole animals is needed before themetameres are incubated in the presence of [35S]methionine.Two-dimensional electrophoresis and fluorography techniquesindicated synthesis by the worms of at least 15 stress pro-teins, including the universal one referred to as stress 70 anda lot of low molecular mass proteins known to be species-specific (Lindquist and Craig 1988). Injection of whole stressedanimals with [35S]methionine produced the same typical stressproteins as those observed with metameres for both stress-ors. Stress 70, synthesized byN. diversicolorin response toboth stressors, appeared on fluorograms as an array of threecharge isomers. Stress 70 proteins belong to the ubiquitousgroup of chaperones involved in normal cellular processessuch as protein folding and assembling and translocation ofprotein precursors from cytosol to organelles (Gething andSambrook 1992).

In N. diversicolor, low molecular mass stress proteins arewidely distributed, varying between 20 and 45 kDa, the mostcommon being 22 kDa. The present authors observed thatmost of the low molecular mass stress proteins built up inresponse to heat shock were different from those observedafter cadmium exposure.

3.3.2.Metal-binding proteins

3.3.2.1.Metallothioneins

3.3.2.1.1.Structure and properties—Metallothioneins (MTs)are metallo-derivates of the sulphur-rich protein thionein.MTs are a group of low molecular mass, heavy metal bindingproteins; they contain a high proportion of cysteine residues,which do not form disulfide bridges (George and Olsson1994). MTs are also characterized by absorbance at 254 nm,owing to the presence of cadmium–mercaptide bonds, andare remarkably stable (in Nejmeddine et al. 1992). MTs havehistorically been split into two classes: class I consists ofthose with sequence homology to equine MT (this includesmost vertebrate MTs) and class II consists of unrelatedcysteine-rich sequences. This group includes invertebrateMTs. It was suggested that the role of MTs is protectionfrom the toxic effects of metal exposure, and physiologicaltolerance of heavy metals was attributed, at least in part, tothe induction of metal-chelating proteins. This led to the as-sumption that the genes responsible for this mechanism willbe up-regulated or differentially expressed during exposure.These metal-chelating gene products include MTs.

Recently, Stürzenbaum et al. (1998), combining standardgel chromatographic techniques and novel molecular tech-nologies (directed differential display and quantitative PCR),

© 2001 NRC Canada

242 Can. J. Zool. Vol. 79, 2001



© 2001 NRC Canada


were the first to sequence two isoforms of a true metallo-thionein from an annelid, the earthwormL. rubellus. Bothproteins are characterized by a high cysteine content (24.5%)and a lack of histidine, methionine, and aromatic residues(Fig. 1).

Analysis of the two earthworm isoforms reveals that al-though they are slightly longer than the archetypal class IMTs, in all structural features they conform to a class I MT.Analysis of the derived amino acid sequence of isoform 2identified two putativeN-glycosylation signal sequences, sug-gesting that the two isoforms may have different subcellulardistributions and functions.

3.3.2.1.2.Inducibility of L. rubellusMT—Stürzenbaum etal. (1998) confirmed responsiveness to metals by determin-ing MT-specific expression profiles in earthworms exposedto soils with differing heavy-metal concentrations. Expres-sion profiles of MT were quantified by those authors bymeans of a fully quantitative fluorescence-based PCR tech-nique in normal earthworms or in those exposed to soilswith different heavy-metal concentrations. MT levels, whichwere low in the unpolluted control population, were 5-foldhigher in worms living in a copper mine. Levels rose by afactor of over 1750 in the population of earthworms nativeto a heavily cadmium-polluted lead and zinc mine. More-over, those authors showed that metallothionein transcriptsincreased 30-fold in earthworms maintained on artificial soilsupplemented with a cadmium salt (6 mg CdCl2/g dry soil)over that in earthworms maintained on artificial soil only.

3.3.2.2.Metalloproteins

3.3.2.2.1.Metallothionein-like proteins—In annelids, metal-binding proteins sharing characteristics with vertebrate MTs(inducibility and affinity to heavy metals, elevated cysteinecontent, low molecular mass, and high absorbance at 254 nm)have been reported to play a role in the detoxification process.These MT-like molecules, however, differ by their highercontents of glycine (up to 18%) and aromatic amino acids(Stone and Overnell 1985).

Suzuki et al. (1980) found a low molecular mass cadmium-binding protein (apparent molecular mass about 10 kDa) inthe earthwormE. fetida. Furst and Nguyen (1989) found thatthe common earthwormL. terrestris produced a cadmium-binding protein (MT-like) after the injection of cadmium salts.Two isoforms of this protein were detected by gel perme-ation. Morgan et al. (1989) isolated low molecular mass pro-teins from the posterior alimentary tract of two earthwormsspecies,D. rubidus (apparent molecular mass 27.5 kDa) andL. rubellus(apparent molecular mass 24 and 27.5 kDa). Bauer-Hilty et al. (1989) isolated a low molecular mass cadmium-binding protein from the limnal oligochaeteLumbriculusvariegatus. This protein has an apparent molecular massof 19 kDa and has a high cysteine content (16.5%) AfterSDS-PAGE, three distinct bands with molecular masses of19, 11.5, and 10.2 kDa were found. The three bands are as-sumed to represent a functional dimer, a monomer, and apartially carboxymethylated monomer. According to thoseauthors, the high capacity for cadmium accumulation possessedby many oligochaetes may be explained by the presence ofsuch cadmium-binding proteins, which probably play an im-portant role in the detoxification of this metal.

Proteins with similar features were also characterized in

polychaetes. Young and Roesijadi (1983) reported the exis-tence of an inducible copper-binding protein in the sabellidpolychaete Eudistyla vancouveri. Eriksen et al. (1988)investigated four groups of polychaete species and found acadmium-binding protein inLumbrineris fragilisthat resem-bled mammalian metallothionein. Eriksen et al. (1989) alsoinvestigated the binding of some metals in five species ofpolychaetes and suggested that metallothionein-like proteinswere present in two of the species investigated (Orbinianorvegica and Goniada maculata). Marcano et al. (1996)studied the patterns of accumulation and elimination ofcopper and zinc in the tropical polychaete wormEurythoecomplanatain relation to levels of metal-binding proteinsduring sublethal exposure. During the uptake and depurationphases, these authors found metallothionein-like proteinswith molecular masses between 10 and 20 kDa with a higheraffinity for zinc than for copper. At present, however, nei-ther amino acid composition analysis nor sequence analysisof MT-like proteins from polychaetes was performed.

Fig. 1. Schematic diagrams depicting the amino-acid sequence ofLumbricus rubellusmetallothionein isoforms 1 and 2 (afterStürzenbaum et al. 1998).



© 2001 NRC Canada

244 Can. J. Zool. Vol. 79, 2001

3.3.2.2.2.Non-metallothionein-like proteins—In annelids,in addition to metallothionein-like proteins, higher and lowermolecular mass metalloproteins that bind heavy metals werefound. In the earthwormE. fetida, Yamamura et al. (1981)studied the distribution profile of cadmium among the solu-ble proteins; in addition to an MT-like fraction (apparentmolecular mass about 10 kDa), cadmium was also found inhigher and lower molecular mass fractions (estimated molec-ular masses 63–70 kDa and less than 2 kDa, respectively).During their study of the binding of some metals in five spe-cies of polychaetes, Eriksen et al. (1989) showed that copperwas primarily associated with components of high and verylow molecular mass in addition to proteins of a size similarto mammalian metallothionein (25 kDa), whereas zinc wasmainly recovered from the high molecular mass pool (35 kDa)and most of the nickel present eluted with very low molecu-lar mass components (4 kDa). In the same manner, Marcanoet al. (1996) found a heavier metalloprotein (60 kDa) in thepolychaeteE. complanatain addition to metallothionein-likeproteins. This molecule displayed a higher affinity for cop-per, suggesting that different biochemical mechanisms under-lie the control of zinc and copper metabolism through theactivity of low and high molecular mass metal-bindingproteins.

On the other hand, in other annelids, a variety of othermetal-binding proteins similar in molecular mass to MTswere isolated and characterized, and all were suggested to beinvolved in heavy-metal detoxification. In contrast to MTs,these metalloproteins have a low cysteine content (1–4%)and a high aromatic amino acid content, as well as a loweraffinity for cadmium, than MTs (in Nejmeddine et al. 1992).

Following experimental exposure of the polychaeteN. diversicolor to cadmium from seawater, the metal wasbound to two forms of cadmium-binding protein: a high mo-lecular mass form (MP I; over 67 kDa) and a low molecularmass form (MP II; about 20 kDa in its native state and13.7 kDa when denatured) (Nejmeddine et al. 1988;Demuynck et al. 1993). MP I was demonstrated to representthe extracellular hemoglobin of this annelid (Demuynck andDhainaut-Courtois 1993). MP II has a cysteine content ofonly 0.9% and possesses aromatic amino acids. The primarysequence of this molecule has been determined by Demuyncket al. (1993). This protein is composed of 119 amino acidsand shows 80.8% identity with theN. diversicolor myo-hemerythrin (Takagi and Cox 1991; Demuynck et al. 1991,1993). To investigate the role of metal-binding proteins intoxic metal metabolism inN. diversicolor, Demuynck andDhainaut-Courtois (1994) studied metal movements at thesubcellular level, performing repetitive exposures to a sublethaldose of cadmium. Those authors demonstrated the predomi-nant role of the component MP I over MP II in the bindingof cadmium. The accumulation of cadmium bound to MP Iwas shown to occur mainly between 24 and 48 h of expo-sure. The intervention of MP II in the binding of cadmiumwas seen to be very effective after 48 h of exposure and it issuggested that it represents a later step in cadmium metabo-lism in N. diversicolor.

Nejmeddine et al. (1992) isolated and characterized acadmium-binding protein from the terrestrial oligochaeteA. caliginosathat was termed Cd-BP14 because of its mo-lecular mass, 14 kDa, which was determined for the purified

protein by SDS-PAGE and gel filtration chromatographyunder native conditions. This protein is a monomer and hasan isoelectric point of 6.5. Cysteine was not present in thismolecule. Cd-BP14 contains a high level of aromatic acids(11%) and histidine (6%). Subsequently, Nejmeddine et al.(1997) isolated two isoforms called Cd-BP14a and Cd-BP14b. The complete amino acid sequence of the majorisoform Cd-BP14a (119 residues) and the amino-terminalsequence (57 residues) of Cd-BP14b were determined. Cd-BP14a and Cd-BP14b differ from each other at positions 19,21, and 41 in the first 57 residues. The substitutions at posi-tions 19 and 21 are conservative, whereas that at position 41consists of the replacement of an aspartate residue inisoform Cd-BP14a by a lysine residue in isoform Cd-BP14b.Amino-acid homology revealed a high degree of homologybetween Cd-BP14, MP II, and proteins of the hemerythrinfamily, non-hemeiron-binding proteins found in marine in-vertebrate phyla (Fig. 2).

In general, some closely related species possess either MTor MT-like proteins, some possess MT-like and higher and(or) lower molecular mass metalloproteins, and others pos-sess only non-MT-like metalloproteins. Thus, the presenceof metal-binding-proteins and their relationship with ambi-ent heavy metal availability seems to differ greatly amongannelid taxa. Their function in metal metabolism and in thecontrol of metal toxicity remains unclear and is not fully un-derstood in these organisms, though it is inferred that theyare important elements in the organisms’ resistance to poten-tially toxic metal concentrations.

3.3.2.3.Non-metallothionein cysteine-rich protein

Willuhn et al. (1996) showed that the expression of acadmium-inducible gene encoding a 25-kDa non-metallothioneincysteine-rich protein (CRP) in the enchytraeid wormEnchy-traeus buchhholzidepends on the cadmium concentration.This protein contains 27% Cys (Willuhn et al. 1994) and 8tandemly arranged repeats of 31 amino acids with 9 cysteineresidues in each one. One repeat contains one Cys-Cys motifand two Cys-Xaa-Cys motifs (Fig. 3) in a sequence resemblingthat of heavy-metal-binding proteins such as metallothionein(Willuhn et al. 1994, 1996).

The amount of CRP mRNA present was correlated withthe environment (worms were exposed to different cadmiumconcentrations in a fluid medium) as well as with the intra-worm cadmium concentration. CRP mRNA is expressed within2 h of the worms’ exposure to cadmium, and even a subtoxicconcentration, 100 mg cadmium/L, induced CRP gene ex-pression. Other heavy metals or stress conditions induce lit-tle or even no CRP mRNA expression.

It appears that CRP is certainly involved in the cadmium-detoxification process in this enchytraeid worm, but its exactfunction in this process is still unknown.

3.4. Enzymes involved in the detoxification ofxenobiotics

Annelids that inhabit terrestrial (oligochaetes and achetes),freshwater (oligochaetes and achetes), and marine ecosys-tems (polychaetes and achetes) may play a very importantrole in the degradation of xenobiotics contaminating thesehabitats.

Some of the common biotransformation enzymes have been



studied in polychaetes and oligochaetes, while nothing isknown about the systems responsible for the biotransformationof xenobiotics in achetes. Of the biotransformation routes,the most important for most animals is oxidation by thecytochrome P-450 system (phase 1) and conjugation toglutathione (phase 2).

3.4.1.The mixed-function oxidase enzyme system

Oxidation is a common route that makes many lipophilicxenobiotics more water soluble, and animals have developeda complicated system involving microsomal monooxygenasesor mixed-function oxidases (MFOs) to achieve this. The roleof this system is to catalyze the oxidation of oil-soluble sub-stances. The properties and role of the MFO system are doc-

umented only for some species of marine polychaetes andterrestrial oligochaetes.

3.4.1.1.Phase 1 cytochrome P-450 mediated MFO

The first authors to demonstrate the presence of a MFOsystem in annelids were Nelson et al. (1976), who found thatthe pesticide aldrin was converted to dieldrin by the commonearthworm L. terrestris. The requirement for oxygen andNADPH and sensitivity to inhibition by carbon monoxidesuggested to those authors that the aldrin epoxydase of thisearthworm is a typical microsomal MFO involving the hemo-protein cytochrome P-450. The highest aldrin epoxidaseactivity was found in the endoplasmic reticulum of the gutwall and the typhlosole (Nelson et al. 1976).

© 2001 NRC Canada


Fig. 2. Comparison of amino-acid sequences of isoform Cd-Bp14a and the N-terminal sequence of isoform Cd-Bp 14b withmyohemerythrin and hemerythrin sequences. 1 and 2 are isoforms Cd-Bp14a and Cd-Bp14b ofAllolobophora caliginosa, respectively.The underlined amino acids in the amino-terminal half of isoform Cd-Bp14b denote changes with respect to isoform Cd-Bp14a; 3, 4,and 5 are myohemerythrins ofPhascolopsis gouldii(sipunculid),Themiste zostericola(sipunculid), andNereis diversicolor, respec-tively. 7, 8, and 9 are hemerythrins ofT. zostericola, Themiste dyscritum(sipunculid), andP. gouldii. Conserved amino acids that areidentical in all members of the myohemerythrin subfamily are shown at line C1 and conserved amino acids that are identical in allproteins are shown at line C2. An asterisk indicates a conservative replacement. The amino acids in boldface type are involved in thecoordination of iron atoms. The four known helical regions inT. dyscritumhemerythrin andT. zostericolaare indicated (afterNejmeddine et al. 1997).



3.4.1.1.1.Isolation and characterization of the cytochromeP-450 dependent mono-oxygenase system—Nelson et al.(1976) were not able to characterize cytochrome P-450oxidase because in earthworms, the presence of a giant hemo-globin molecule similar in density and size to microsomes,makes studying cytochrome P-450 difficult because hemo-globin itself may catalyze a wide variety of mono-oxygenasereactions (Mieyal 1985). By using gel filtration chromatog-raphy on Sepharose 2B, Liimatainen and Hanninen (1982)separated the interfering earthworm blood pigments from themicrosomes and partially purified the cytochrome P-450 ofL. terrestris. Berghout et al. (1991) isolated, partly purified,and characterized the cytochrome P-450 dependent mono-oxygenase system from the midgut of the same species.Those authors identified at least 3 different cytochrome P-450s with apparent molecular masses of 48, 51, and 53 kDa,estimated by means of SDS-PAGE. The apparent molecularmass of the NADPH cytochrome P-450 reductase was63 kDa. According to those authors, at present time the na-ture of the physiological substrate of the cytochrome P-450dependent mono-oxygenase of the earthworm has yet to bedetermined.

Lee et al. (1979) characterized the MFO system of thepolychaeteN. virensand showed that it was similar to thoseof other invertebrate and vertebrate groups (Fries and Lee1984); it was composed of phospholipid, NADPH cytochromeP-450 reductase, and cytochrome P-450. The highest MFOactivity was associated with the intestine.

Induction of the cytochrome P-450 dependent mono-oxygenase system: Milligan et al. (1986) were unable todetect an increased amount of cytochrome P-450 in theoligochaeteDendrobaena venetaafter pretreatment with ei-ther 3-methylcholantrene or phenobarbitol, which are knownto be good inducers of cytochrome P-450 in mammals, andthey concluded that non-inducibility may be a general char-acteristic of earthworms.

In contrast, Lee et al. (1981) showed that MFO(benzo[a]pyrene hydroxylase) activity and cytochrome P-450 content increased in the polychaeteN. virensafter in vi-tro exposure to PCBs. Worms exposed to food containingbenzo[a]pyrene or a mixture of PCBs (Arocolor 1254) hadsignificantly higher MFO activity and cytochrome P-450content than controls (Fries and Lee 1984). However,benzo[a]pyrene hydroxylase activity did not increase after

injection with the inducing agent 3-methylcholanthrene orexposure to benz[a]anthracene in food, water, or sediment(McElroy 1990). Hahn and Stegeman (1992) suggested thatN. virensmay lack the receptor that mediates the inductionof the aromatic-hydrocarbon-inducible isoform of cytochromeP-450 in other species. Thus, induction of cytochrome P-450by aromatic hydrocarbons in polychaetes, if it exists, may bemechanistically different from induction in vertebrates. Re-cent studies suggest that metabolism of benzo[a]pyrene wasnot induced in various polychaetes, includingL. fragilis,N. diversicolor, and Scolecolepides(Marenzellaria) viridis(Driscoll and McElroy 1996) from contamined sites.Moreover, only one species,S. (M.) viridis, showed a smallincrease in thepercentage of total tissue benzo[a]pyrenethat was biotransformed after the worms were exposed to3-methylcholanthrene in the laboratory.

Owing to the fact that investigations have focused on onlya limited number of species of polychaetes and oligochetesand no species of achetes, and because the results of induc-tion experiments clearly indicate that these are large species-specific differences among polychaetes, further experimentson cytochrome P-450 mediated metabolism of lipophilicxenobiotics should be carried out with more species beforeconclusions are drawn concerning the role of cytochromeP-450 in annelids.

3.4.1.2.Phase 2 conjugation enzyme: glutathione S-transferases

3.4.1.2.1.Isolation and characterization of glutathioneS-transferases—The glutathioneS-transferases (GSTs) catalyzethe reaction of the tripeptide glutathione with hydrophobicsubstances bearing an electrophilic center. Glutathione con-jugation to electrophilic xenobiotics is a primary detoxifica-tion mechanism catalyzed by GTS (EC 2.5.1.18), responsiblefor glutathione-mediated dealkylation, dearylation anddehalo-genation of insecticides, and elimination of many epoxidesformed by the MFO system. GSTs have been isolated andcharacterized only from oligochaetes.

GST isoenzymes from cytosolic extracts of several earth-worm species have been separated by isoelectric focusing(Stenersen et al. 1979), anion-exchange and hydroxyl apatiteadsorption chromatography (Stenersen and Oien 1981), oraffinity chromatography and electrophoresis (Stenersen et al.1987). The different types present inE. f. andrei weresubstrate-specific. Recently, Borgeraas et al. (1996) purifiedthe GSTs ofE. f. andreiandE. venetaand divided them into6 and 5 groups, respectively. According to their structural,catalytical, and immunological properties, Borgeraas et al.(1996) found a high level of homogeneity with the pi classof mammalian GST.

Nevertheless, the physiological role of GSTs in earthwormsis obscure because each one characterized had narrow sub-strate specificity, GST activities showed great species differ-ences, and some substrates were not, whereas other were,conjugated and some substrates were conjugated in somespecies only. Typical “methyl transferase” activity was notfound in earthworms (Stenersen and Oien 1981), and ac-cording to Stenersen (1984) they probably lack the ability todemethylate organophosphorous insecticides like bromophosand parathion methyl; consequently, their intrinsic functionmay be to support excretion of endogenous catabolic prod-ucts (higher GST activity was detected by Stenersen and

© 2001 NRC Canada

246 Can. J. Zool. Vol. 79, 2001

Fig. 3. Consensus sequence of the CRP repeat. The sequencewas deducted from the nucleotide sequence of CRP cDNA. Xaaindicates variable amino acid residues (after Willuhn et al. 1996).



Oien (1981) in the nephridia than in other tissues), ratherthan to support detoxication of xenobiotics.

3.4.1.2.2.Inducibility of GSTs—Hans et al. (1993) showedthat total GST activity in the earthwormPheretima posthumaincreased considerably in the early stages of exposure to1 mg/g of aldrin, endosulphan, or lindane, and activity sub-sequently declined to near control levels after 4 weeks. Bycontrast, Borgeraas et al. (1996) showed that exposure ofE. fetida and E. venetato trans-stilbene oxide, 3-methyl-cholanthrene, and phenobarbital for 3 weeks did not elevatethe activity of GST.

A recent study by Grelle and Descamps (1998) showedthat GST activity in the earthwormE. fetida is not affectedby heavy metals (cadmium, copper, lead, zinc) either in vivoor in vitro. According to these authors, the original functionof GST in oligochaetes in not yet clear but its non-inducibility, as is also the case for the MFO system by or-ganic compounds, seems to be a common phenomenon(Milligan et al. 1986; Stokke and Stenersen 1993; Borgeraaset al. 1996). It may be suggested that the non-inducibility,relative to plant-eating animals, is due to their diet of detri-tus. The capacity for enzyme induction, which represents animportant defense mechanism in some animals, has probablyevolved for detoxifying secondary plant metabolites in theconstant “chemical warfare” between herbivores and plants(Brattsten et al. 1977; Yu 1982; Nebert et al. 1989; Stenersen1992).

To summarize, though GSTs are present in annelids, thecurrent data do not allow one to assign to them a clear rolein the defensive mechanisms of annelids.

3.4.2.Oxidative stress and antioxidant defenses

The formation of highly reactive oxygen species (ROSs)is a normal consequence of essential biochemical reactions,including mitochondrial and microsomal electron transportsystems, phagocytosis, xenobiotic-enhanced redox cycling,and transition metal chemistry. As a consequence of the in-stability of these ROSs and their potential to damage cellsand tissues, there are both enzymes and low molecular massmolecules with antioxidant capability that can protect againstthe adverse effects of ROSs (in Saint-Denis et al. 1998).When rates of production of ROSs exceed their rates ofremoval, oxidative damage, such as lipid peroxidation, oc-curs. Among the antioxidants are enzymes that reduce ROSsin order to move stable compounds. Catalase (CAT, EC1.11.1.6) and glutathione peroxidase (GPX, EC 1.11.1.9) in-tervene in hyperoxide detoxification (preventing either ROStoxicity or the chain-propagation reactions involved in lipidperoxidation). CAT is one of the main antioxidant enzymesthat converts hydrogen peroxide to nontoxic oxygen and wa-ter at a very rapid rate (H2O2 → 2H2O + O2

−). Glutathioneperoxidase reduces peroxides (ROOH; H2O2 or organic per-oxides) to their corresponding alcohols (ROH) or water viathe reaction 2GSH + ROOH→ GSSG + ROH + H2O.Superoxide dismutase (SOD, EC 1.15.1.1) reduces oxygenvia the reaction 2O2

− + 2H+ → H2O2 + O2. Glutathione alsointervene in cell defense against ROSs and xenobiotics be-cause it is the substrate for GPX enzymes, it can be conju-gated with electrophilic xenobiotics as a result of GSTactivity, and it can link directly to pro-oxidants. In the cells,

there is an equilibrum between the reduced (GSH) and theoxidized (GSSG) forms of glutathione. However, the onlychemically active form is the reduced one. Thus, as withGPX activities, GST activities cause a reduction in the GSHlevel and, thus, a decrease in the cellular antioxidant status.The glutathione reductase enzyme (GR, EC 1.6.4.2), then,plays an important role in cell protection by reducing GSSGto GSH via the reaction GSSG + NADPH + H+ → 2 GSH +NADP+ (Saint-Denis et al. 1998).

Very few studies of antioxidant enzymes in annelids havebeen performed. However, there is recent evidence that oxi-dative stress may occur in oligochaetes. Little is knownabout oxidative stress and antioxidant defenses in otherclasses of annelids. Stenersen et al. (1979) and Stenersenand Oien (1981) showed the existence of an operative sys-tem involving glutathione and GST in earthworms. Chen etal. (1994) studied the effects of 1 mM cadmium(II) aloneand in combination with several concentrations of iron(II) onin vivo CAT activity in the freshwater oligochaeteTubifextubifex. Those authors reported that CAT activity can in-crease after individuals are treated with certain concentra-tions of cadmium(II), iron(II), or cadmium(II) with iron(II)for 48 h. In contrast, metals, especially iron, have an inhibi-tory effect on purified CAT in vitro and immediately (6 h)post exposure in vivo. A mixture of cadmium(II) and iron(II)increases CAT activity in vivo after 2 days’ exposure, andcertain concentrations of iron(II) are protective against cad-mium(II) toxicity. According to those authors, iron could in-hibit cadmium uptake and transfer; moreover, iron, which isan integral part of the CAT molecule, may enhance the syn-thesis of CAT and could protect theT. tubifexworms againstthe toxic effects of cadmium(II). Furthermore Labrot et al.(1996) showed that in vitro exposure to uranium inhibitsCAT and GPX activities in the earthwormE. f. andrei, whereaslead inhibits only CAT activity. The same results were ob-tained during in vivo exposure. So lead and uranium havethe power to inactivate enzymes. More recently, Saint-Deniset al. (1998) studied the activities of the main enzymes(CAT, GPX, GR, and GST) involved in the antioxidant de-fenses of the same species. The four enzymes were localizedmainly in the cytosolic fraction of cells. CAT distributionwas unusual, as it was not associated with peroxisomes. TheCAT of E. f. andreicatalyzes both catalytic and peroxidativereactions; thus, its properties are consistent with a catalase–peroxidase rather than a true CAT. The authors’ observationsindicate the presence of a distinct cytosolic selenium-dependentGPX (Se-GPX) and a possible microsomal Se-GPX. Strongnon-Se-GPX activity was measured in the CF and the coelom-ocytes, which could be linked to the peroxidase activity offetidins secreted by coelomocytes (Milochau 1997) and withROS production in these cells. According to that authorE. andreiis well equipped for the metabolism of electrophilicand pro-oxidants through glutathione.

4. Conclusion

Annelida are among the first coelomates and are thereforeof special phylogenetic interest. Polychaeta are restricted tothe marine domain and are considered the most primitiveannelids, based on morphology, physiology, and develop-

© 2001 NRC Canada




ment. Oligochaeta, especially the ectoparasitic Hirudinea, aremore evolved.

The immune defenses of these animals partially reflectthis situation. The primary protection against bacterialaggressionis provided by the mucus covering the body wall,especially in Polychaeta. The immune defenses of Annelidahave developed principally as cellular immunity. Phagocytosis,an ancestral mechanism that first appeared in Protozoa, rep-resents an active way of engulfing and destroying microor-ganisms. Encapsulation is more efficient for protectionagainst parasites. The building of the capsule is an interest-ing model of cooperation between several coelomocyte pop-ulations. This process likely involves emission of signals,but the nature of these molecules remains unknown.

Cell cytotoxicity has been well studied in Oligochaeta bythe method of graft rejection. Subsequent xenografts are re-jected faster than the first implant. Accelerated rejection,weak specificity, and short-term “memory” (a few days) arethe three characteristics of the earthworm’s cellular immunedefense system. In contrast to the first-set xenograft, thesecond-set xenograft accelerates the rise in coelomocytenumbers. The small-coelomocyte population becomes themost abundant, whereas larger coelomocytes are not stimu-lated by grafts. This cell specialization could be the evolu-tionary origin of the memory cells of vertebrates, but thesecytotoxic cells could also be the hypothetical evolutionaryprecursor of the NK cells. Polychaeta also contain cytotoxiccoelomocytes that are morphologically similar to the smallcoelomocytes of Oligochaeta. These cells are capable of kill-ing xenogenic cells by a contact-dependent cytolytic pro-cess, releasing an adhesive and lytic factor (perforin-like)against the target cells.

Invertebrates have no humoral components that possessspecificity analogous to vertebrate antibodies. However, theCF contains molecules (agglutinins) that have the capacity toaggregate foreign particles, especially bacteria, through af-finity with the sugars of the microorganism wall. This pro-cess facilitates phagocytosis or encapsulation of pathogens.Prestimulation by bacteria or erythrocytes facilitates (up to7-fold) the release of this agglutinin. The presence of othermolecules (antigen-binding proteins) in Oligochaeta afterhaptened proteins were injected into the CF has been men-tioned. Experimentally, binding with the antigen was signifi-cantly greater when the same antigen was used for sucessivestimulations, but specificity remained considerably lowerthan that of vertebrate immunoglobulins. Oligochaeta alsopossess a hemolytic and antibacterial protein (fetidin) thatconstitutes about 75% of the bacterial activity of the CF.Polychaeta seem to have less active humoral immunity, per-haps because cellular activity seems to be enhanced in thismore primitive group. However, an antibacterial protein act-ing against bacteria by sideroprivation has been detected.

By contrast with the deuterostome coelomates (tunicates,vertebrates), the Protostomia (Annelida, Mollusca, Arthropoda)have not developed specifity in the course of evolution. How-ever, the insects, the most evolved group of Protostomia,have acquired many antibacterial peptides that are availablefor use against several strains of bacteria. Except for theevolved group of Hirudinea, in which an antibacterialpeptide (enkelitin) has been discovered, no other similarmolecules have been mentioned in Annelida.

Annelida react to physical and chemical insults by meansof various processes, principally the synthesis of stress-inducedproteins and modulation (inhibition or stimulation) of en-zyme activities. Moreover, these responses are frequentlydifferent from those of vertebrates, particularly in the natureof the inducers. In other respects, these responses are ex-tremely variable in the phylum Annelida, even closely re-lated species. Our knowledge of defense mechanisms inAnnelida remains very poor, and it is necessary to carry outfurther studies with more species, especially with hirudeans,before wide-ranging conclusions can be drawn concerningthe nature of defense mechanisms in this phylum.

5. References

Andersen, R.A., Aune, T., and Barstad, J.A.B. 1978. Characteris-tics of cholinesterase of the earthwormEisenia foetida. Comp.Biochem. Physiol. C,61: 81–87.

Anderson, R.S. 1980. Hemolysins and haemagglutinins in thecoelomic fluid of a polychaete annelid,Glycera dibranchiata.Biol. Bull. (Woods Hole, Mass.),159: 259–268.

Augustinsson, K.B. 1963. Classification and comparative enzymologyof the cholinesterases and methods for their determination. Handb.Exp. Pharmacol.15: 90–128.

Baert, J.L., and Slomianny, M.C. 1987. Heterosynthetic origin ofthe major yolk protein, vitellin, in a nereid,Perinereis cultrifera(Polychaete Annelida). Comp. Biochem. Physiol. B,88: 1191–1199.

Baskin, D.G. 1974. The coelomocytes of nereid polychaetes.InContemporary topics in immunobiology. Vol. 4.Edited byE.L.Cooper. Plenum Publishing Corp., New York. pp. 55–64.

Bauer-Hilty, A., Dallinger, R., and Berger, B. 1989. Isolation andpartial characterization of a cadmium-binding protein fromLumbriculus variegatus(Oligochaeta, Annelida). Comp. Biochem.Physiol. C, 94: 373–379.

Berghout, A.G.R.V., Wenzel, E., Büld, J., and Netter, K.J. 1991.Isolation, partial purification and characterization of the cyto-chrome P-450-dependent monooxygenase system from themidgut of the earthwormLumbricus terrestris. Comp. Biochem.Physiol. C,100: 389–396.

Bierne, J., and Langlet, C. 1974. Recherches sur l’immunité degreffe chez les Némertiens du genreLineus: étude de la réponseprimaire à la transplantation hétérospécifique. C. R. Acad. Sci.Paris,278: 1445–1447.

Bilej, M., Brys, L., Beschin, A., Lucas, R., Vercauteren, E., Hanusova,R., and De Baetselier, P. 1995a. Identification of a cytolytic proteinin the coelomic fluid ofEisenia foetidaearthworms. Immunol.Lett. 45: 123–128.

Bilej, M., Tuckova, L., and Romanovsky, A. 1995b. Characteriza-tion of the limited specificity of antigen recognition in earth-worms. Folia Microbiol.40: 436–440.

Boiledieu, D., and Valembois, P. 1977. Natural cytotoxicity activityof sipunculid leukocytes on allogeneic and xenogenic erythro-cytes. Dev. Comp. Immunol.1: 207–216.

Boilly-Marer, Y. 1979. Mise en évidence des réactions de rejetsaprès transplantation chez les Néréidiens (Annélides Polychètes).C. R. Acad. Sci. Paris,288: 1411–1414.

Borgeraas, J., Nilsen, K., and Stenersen, J. 1996. Methods for puri-fication of glutathione transferases in the earthworm genusEiseniaand their characterization. Comp. Biochem. Physiol. C,114: 129–140.

Brasil, L. 1904. Arch. Zool. exp. gen.2: 91–255.Brattsten, L.B., Wilkinson, C.F., and Eisner, C.F. 1977. Herbivore

plant interations: mixed function oxidases and secondary plantsubstances. Science (Washington, D.C.),196: 1349–1352.

© 2001 NRC Canada

248 Can. J. Zool. Vol. 79, 2001



© 2001 NRC Canada


Braunbeck, T., and Dales, R.P. 1984. The role of the heart-bodyand of the extravasal tissue in disposal of foreign cells in twopolychaete annelids. Tissue Cell,16: 557–564.

Canicatti, C., and Roch, P. 1993. Erythrocyte membrane structuralfeatures that are critical for the lytic reaction ofSpirographisspallanzanicoelomic fluid hemolysin. Comp. Biochem. Physiol.C, 105: 401–407.

Canicatti, C., Ville, Pagliara, P., and Roch, P. 1992. Hemolysinfrom mucus ofSpirographis spallanzani(Polychaeta Sabellidae).Mar. Biol. (Berl.), 114: 453–458.

Caullery, M., and Mesnil, F. 1898. Les formes épitoques etl’évolution des Cirratuliens. Ann. Univ. Lyon,39: 1–200.

Chain, B.M., and Anderson, R.S. 1983a. Antibacterial activity ofthe coelomic fluid of the polychaeteGlycera dibranchiata. I.The kinetics of the bactericidal reaction. Biol. Bull. (WoodsHole, Mass.),163: 20–40.

Chain, B.M., and Anderson, R.S. 1983b. Antibacterial activity ofthe coelomic fluid of the polychaeteGlycera dibranchiata. II.Partial purification and biochemical characterization of the ac-tive factor. Biol. Bull. (Woods Hole, Mass.),163: 41–49.

Chen, T., Furst, A., and Chien, P.K. 1994. The effects of cadmiumand iron on catalase activities inTubifex. J. Am. Coll. Toxicol.13: 112–120.

Cooper, E.L. 1971. Phylogeny of transplantation immunity: graftrejection in earthworms. Transplant. Proc.3: 214–216.

Cooper, E.L. 1980. Specificity and memory in invertebrates.InPhylogeny of immunological memory.Edited byM.G. Manning.Elsevier – North Holland Biomedical Press, Amsterdam. pp. 245–259.

Dales, R.P. 1982. Haemagglutinins and haemolysins in the bodyfluids of Neoamphitrite figulusandArenicola marina(AnnelidaPolychaeta). Comp. Biochem. Physiol. A,73: 663–667.

Dales, R.P. 1983. Observations on granulomata in the polychaetousNereis diversicolor. J. Invertebr. Pathol.42: 288–291.

Dales, R.P., and Dixon, L.R.J. 1980. Responses of polychaeteannelids tobacterial infection. Comp. Biochem. Physiol. A,67:391–396.

Dales, R.P., and Dixon, L.R.J. 1981. Polychaete.In Invertebrateblood cells.Edited byN.A. Ratcliffe and A.F. Rowley. Vol. 1.Academic Press, New York. pp. 35–74.

Dales, R.P., and Kalac, Y. 1992. Phagocytic defense by the earth-worm Eisenia foetidaagainst certain pathogenic bacteria. Comp.Biochem. Physiol. A,101: 487–490.

Decker, J.M., Elmholt, A., and Muchmore, V. 1981. Spontaneouscytotoxicity mediated by invertebrate mononuclear cells towardnormal and malignant vertebrate targets: inhibition by definedmono- and disaccharidedes. Cell. Immunol.59: 161–170.

Dehorne, A., and Defretin, R. 1933. Phagocytose active des sarcolytesamphioxes chezHeteronereis pelagicaL. C. R. Seances Soc.Biol. 113: 667–680.

Demuynck, S., and Dhainaut-Courtois, N. 1993. Identification ofextracellular haemoglobin as the major high molecular weightcadmium-binding protein of the polychaeteNereis diversicolor.Comp. Biochem. Physiol. C,106: 467–472.

Demuynck, S., and Dhainaut-Courtois, N. 1994. Metal-protein bind-ing patterns in the polychaete wormNereis diversicolorduringshort-term acute cadmium stress. Comp. Biochem. Physiol. C,108: 59–64.

Demuynck, S., Sautière, P., van Beemen, J., and Dhainaut-Courtois,N. 1991. Homologies entre les hémérythrines des sipunculienset une métalloprotéine complexant le cadmium (MPII) d’uneannélide polychaeteNereis diversicolor. C. R. Acad. Sci. Ser. IIISci. Vie, 312: 317–322.

Demuynck, S., Li., K.W., Van der Schors, R.V., and Dhainaut-

Courtois, N. 1993. Amino acid sequence of the small cadmium-binding protein (MP II) from Nereis diversicolor(Annelida,Polychaeta): evidence for a myohemerythrin structure. Eur. J.Biochem.217: 151–156.

Dhainaut, A. 1966. Etude ultrastructurale de l’évolution des éléocyteschezNereis pelagicaL. (Annélide polychète) à l’approche de lamaturité sexuelle. C. R. Acad. Sci. Paris,262: 2740–2743.

Dhainaut, A. 1984a. Aspects cytophysiologiques des coelomocytesde Néréidiens (Annélides polychètes). Arch. Anat. Microsc.Morphol. Exp.73: 133–150.

Dhainaut, A. 1984b. Oogenesis in polychaetes: ultrastructural dif-ferentiation and metabolism of nereid oocytes. Fortrschr. Zool.29: 183–205.

Dhainaut, A., and Porchet-Henneré, E. 1988. Haemocytes andcoelomocytes.In The ultrastructure of the Polychaeta.Edited byW. Westheide and C.O. Hermans. G. Fischer Verlag, Stuttgartand New York. pp. 215–230.

Dhainaut, A., Raveillon, B., M’Beri, M., Porchet-Henneré, E., andDemuynck, S. 1989. Purification of an antibacterial protein inthe coelomic fluid ofNereis diversicolor(Annelida Polychaeta):similitude with a cadmium-binding protein. Comp. Biochem.Physiol. C,94: 555–560.

Dhainaut-Courtois, N., Nejmeddine, A., Baert, J.L., and Dhainaut,A. 1987. Localisation immunocytochimique d’une protéinecomplexant le cadmium chez un ver marin (Nereis diversicolor).C. R. Acad. Sci. Paris,305: 237–241.

Driscoll, S.K., and McElroy, A.E. 1996. Bioaccumulation and metab-olism of benzo[a]pyrene in three species of polychaete worms.Environ. Toxicol. Chem.15: 1401–1410.

Eckelberger, K.J. 1976. Origin and developpement of the amoeb-ocytes of Nicolea zostericola(Polychaeta: Terebellidae). Mar.Biol. (Berl.), 35: 353–370.

Eriksen, K.D.H., Daae, H.L., and Andersen, R.A. 1988. Evidenceof presence of heavy-metal binding proteins in polychaete spe-cies. Comp. Biochem. Physiol. C,91: 377–384.

Eriksen, K.D.H., Andersen, T., and Gray, J.S. 1989. Metal-bindingin polychaetes: quantitative and qualitative studies of five spe-cies. Mar. Environ. Res.28: 167–171.

Eue, I., Kauschke, E., Mohrig, W., and Cooper, E.L. 1998. Isola-tion and characterization of earthworm hemolysins and aggluti-nins. Dev. Comp. Immunol.22: 13–25.

Fauchald, K., and Rouse, G. 1997. Polychaete systematics: pastand present. Zool. Scr.26: 71–138.

Fischer, A., and Dhainaut, A. 1985. The origin of yolk in theoocytes ofNereis virens(Annelida Polychaeta): electronmicroscopicand autoradiographic studies by use of unspecific and yolk-specific markers. Cell Tissue Res.240: 67–76.

Fitzgerald, S.W., and Ratcliffe, N.A. 1982. Evidence for the pres-ence of subpopulations ofArenicola marinacoelomocytes iden-tified by their selective response towards Gram+ve and Gram–vebacteria. Dev. Comp. Immunol.6: 23–34.

Franceschi, C., Cossariza, D., Monti, D., and Ottaviani, E. 1991.Cytotoxicity and immunocyte markers in cells from the freshwatersnail Planorbarius corneusL. (Gasteropoda Pulmonata): impli-cation for the evolution of natural killer cells. Eur. J. Immunol.21: 489–493.

Fries, C.R., and Lee, R.F. 1984. Pollutant effects of the mixedfunction oxygenase (MFO) and reproductive systems of the ma-rine polychaeteNereis virens. Mar. Biol. (Berl.), 79: 187–193.

Fugère, N., Brousseau, P., Krzystyniak, K., Coderre, D., and Fournier,M. 1996. Heavy metal-specific inhibition of phagocytosis anddifferent in vitro sensitivity of heterogeneous coelomocytes fromLumbricus terrestris(Oligochaeta). Toxicology,109: 157–166.

Furst, A., and Nguyen, Q. 1989. Cadmium-induced metallothionein



© 2001 NRC Canada

250 Can. J. Zool. Vol. 79, 2001

in earthworms (Lumbricus terrestris). Biol. Trace Elem. Res.21: 81–85.

George, S.G., and Olsson, P.-E. 1994. Metallothioneins as indica-tors of trace metal pollution.In Biomonitoring of coastal watersand estuaries.Edited byK.J.M. Kramer. CRC Press Inc., BocaRaton, Fla. pp. 151–171.

Gething, M.J., and Sambrook, J. 1992. Protein folding in the cell.Nature (Lond.),355: 323–45.

Goumon, Y., Strub, J.M., Moniatte, M., Nullans, G., Poteur, L.,Hubert, P., Vandorsselaer, A., Aunis, D., and Metz-Boutique,M.Z. 1996. The C-terminal biophospholated proenkephalin-A-(209–237)-peptide from adrenal medullary chromaffin granulesposseses antibacterial activity. Eur. J. Biochem.235: 516–525.

Goven, A.J., Fitzpatrick, L.C., and Venables, B.J. 1994. Chemicaltoxicity and host defense in earthworms: an invertebrate model.Ann. N.Y. Acad. Sci.712: 280–300.

Grelle, C., and Descamps, M. 1998. Heavy metal accumulation byEisenia fetida and its effects on glutatahioneS-transferase.Pedobiologia,42: 289–297.

Hahn, M.E., and Stegeman, J.J. 1992. Phylogenetic distribution ofthe Ah receptor in non-mammalian species: implications for di-oxin toxicity and Ah receptor evolution. Chemosphere,25: 931–937.

Hans, R.K., Khan, M.A., Farooq, M., and Beg, M.U. 1993.GlutathioneS-transferase activity in an earthworm (Pheretimaposthuma) exposed to three insecticides. Soil Biol. Biochem.25:509–511.

Hirigoyenberry, F., Lassalle, F., and Lassegues, M. 1990. Antibacte-rial activity of Eisinia fetida andreicoelomic fluid: transcriptionand translation regulation of lysozyme and proteins evidencedafter bacterial infestation. Comp. Biochem. Physiol. B,95: 71–75.

Hoeger, U. 1991. Hydrolytic enzymes in the coelomic cells of thepolychaeteNereis virensduring sexual maturation. Mar. Biol.(Berl.), 110: 7–12.

Hunt, R., and Taveaux, R. 1906. On the physiological action ofcertain choline derivatives and new methods for detecting choline.Br. Med. J.11: 1788–1791.

Jamieson, B.G.M. 1981. Coelomocytes.In The ultrastructure of theOligochaeta. Academic Press, London and New York. pp. 62–118.

Johansson, M.W., and Soderhall, K. 1989. Cellular immunity incrustaceans and the Propo system. Parasitol. Today,5: 171–176.

Jolles, P., and Zuili, S. 1960. Purification et étude comparée denouveaux lysozymes : extrait de poumon et deNephthys hombergii.Comp. Biochem. Biophys.39: 212–217.

Kaloustian, K.V. 1981. Changes in acetylcholinesterase isozymepattern in estivatingAllobophora caliginosa(Savigny). Comp.Biochem. Physiol. B,70: 157–160.

Kauschke, E., Pagliara, P., Stanbili, L., and Cooper, E.L. 1997.Characterization of proteolytic activity in coelomic fluid ofLumbricus terrestrisL. (Annelida, Lumbricidae). Comp. Biochem.Physiol. B,116: 235–242.

Labrot, F., Ribera, D., Saint Denis, M., and Narbonne, J.F. 1996. Invitro and in vivo studies of potential biomarkers of lead and ura-nium contamination: lipid peroxidation, acetylcholinesterase andglutathione peroxidase activities in three non-mammalian spe-cies. Biomarkers,1: 21–28.

Lai, P.S., So, L.P., and Russel, C.S. 1989. A lipid-associated sulfatedproteoglycan fromNereis coelomic fluid is a hemagglutinin.Comp. Biochem. Physiol. B,93: 859–865.

Langlet, C., and Bierne, J. 1977. The immune response to xenograftsin nemertines of the genusLineus. In Developmental immuno-

biology. Edited by J.B. Salomon and J.D. Horton. Elsevier –North Holland Biomedical Press, Amsterdam. pp. 17–26.

Lassalle, F., Lassegues, M., and Roch, P. 1988. Protein analysis ofeathworm coelomic fluid. IV. Evidence, activity induction andpurification of Eisenia fetida andreilysozyme (Annelida). Comp.Biochem. Physiol. B,91: 187–192.

Lassegues, M., Milochau, A., Doignon, F., Du Pasquier, L., andValembois, P. 1997. Sequence and expresdsion of anEiseniafetida-derived cDNA clonr that encodes the 40-kDa fetidin anti-bacterial protein. Eur. J. Biochem.678: 1–7.

Laulan, A., Morel, A., Lestage, J., Dalaage, M., and Chateaureynaud-Duprat, P. 1985. Evidence of synthesis byLumbricus terrestrisof specific substances in response to an immunization with asynthetic hapten. Immunology,56: 751–758.

Laulan, A., Lestage, J., Bouc, A.M., and Chateaureynaud-Duprat,P. 1988. The phagocytic activity ofLumbricus terrestrisleuko-cytes is enhanced by the vertebrate opsonins/IgG and comple-ment C3b fragment. Dev. Comp. Immunol.12: 269–277.

Lee, R.F., Singer, S.C., Tenore, W.S., Gardner, R.M., and Philpot,R.M. 1979. Detoxifying system in polychaete worms: impor-tance in the degradation of sediment hydrocarbons.In Marinepollution: functional responses.Edited byW.B. Vernberg, F.P.Thurberg, A. Calabrese, and F.J. Vernberg. Academic Press,New York. pp. 23–27.

Lee, R.F., Singer, S.C., and Page, D.S. 1981. Responses of cytochromeP-450 systems in marine crab and polychaetes to organic pollut-ants. Aquat. Toxicol.1: 355–365.

Liimatainen, A., and Hanninen, O. 1982. Occurrence of cytochromeP-450 in the earthwormLumbricus terrestris. In Cytochrome P-450: biochemistry, biophysics and environmental implications.Edited byE. Hietanen, M. Laitinen, and O. Hanninen. Elsevier,Amsterdam. pp. 255–258.

Lindquist, S., and Craig, E.A. 1988. The heat protein. Annu. Rev.Genet.22: 631–677.

Linthicum, D.S., Stein, E.A., Marks, D.H., and Cooper, E.L. 1977a.Electron-microcroscopic observations of normal coelomocytesfrom the earthworm,Lumbricus terrestris.Cell Tissue Res.185:315–330.

Linthicum, D.S., Marks, D.H., Stein, E.A., and Cooper, E.L. 1977b.Graft rejection in earthworms: an electron microscopic study.Eur. J. Immunol.7: 871–876.

Marcano, L., Nusetti, O., Rodriguez-Grau, J., and Vilas, J. 1996.Uptake and depuration of copper and zinc in relation to metal-binding protein in the polychaeteEurythoe complanata. Comp.Biochem. Physiol. C,114: 179–184.

M’Beri, M. 1988. Dégranulation des coelomocytes au cours desréactions immunitaires d’un invertébré marin :Nereis diversicolor(Annélide polychète), caractérisaton des coelomocytes d’aprèsleurs récepteurs membranaires, identification et rôle biologiquedes produits. Ph.D. dissertation, Université de Lille I, France.

M’Beri, M., Debray, H., Dhainaut, A., and Porchet-Henneré, E.1988. Distribution and nature of membrane receptors for differentplant lectins in the coelomocyte subpopulations of the annelidNereis diversicolor. Dev. Comp. Immunol.12: 1–15.

McElroy, A.E. 1990. Polycyclic aromatic hydrocarbon metabolismin the polychaeteNereis virens. Aquat. Toxicol.18: 35–50.

Mieyal, J.J. 1985. Monooxygenase activity of hemoglobin andmyoglobin. Rev. Biochem. Toxicol.7: 1–66.

Milligan, D.L., Babish, J.G., and Neuhauser, E.F. 1986. Noninducibilityof cytochrome P-450 in the earthwormDendrobaena veneta.Comp Biochem. Physiol. C,85: 85–87.

Milochau, A. 1997. Purification d’une nouvelle classe de protéinesantibactériennes, les fétidines isolées chez l’annélideEiseniafetida andrei: étude de leurs activités biologiques, caractéristiques



biochimiques et séquence primaire : comparaison avec une protéinerecombinée codée par un ADNc cloné dansEscherichia coli.Ph.D. dissertation, University of Bordeaux I, Bordeaux, France.pp. 1–87.

Milochau, A., Lassegues, M., and Valembois, P. 1997. Purification,characterization and activities of two hemolytic and antibacterialproteins from coelomic fluid of the annelidEisenia fetida andrei.Biochem. Biophys. Acta,1337: 123–132.

Mohrig, W., Eue, I., Kauschke, E., and Hennicke, F. 1996. Crossreactivity of hemolytic and hemagglutinating proteins in thecoelomic fluids of Lumbricidae (Annelida). Comp. Biochem.Physiol. A, 115: 19–30.

Morgan, J.E., Norey, C.G., Morgan, A.J., and Kay, J. 1989. A com-parison of the cadmium-binding proteins isolated from the pos-terior alimentary canal of the earthwormsDendrodrilus rubidusandLumbricus rubellus. Comp. Biochem. Physiol. C,92: 15–21.

Nebert, D.W., Nelson, D.R., and Feyereisen, R. 1989. Evaluationof the cytochrome P-450 genes. Xenobiotica,19: 1149–1160.

Nejmeddine, A., Dhainaut-Courtois, N., Baert, J.L., Sautière, P.,Fournet, B., and Boulanger, P. 1988. Purification and character-ization of a acdmium-binding protein fromNereis diversicolor(Annelida, Polychaeta). Comp. Biochem. Physiol. C,89: 321–326.

Nejmeddine, A., Sautière, P., Dhainaut-Courtois, N., and Baert,J.L. 1992. Isolation and characterization of a Cd-binding proteinfrom Allolobophora caliginosa(Annelida, Oligochaeta): distinc-tion from metallothioneins. Comp. Biochem. Physiol. C,101:601–605.

Nejmeddine, A., Wouters-Tyrou, D., Baert, J.L., and Sautière, P.1997. Primary structure of a myohemerythrin-like cadmium-binding protein, isolated from a terrestrial annelid oligochaete.C. R. Acad. Sci. Ser. III Sci. Vie,320: 459–468.

Nelson, P.A., Stewart, R.R., Morelli, M.A., and Nakatsugawa, T.1976. Aldrin epoxidation in the earthwormLumbricus terrestris.L. Pestic. Biochem. Physiol.6: 243–253.

Organization for Economic Cooperation and Development (OECD).1984. OECD guidelines for testing chemicals. Section 2: Effectson biotic systems. Method 207. Earthworms, acute toxicity test.Organization for Economic Cooperation and Development, Paris.

Paemen, L.R., Porchet-Henneré, E., Masson, M., Leung, M.K.,Hughes, T.K., and Stefano, G.B. 1992. Glial localization of glialinterleukin-1 in invertebrate ganglia. Cell Mol. Neurobiol.12:463–471.

Parrinello, N., and Rindone, D. 1981. Studies on the natural hemolyticsystem of the annelid wormSpirographis spallanzaniViviani(Polychaeta). Dev. Comp. Immunol.5: 33–42.

Perin, J.P., and Jolles, P. 1972. The lysozyme fromNepthys hombergi(Annelid). Biochem. Biophys. Acta,263: 683–689.

Porchet-Henneré, E. 1969. Corrélations entre le cycle biologiqued’une Coccidie :Coelotropha durchoni; et celui de son hôte :Nereis diversicolor(Annélide Polychète). Z. Parasitenkd.31:299–314.

Porchet-Henneré, E. 1990. Cooperation between different coelom-ocyte populations during the encapsulation response ofNereisdiversicolor demonstrated by using monoclonal antibodies. J.Invertebr. Pathol.56: 353–361.

Porchet-Henneré, E., and Dugimont, T. 1992. Adaptability of thecoccidianCoelotrophato parasitism. Dev. Comp. Immunol.16:263–274.

Porchet-Henneré, E., and Vernet, G. 1992. Cellular immunity in anannelid (Nereis diversicolor, Polychaeta): production of melanin bya subpopulation of granulocytes. Cell Tissue Res.269: 167–174.

Porchet-Henneré, E., Nejmeddine, A., Baert, J.L., and Dhainaut-Courtois, N. 1987a. Selective immunostaining of type I granulo-

cytes of the polychaete annelidNereis diversicolorby a mono-clonal antibody against a cadmium-binding protein (MP II). Biol.Cell. 60: 259–261.

Porchet-Henneré, E., M’Beri, M., Dhainaut, A., and Porchet, M.1987b. Ultrastructural study of the encapsulation response of thepolychaete annelidNereis diversicolor. Cell Tissue Res.248:463–471.

Porchet-Henneré, E., Dugimont, T., and Fischer, A. 1992. Naturalkiller cells in a lower invertebrate,Nereis diversicolor. Eur. J.Cell Biol. 58: 99–107.

Principato, G.B., Ambrosini, M.V., Menghini, A., Giovannini, E.,and Dell’Agata, M. 1978. Multiple forms of acetylcholinesterasein Allolobophora caliginosa: purification and partial character-ization. Comp. Biochem. Physiol. C,61: 147–151.

Principato, G.B., Ambrosini, M.V., and Giovannini, E. 1979. Kineticstudies on the acetylcholinesterase (AChE) fromAllolobophoracaliginosa. Comp. Biochem. Physiol. C,62: 173–180.

Principato, G.B., Ambrosini, M.V., Liotti, F.S., and Giovannini, E.1981. Propionylcholinesterase inHirudo medicinalis: purifica-tion, partial characterization and comparative study with a mam-malian acetylcholinesterase. Comp. Biochem. Physiol. C,70:209–213.

Principato, G.B., Contenti, S., Talesa, V., Mangiabene, G., Pascolini,R., and Rosi, G. 1989. Propionylcholinesterase fromAllolobophoracaliginosa. Comp. Biochem. Physiol. C,94: 23–27.

Principato, G.B., Rosi, G., Biagioni, M., and Giovannini, E. 1983.Kinetic studies on the reaction mechanism of the propionyl-cholinesterase fromHirudo medicinalis. Comp. Biochem. Physiol.C, 75: 185–192.

Ratcliffe, N.A., Rowley, A.F., Fitzgerald, S.W., and Rhodes, C.P.1985. Invertebrate immunity: basic concepts and recent advances.Int. Rev. Cytol.97: 183–350.

Roch, P., Valembois, P., Davant, N., and Lassegues, M. 1981. Pro-tein analysis of earthwormEisenia fetida andreifactor (EFAF).Comp. Biochem. Physiol. B,69: 829–836.

Roch, P., Giangrande, A., and Canicatti, C. 1990. Comparison ofhemolytic activity of eight species of polychaetes. Mar. Biol.(Berl.), 107: 199–203.

Roch, P., Stabili, L., and Pagliara, P. 1991. Purification of threeserine proteases from the coelomic cells of earthworms (Eiseniafetida). Comp. Biochem. Physiol. B,98: 597–602.

Roch, P., Ville, P., and Cooper, E.L. 1998. Characterization of a14 kDa plant-related serine protease inhibitor and regulation ofcytotoxic activity in earthworm coelomic fluid. Dev. Comp.Immunol. 22: 1–12.

Romieu, M. 1923. Recherches histophysiologiques sur le sang et lecorps cardiaque des Annélides polychètes: contribution à l’histologiecomparée du sang. Arch. Morphol. Gen. Exp.17: 1–336.

Ruffin, P., Demuynck, S., Hilbert, J.L., and Dhainaut, A. 1994.Stress induced proteins in the polychaete annelidNereisdiversicolor induced by heat shock or cadmium exposure.Biochimie, 76: 423–427.

Russel, C.S., Rodriguez, J., and Lai, P.S. 1983. Hemagglutininactivity in Nereiscoelomic fluid. Comp. Biochem. Physiol. A,75: 57–64.

Saint-Denis, M., Labrot, F., Narbonne, J.F., and Ribera, D. 1998.Glutathione, glutathione related enzymes, and catalase activitiesin the earthwormEisenia fetida andrei. Arch. Environ. Contam.Toxicol. 35: 602–614.

Salzet, M. 2001. The neuroendocrine system of annelids. Can. J.Zool. 79: 175–191.

Salzet-Raveillon, B., Rentier-Delrue, F., and Dhainaut, A. 1993.Detection of mRNA encoding an antibacterial-metalloprotein(MPII) by in situ hybridization with a cDNA probe generated

© 2001 NRC Canada




by polymerase chain reaction in the wormNereis diversicolor.Cell. Mol. Biol. 39: 105–114.

Scaps, P., Demuynck, S., Descamps, M., and Dhainaut, A. 1996.Biochemical and enzymatic characterization of an acetyl-cholinesterase fromNereis diversicolor(Annelida, Polychaeta):comparison with the cholinesterases ofEisenia fetida(Annelida,Oligochaeta). Biol. Bull. (Woods Hole, Mass.),190: 396–402.

Scaps, P., Demuynck, S., Descamps, M., and Dhainaut, A. 1997a.Effect of organophospahte and carbamate pesticides on acetyl-cholinesterase and choline acetyltransferase activities of the poly-chaeteNereis diversicolor. Arch. Environ. Contam.33: 203–208.

Scaps, P., Demuynck, S., Descamps, M., and Dhainaut, A. 1997b.Cadmium and lead accumulation in the earthwormEisenia fetida(Savigny) and its impact on cholinesterase and metabolic path-way enzyme activity. Comp. Biochem. Physiol. C,116: 233–238.

Schubert, I., and Messner, B. 1971. Untersuchungen über dasVorkommen von Lysozym bei Anneliden. Zool. Jahrb. Physiol.76: 36–50.

Stefano, G.B., and Salzet, M. 1999. Invertebrate opioid precursors:evolutionary conservation and the significance of enzymatic pro-cessing. Int. Rev. Cytol.187: 261–285.

Stein, E.A., and Cooper, E.L. 1981. The role of opsonins in phago-cytosis by coelomocytes of the earthworm,Lumbricus terrestris.Dev. Comp. Immunol.5: 415–425.

Stein, E.A., and Cooper, E.L. 1983. Carbohydrate and glycoproteinsinhibitors of naturally occurring and induced agglutinins in theearthwom,Lumbricus terrestris. Comp. Biochem. Physiol. B,76: 197–206.

Stein, E.A., Avtalion, R.R., and Cooper, E.L. 1977. The coelomocytesof the earthwormLumbricus terrestris: morphology and phagocyticproperties. J. Morphol.153: 467–478.

Stein, E.A., Wodjani, A., and Cooper, E.L. 1982. Agglutinins inthe earthworm,Lumbricus terrestris: naturally occurring or in-duced? Dev. Comp. Immunol.6: 407–422.

Stein, E.A., Younai, S., and Cooper, E.L. 1986. Bacterial aggluti-nins of the earthworm,Lumbricus terrestris. Comp. Biochem.Physiol. B,84: 409–415.

Stein, E.A., Moravati, A., Rahimian, P., and Cooper, E.L. 1989.Lipid agglutinins from coelomic fluid of the earthworm,Lumbricusterrestris. Comp. Biochem. Physiol. B,94: 703–707.

Stein, E.A., Younai, S., and Cooper, E.L. 1990. Separation and par-tial purification of agglutinins from coelomic fluid of the earth-worm, Lumbricus terrestris. Comp. Biochem. Physiol. B,97:701–705.

Stenersen, J. 1979. Action of pesticides on earthworms. Part II:elimination of parathion by the earthwormEisenia foetidaSavigny.Pestic. Sci.10: 104–112.

Stenersen, J. 1980a. Esterases of earthworms. Part I: characteriza-tion of the cholinesterases inEisenia foetida(Savigny) by sub-strates and inhibitors. Comp. Biochem. Physiol. C,66: 37–44.

Stenersen, J. 1980b. Esterases of earthworms. Part II: characteriza-tion of the cholinesterases inEisenia foetida(Savigny) by ionexchange chromatography and electrophoresis. Comp. Biochem.Physiol. C,66: 45–51.

Stenersen, J. 1984. Minireview: detoxification of xenobiotics byearthworms. Comp. Biochem. Physiol. C,78: 249–252.

Stenersen, J. 1992. Uptake and metabolism of xenobiotics byearthworms.In Ecotoxicology of earthworms.Edited by P.W.Greigh-Smith, H. Becker, P.J. Edwards, and F. Heimbach. Inter-cepts Ltd., Andover, U.K. pp. 129–139.

Stenersen, J., and Oien, N. 1981. Glutathione S-transferase in earth-worms (Lumbricidae): substrate specificity, tissue and speciesdistribution and molecular weight. Comp. Biochem. Physiol. C,69: 243–252.

Stenersen, J., Guthenberg, C., and Mannervik, B. 1979. GlutathioneS-transferase in earthworms (Lumbricidae). J. Biochem.181:47–50.

Stenersen, J., Kobro, S., Bjerke, M., and Arend, U. 1987. Glutathionetransferases in aquatic and terrestrial animals from nine phyla.Comp. Biochem. Physiol. C,86: 73–82.

Stenersen, J., Brekke, E., and Engelstad, F. 1992. Earthworms fortoxicity testing; species differences in response towardscholin-esterase inhibiting pesticides. Soil Biol. Biochem.24: 1761–1764.

Stokke, K., and Stenersen, J. 1993. Non-inducibility of the glutathionetransferases of the earthwormEisenia andrei. Comp. Biochem.Physiol. C,106: 753–756.

Stone, H.C., and Overnell, J. 1985. Minireview: non-metallothioneincadmium-binding protein. Comp. Biochem. Physiol. C,80: 9–14.

Stürzenbaum, S.R., Kille, P., and Morgan, A.J. 1998. The identifica-tion, cloning and characterization of earthworm metallothionein.FEBS Lett.431: 437–442.

Suzuki, K.T., Yamamaura, M., and Mori, T. 1980. Cadmium-bindingproteins induced in the earthworm. Arch. Environ. Contam. Toxicol.9: 415–424.

Suzuki, M., Cooper, E.L., Eyambe, G.S., Goven, A.G., Fitzpatrick,L.C., and Venables, B.J. 1995. Polychlorinated biphenyls (PCBs)depress allogeneic natural cytotoxicity by earthworm coelomocytes.Environ. Toxicol. Chem.14: 1697–1700.

Suzuki, M.M., and Cooper, E.L. 1995a. Spontaneous cytotoxicearthworm leukocytes kill K562 tumor cells. Zool. Sci. (Tokyo),12: 443–451.

Suzuki, M.M., and Cooper, E.L. 1995b. Allogenic killing by earth-worm effector cells. Nat. Immunol.14: 11–19.

Svendsen, C., Meharg, A.A., Freestone, P., and Weeks, J.M. 1996.Use of an earthworm lysosomal biomarker for the ecological as-sessment of pollution from an industrial plastic fire. Appl. SoilEcol. 3: 99–107.

Takagi, T., and Cox, J.A. 1991. Primary structure of myohemerythrinfrom the annelidNereis diversicolor. FEBS Lett.285: 25–27.

Talesa, V., Grauso, M., Giovannini, E., Rosi, G., and Toutant, J.P.1995. Solubilization, molecular forms, purification and substratespecificity of two acetylcholinesterases in the medical leech(Hirudo medicinalis). J. Biochem.306: 687–692.

Thomas, J.A. 1930. Etudie d’un processus néoplasique chezNereisdiversicolor O.F.M. dû à la dégenérescence des ovocytes etquelquefois des soies. Arch. Anat. Microsc.26: 251–333.

Toupin, J., and Lamoureaux, G. 1976. Coelomocytes of earthworms:phytohemagglutinin (PHA) responsiveness.In Phylogeny ofthymus andbone marrow-bursa cells.Edited byR.K. Whrightand E.L. Cooper. Elseiver – North Holland Biomedical Press,Amsterdam. p. 19.

Tuckova, L., Rejnek, J., and Sima, P. 1988. Response to parentstimulation in earthwormLumbricus terrestrisandEisenia foetida.Dev. Comp. Immunol.12: 287–296.

Tuckova, L., Rejnek, J., Bilej, M., and Pospisil, R. 1991a. Charac-terization of antigen-binding protein in earthwormsLumbricusterrestrisandEisenia fetida. Dev. Comp. Immunol.15: 263–268.

Tuckova, L., Rejnek, J., Hajkova, H., and Romanowsky, A. 1991b.Monoclonal antibodies to antigen binding protein of annelid(Lumbricus terrestris). Comp. Biochem. Physiol. B,100: 19–23.

Valembois, P. 1971. Etude ultrastructurale des coelomocytes dulombricienEisenia fetida(Sav). Bull. Soc. Zool. Fr.96: 59–72.

Valembois, P. 1974. Cellular aspects of graft rejection in earth-worms and some other Metazoa.In Contemporary topics inimmunobiology. Vol. 4.Edited byE.L. Cooper. Plenum Press,New York.

Valembois, P., and Lassegues, M. 1995. In vitro generation of reac-tive oxygen species by free coelomic cells of the annelidEisenia

© 2001 NRC Canada

252 Can. J. Zool. Vol. 79, 2001



© 2001 NRC Canada


fetida andrei: an analysis by chemiluminescence and nitro bluetetrazolium reduction. Dev. Comp. Immunol.19: 195–204.

Valembois, P., Roch, P., and Boiledieu, D. 1980. Natural and in-duced cytotoxicities in sipunculids and annelids.In Phylogenyof immunological memory.Edited byM.G. Manning. Elsevier –North Holland Biomedical Press, Amsterdam. pp. 47–55.

Valembois, P., Roch, P., Lassegues, M., and Davant, N. 1982.Bacteriostatic activity of a chloragogen cell secretion. Pedo-biologia, 24: 191–195.

Valembois P., Roch, P., and Lassegues, M. 1984. Simultaneous ex-istence of hemolysins and hemagglutinins in the coelomic fluidand in the cocoon albumen of the earthwormEisenia fetidaandrei. Comp. Biochem. Physiol. A,78: 141–145.

Valembois, P., Lassegues, M., and Roch, P. 1992. Formation ofbrown bodies in the coelomic cavity of the earthwormEiseniafetida andreiand attendant changes in shape and adhesive ca-pacity of constitutive cells. Dev. Comp. Immunol.16: 95–101.

Valembois, P., Lassegues, M., Hirigoyenberry, F., and Seymour, J.1993. Clearance and brekdown of pathogenic bacteria injectedinto the body cavity of the earthwormEisenia fetida andrei.Comp. Biochem. Physiol. C,106: 255–260.

Valembois, P., Seymour, E., and Roch, P. 1994. Evidence oflipofuscin and melanin in the brown body of the earthwormEisenia fetida. 277: 183–188.

Ville, P., Roch, P., Cooper, E.L., Masson, P., and Narbonne, J.F.1995. PCBs increase molecular-related activities (lysozyme, anti-bacterial hemolysis, proteases) but inhibit macrophage-relatedfunctions (phagocytosis, wound healing) in earthworms. J.Invertebr. Pathol. 65: 217–224.

Vivier, E., and Porchet-Henneré, E. 1964. Cytologie, cycle et affinités

de la CoccidieCoelotropha durchoni, parasite deNereis diversicolor(Annélide Polychète). Bull. Biol. Fr. Belg.18: 153–206.

Weeks, J.M., and Svendsen, C. 1996. Neutral red retention bylysosomes from earthworm (Lumbricus rubellus) coelomocytes:a simple biomarker of exposure to soil copper. Environ. Toxicol.Chem.15: 1801–1805.

Willuhn, J., Otto, A., Schmitt-Wrede, A.P., Greven, H., andWunderlich, F. 1994. cDNA cloning of a cadmium-induciblemRNA encoding a novel cysteine-rich, non-metallothionein25-kDa protein in an enchytraeid earthworm. J. Biol. Chem.269: 24 688 – 24 691.

Willuhn, J., Otto, A., Schmitt-Wrede, A.P., and Wunderlich, F.1996. Earthworm gene as indicator of bioefficacious cadmium.Biochem. Biophys. Res. Commun.220: 581–585.

Winners, W., Neef, J., and Van Wachtendonk, D. 1978. Distribu-tion of cholinesterases in haemolymphs and smooth muscles ofmolluscs. Comp. Biochem. Physiol. C,61: 121–131.

Yamamura, M., Mori, T., and Suzuki, K.T. 1981. Metallothioneininduced in the earthworm. Experientia,37: 1187–1189.

Yoshino, T.P. 1986. Surface membranes components of circulatinginvertebrate blood cells and their role in internal defence.InImmunity of invertebrates.Edited by M. Brehelin. Springer-Verlag, Berlin and Heidelberg. pp. 12–24.

Young, J.S., and Roesijadi, G. 1983. Reparatory adaptation tocopper-induced injury and occurrence of a copper-bindingprotein in the polychaeteEudistyla vancouveri. Mar. Pollut.Bull. 14: 30–32.

Yu, S.T. 1982. Host plant induction of glutathioneS-transferase inthe fall armyworm. Pestic. Biochem. Physiol.18: 101–106.



immune defense and biological responses induced by toxics ...€¦ · annelida react to physical...

Documents