Pedobiologia 47, 676–688, 2003

© Urban & Fischer Verlag

http://www.urbanfischer.de/journals/pedo

The 7th international symposium on earthworm ecology · Cardiff · Wales · 2002

Earthworm immunity: a model of immune competence

Edwin L. Cooper1* and Philippe Roch2

1 Laboratory of Comparative Immunology, Department of Neurobiology, David Geffen School of Medicine at UCLA,
University of California, Los Angeles, Los Angeles, California 90095-1763, USA
2 Université de Montpellier 2, Défense et Résistance chez les Invertébrés Marins (DRIM), cc 080, Place E. Bataillon,
34095 Montpellier cedex 5, France

Submitted September 6, 2002 · Accepted May 16, 2003

Summary
From the 1960s, work on the immune system of earthworms was concerned with inflammatory and cellular responses af-
ter tissue transplantation. Later, interest in the humoral immune system emphasized those molecules that cause death of
the targets or regulate immune-related activities. Specific interest was on cytolysins, agglutinins, proteases, protease in-
hibitors and phenoloxidase. Responses of the immune system were used as markers to evaluate the effects of xenobiotics
on the environment. Earthworms were confronted with three contaminants (Arochlor 1254, 2,4 D, and carbaryl) and the
T2 toxin. Lysozyme was enhanced by Arochlor, but inhibited by carbaryl and T2 toxin, whereas 2,4 D had no effect. Only
Arochlor and carbaryl significantly increased intracellular serine protease activity. Plasma serine protease inhibitory activ-
ity was completely suppressed by carbaryl and also reduced by T2 toxin. Phagocytosis was dramatically depressed and the
effect can be observed on macrophage morphology. All xenobiotics increased plasma cytolytic activity. Detoxification me-
tabolism was stimulated by carbaryl but not by T2 toxin. Since T2 toxin did not increase cytochrome P450 detoxification
pathway, this may explain its inhibitory effect on phagocytosis, serine protease inhibitors and lysozyme. We hypothesize
that long-term effects of environmental contamination by toxic substances may interfere with the earthworm populations
through their immune capacities.

Key words: Earthworms, Lumbricus, Eisenia, immunity, xenobiotics, contaminants

The earthworm’s innate immune system

Earthworm innate immunity has received less attention
than that of the “model” invertebrate, Drosophila. Earth-
worm immunity is intrinsically interesting, however, from
the dual perspective of both immune system function and
of ecological importance. The capacity of oligochaetes to
destroy bacteria and other microbes apparently exists in
parallel with the capacity to lyse foreign, eukaryotic cells,
two responses found in many invertebrates. Earthworm’s
body cavity contains coelomic fluid and leukocytes, mor-
phologically as varied as they are in other equally com-
plex invertebrates. In fact, they are structurally and func-
tionally similar to vertebrate leukocytes (Vetvicka et al.
1994) with respect to opsonization, phagocytosis, encap-
sulation, inflammation, agglutination, mitogenesis, lysis
and destruction of allogeneic, xenogeneic but not auto-
geneic targets in vivo and in vitro.

*E-mail corresponding author: cooper@mednet.ucla.edu

0031-4056/03/47/05–06–676 $15.00/0

Table 1. Innate immune components in invertebrates (modified and completed from Cooper et al. 2002)
COMPONENTS / CHARACTERISTICS
Encapsulating-relating proteins (ERPs)
Eicosanoïds
Inducible inflammatory system (Toll, NFΚB)
Acute phase proteins: Pentraxins, Ig superfamily, complement
α2-macroblobulin family (protease inhibitor)
C3 homologue
Collectin (collagenous, carbohydrate binding protein)
Lectin-mediated complement pathway
Mannose binding protein (MBP) –Lectin (MBL)
Mannose associated serine protease (MASP)
Lipoprotein-receptor related protein (LRP/α2 M-R)
Antimicrobial peptides
Lytic and antimicrobial components
Pattern recognition molecule (CCF)
Reactive oxygen intermediates (ROIs)
Fibrinogen related proteins (FREPS)
NK-like cells associated with CD markers
Acute phase proteins: C-reactive protein, LPS-binding protein (LBP)
Cellular activities are based mainly upon phagocy-
tosis and leukocyte cell-to-cell recognition (Roch
1996). The latter leads to cytotoxicity (Valembois et al.
1980a) mixed lymphocyte stimulation like-reactions
(Valembois et al. 1980b) and cellular cooperation
(Cooper & Roch 1984). Humoral activities include
lysozyme (Lassalle et al. 1988), synthesis and secre-
tion of agglutinins (Stein et al. 1990), a phenoloxi-
dase/peroxidase system (Valembois et al. 1991), and
synthesis and expression of the first to be discovered
lytic components, the fetidins (Roch 1979). An explicit
aim of the present paper is to review evidence that
earthworm immune response can be modulated by ex-
posure to xenobiotics.
Inflammatory responses and cellular
immunity in vitro
Earthworm immunobiologists first demonstrated cy-
totoxicity using leukocyte effectors co-cultured with a
variety of targets including allogeneic cells (Valem-
bois et al. 1980a; Cooper 1981, 1994). More recent
trials involving autogeneic cells alone or in mixtures
from the same genus as in allogeneic combinations re-
vealed a correlation between morphology and func-
tion. For instance, small leukocytes lyse classical NK-
Earthworm immunity: a model of immune competence 677
ANIMAL MODELS
Insect (Tenebrio molitor)
Insects
Insects, arthropods,
Caenorhabditis elegans
Insects/arthropods
Nematodes, crustaceans, mollusks, echinoderms, tunicates
Echinoderms, tunicates, cephalochordates, insects
Tunicates, horseshoe crab
Tunicates, horseshoe crab
Caenorhabditis elegans, tunicates
Tunicates
Caenorhabditis elegans
Arthropods, mollusks (mussels), tunicates
Earthworm, mollusks (mussels)
Earthworms
Mollusks, echinoderms, tunicates, crustaceans
Mollusks (snails)
Sipunculids, annelids, mollusks
Crustaceans, horseshoe crab
sensitive cells as well as NK-resistant targets (Cooper
et al. 1995). First, cell contact is essential as revealed
by the intricacies of lysis after binding of effectors to
target cell membranes. Second, killing is effected by
small leukocytes. Third, debris are removed by large
leukocytes that effect phagocytosis and granuloma
formation (Quaglino et al. 1996; Cossarizza et al.
1996).
Transplantation experiments in earthworms of allo-
and xenografts revealed specificity and weak memory
(Cooper 1969; Cooper & Roch 1986). Small leuko-
cytes are dense, not only phagocytic, and are positive
for cell markers CD11a, CD45RA, CD45RO,
CDw49B, CD54, Thy-1 (CD90) and β2-microglobulin
(Cooper & Mansour 1989; Roch et al. 1983). Large
leukocytes do not bind to K562 tumor cells but are
mostly phagocytic and negative for the above markers.
All leukocytes are negative for other CD and MHC
class I and class II markers. Although CD markers are
associated with human lymphocytes, monocytes and
macrophages, those positive earthworm small leuko-
cytes are interpreted as putative (convergent?) evolu-
tionary precursors. Dissociation of phagocytosis and
cytotoxicity, mediated by two different cell types in
Vertebrates, suggests that earthworm leukocytes, and
most likely those of other invertebrates, are multi-
functional and not exclusively phagocytic.
Pedobiologia (2003) 47, 676–688
678 Edwin L. Cooper and Philippe Roch

Humoral immunity: lysins and serine Lysenin

proteases of Eisenia fetida
Fetidins
The fetidins of E. fetida are heat labile, polymorphic
and multifunctional proteins, responsible for cytolysis,
antibacterial reaction and clotting (Valembois et al.
1982, 1988; Roch et al. 1989). Clotting of fetidins
eliminates non-pathogenic bacteria, mediated by ser-
ine protease/serine protease inhibitor equilibrium.
These constitute normal responses that occur at low
but constant rates on the outer surface of earthworms.
Mixed with mucus, fetidins cover the body where they
constitute an external nonspecific antimicrobial barrier
(Valembois et al. 1985). Complexity surrounding E.
fetida lytic components, including fetidins, has been
partially clarified (Table 2).
Fetidins appear to be controlled by two independent
genes: one always expressed and coding for a pI 6.1 mol-
ecule. The other encompasses four alleles that define at
least ten genetic families (Roch et al. 1987). Lysis de-
pends upon the presence of 2 monomeric glycoproteins
of 40 and 45 kDa (Roch et al. 1981). Significant similar-
ities between them are large quantities of aspartic acid,
glutamic acid and glycine (Roch et al. 1981). In addition,
polyclonal as well as monoclonal antibodies cross-re-
acted with the 2 fetidins. The cDNA encoding a 34-kDa
protein that corresponds to the size of deglycosylated fe-
tidins contains a peroxidase signature that confirms per-
oxidase activity of fetidins (Lassegues et al. 1997). The
recombinant protein is also antibacterial since it inhibits
in vitro growth of Bacillus megaterium. Fetidins possess
varying degrees of homology with lysenins 1, 2 or 3.
Meanwhile, there is no significant homology with CCF
nor lumbricin I (Cooper et al. 2002).
Lysenin, a 41-kDa protein isolated from E. fetida, is
a protein sharing biological roles and biochemical
properties with fetidins, and eiseniapore, but charac-
terized by different functional properties (Sekizawa
et al. 1997) (Table 2). Although native and recombi-
nant lysenin had similar contractile activities on rat
smooth muscle, the amino acid sequence revealed
no significant homology to previously characterized
vasoactive substances. Like fetidins, lysenin induces
hemolysis and binds specifically to sphingomyelin
in cellular membranes, including those found in var-
ious spermatozoa. However, lysenin differs in amino
acid sequence and certain biological characteristics
from the other E. fetida lytic proteins. For instance,
lysenin did not bind to other phospholipids, includ-
ing sphingomyelin analogues such as sphingosine,
ceramide, and sphingosyl-phosphorylcholine, sug-
gesting that it recognizes the precise molecular
structure of sphingomyelin (Yamaji et al. 1998).
Sphingomyelin accessibility to lysenin increases
with membrane redistribution induced by choles-
terol.
Northern blot analysis revealed that the lysenin gene
is expressed in coelomic cells (Ohta et al. 2000). Im-
mune reactive lysenin was detected in large leukocytes
and in free large chloragogue cells present in the lumen
of the typhlosole, a depression in the dorsal wall of the
intestine. The coelomic fluid of E. fetida was toxic to
vertebrate cells, but not to cells derived from
42 species belonging to 7 invertebrate phyla
(Kobayashi et al. 2001). The toxicity to fish, tadpoles
and mice was lost after heating the coelomic fluid at
56 °C, as was also demonstrated for fetidins or super-
natants from in vitro coelomic cell cultures.

Table 2. Listing of names and characteristics of Eisenia fetida proteins exerting cytolytic functions (except lumbricin I from Lumbricus rubel-
lus) (modified from Cooper et al. 2002)

NAME PRESENCE IDENTIFIED FUNCTIONS

Fetidin 1, 40 kDa, Coelomic fluid, chloragocytes, increases Bind to sphingomyelin, hemolytic,
Four isoforms after injecting pathogenic bacteria pore-forming, bacteriostatic, agglutination,
Fetidin 2, 45 kDa clotting, opsonization, heme-binding
Monomorphic enzyme, peroxidase
Lysenin, 41 kDa Coelomic fluid, chloragocytes, leukocytes Contract rat smooth muscle
Three isoforms
Eiseniapore, 38 kDa Coelomic fluid and/or cells Bind to sphingomyelin, pore-forming
Coelomic Cytolytic Factor (CCF) 42 kDa Coelomic fluid, leukocytes Opsonization, tumorolytic, pattern
recognition molecule
Lumbricin I, 7.2 kDa Whole worm, constitutive Antimicrobial

Pedobiologia (2003) 47, 676–688


Eiseniapore
Eiseniapore is a protein of 38 kDa with lytic activity
isolated from E. fetida (Lange et al. 1997) (Table 2).
Various lipid vesicles were used to determine the re-
ceptor to eiseniapore. The lysine molecules bind to and
disturb the lipid bilayer only when particular sphin-
golipids, consisting of a hydrophilic head group as
phosphorylcholine or galactosyl and the ceramide
backbone, (e.g. sphingomyelin), are present. As for ly-
senin, cholesterol enhances eiseniapore lytic activity
toward sphingomyelin-containing vesicles. Leakage of
vesicles was most efficient when the lipid composition
resembled that of the outer leaflet of human erythro-
cytes.
The secondary structure of eiseniapore did not
change upon binding to lipid membranes. Meanwhile,
lytic activity was completely abolished after denatura-
tion or pre-incubation with polyclonal antibodies, sug-
gesting that i) the presence of specific sphingolipids is
sufficient for eiseniapore mediated lytic activity, or ii)
membrane glycoproteins, are not required as long as
specific sphingolipids are present. Electron micro-
scopic observations revealed several ring shaped struc-
tures (pores) on erythrocyte membranes, composed of
a central channel (3 nm) surrounded by an external
ring of 10 nm in diameter. The channel complex con-
sists of six monomers as deduced from the molecular
mass of 228 kDa (Lange et al. 1999). Almost identical
morphological structures and polymerisation were re-
ported also for fetidins concluding that erythrocyte ly-
sis necessitates more than 10,000 pores per square mi-
crometer (Roch et al. 1989).
Coelomic cytolytic factor (CCF)
Bilej et al. (1995) identified a coelomic cytolytic factor
of 42 kDa from E. fetida. This protein is not hemolytic,
but able to lyse mammalian TNF-sensitive tumor cell
line in a protease-independent way. Although to lyse
mammalian cells might not be relevant for earth-
worms, CCF participates in immune defense by trig-
gering activation of prophenoloxidase cascade upon
binding to conserved microbial polysaccharides
(Beschin et al. 1998). It also displays functional analo-
gies with the mammalian cytokine TNF, despite amino
acid sequence homology and gene evidence (Bilej et
al. 2001; Olivares Fontt et al. 2002).
Serine proteases:
role in regulating activity of fetidins
Three serine proteases have been isolated from leuko-
cytes of E. fetida, one of which was of the trypsin-
type (Roch et al. 1991). Serine protease activity has
Earthworm immunity: a model of immune competence 679
been reported also in Lumbricus terrestris (Leipner et
al. 1993; Kauschke et al. 1997). According to obser-
vations done in insects, serine proteases are usually
stored inside the cells. They are released upon cell ac-
tivation and pre-existing extra cellular inhibitors reg-
ulate their protease activity. One serine protease in-
hibitor was purified from E. fetida coelomic fluid by
affinity chromatography on trypsin: it is a monomer
of 14 kDa (Roch et al. 1998). Its partial N-terminal
amino acid sequence revealed a basic hydrophobic
fragment that shared 68–75 % homologies and
47–60 % identities with several plant serine protease
inhibitors. A cytotoxic cascade involving the release
of intracellular fetidins, their activation by trypsin-
like proteases and their inhibition by extra cellular
serine protease inhibitor, is likely to occur in E.
fetida.
Immune parameters and toxicity
The most extensively studied xenobiotic detoxifica-
tion reactions are those involving the cytochrome
P450 families that catalyze a series of reactions
whereby water-insoluble drugs and metabolites are
rendered sufficiently water-soluble to be excreted.
Microsomal mono-oxygenases were already known in
the earthworm L. terrestris (Nelson et al. 1976). The
requirement of O2 and NAD (P) H and the inhibition
by CO suggested the participation of cytochrome
P450-dependent mono-oxygenase system in the reac-
tion.
Stimulation of oxidative detoxification pathway
We demonstrated that exposure to carbaryl, using
the contact test (Ville et al. 1995), had an inducing
effect on E. fetida MFO (mixed function oxidase)
system as it had in rats and mice. EROD (ethoxyre-
soufin-O-dealkylase) activity, used as representative
of cytochrome P-450 detoxification activity, in-
creased significantly for carbaryl doses of 0.1 and
0.5 µg/cm2 compared to non-exposed earthworms
(Fig. 1). Meanwhile, carbaryl can act in vitro as a
competitive inhibitor of some cytochrome P-450-de-
pendent activities (Beraud et al. 1989). The presence
of inducible EROD activity also reveals that earth-
worms can develop a phase I xenobiotic metabolism
in reaction to the presence of xenobiotics such as
pesticides and this measurement could therefore
lead to a better understanding of the earthworm’s
ability to adapt to perturbations in the environment.
Using the same technique, we were not able to re-
veal any effect due to T2 toxin on the E. fetida MFO
system (Fig. 1).
Pedobiologia (2003) 47, 676–688
680 Edwin L. Cooper and Philippe Roch
Different sensitivities of lysozyme
The 4 compounds, Arochlor, 2,4 D, carbaryl and T2
toxin, were assayed for interference with lysozyme ac-
tivity. Exposure to 1 µg/cm2 of Arochlor induced a small
but significant enhancement of lysozyme activity (Fig.
2). This increase was confirmed using an exposure dose
of 10 µg/cm2. Even at 3.5 µg/cm2, 2,4 D did not modify
the earthworm lysozyme activity. By contrast, both car-
baryl and T2 toxin inhibited lysozyme for doses as low as
0.1 µg/cm2 (Fig. 2). Increasing carbaryl dose 10-fold, and
the T2 toxin dose 5-fold, reinforced the inhibition.
Lysozyme activity was never completely suppressed in
exposed earthworms, suggesting that the experimental
doses inhibited metabolism without being lethal. Clearly,
lysozyme activity studies indicated different cell targets
and/or different modes of action of the toxicants.
Pedobiologia (2003) 47, 676–688
Fig. 1. Effect of carbaryl and T2 toxin on detoxification metab-
olism of E. fetida evaluated as EROD activity used as represen-
tative of cytochrome P-450 activity. The 7-O-deethylation of 7-
ethoxyresorufin, referring to EROD activity, was assayed at
25 °C by the endpoint fluorimetric method and expressed as
pmol of resorufin synthesized/min/mg of protein measured at
585 nm with excitation at 530 nm (Burke & Mayer 1974). Incu-
bation conditions were optimized and standardized using post-
mitochondrial S9 fraction. * Indicates values statistically signifi-
cantly different from unexposed earthworms (p < 0.01)
Fig. 2. Effect of Arochlor, 2–4 D, carbaryl
and T2 toxin on lysozyme activity of E.
fetida. measured as M. lysodeiktucus cell
wall. Standard assay measured the ca-
pacity to lyse in vitro M. lysodeikticus cell
wall after 4h incubation at 37 °C (Lassalle
et al. 1988). Partially from Ville et al.
1997. * indicates values statistically sig-
nificantly different from unexposed earth-
worms (p < 0.01)
Increased intra-cellular proteases vs inhibition of
plasma protease inhibitors
Many metabolic cascades are regulated by the pro-
tease/anti-protease equilibrium. Activation of pro-
PO (phenol oxidize) (Beschin et al. 1998) and neu-
tralization of cytolytic proteins (Roch et al. 1998)
have been demonstrated in earthworm immune re-
actions. Proteases were located in circulating earth-
worm leucocytes (Roch et al. 1991; Leipner et al.
1993). In addition, protease inhibitors have been
observed in cell-free coelomic fluid (Roch et al.
1998).
Intracellular protease activity was increased after
Arochlor and carbaryl exposures, the stimulating ef-
fect being evident with the lowest dose of carbaryl
(0.1 µg/cm2) (Fig. 3a). Protease activity tended to in-
 Earthworm immunity: a model of immune competence 681

Fig. 3. Effect of Arochlor, 2,4 D, carbaryl

and T2 toxin on protease activity of leuco-
cytes (a) and on protease inhibitory activ-
ity of free-cell coelomic fluid (b) using a
modification of the Maskel & Di Capua
(1988) method for in vitro gelatin lysis. Di-
ameters of lysis were measured after 4h
incubation at 37 °C. Tested liquids were ei-
ther leucocyte lysates (for protease activity
indicated as diameter of gelatin lysis) or
cell-free coelomic fluid previously incu-
bated during with 30 min at 20 °C with a
definite number of trypsin units (for pro-
tease inhibitors indicated as % of remain-
ing trypsin lytic activity). * indicates values
statistically significantly different from un-
exposed earthworms (p < 0.01)

crease with 2,4 D and T2 toxin exposures, but the
changes were statistically insignificant.
In contrast, plasma protease inhibitors were down
regulated by all the tested xenobiotics (Fig. 3b). Expo-
sure to 0.1 µg/cm2 of both carbaryl and T2 toxin dra-
matically inhibited the protease inhibitory activity. Al-
though inhibitory, 2,4 D gave statistically non-signifi-
cant results.
Dramatic inhibition of phagocytosis
heat-killed yeasts. All the xenobiotics examined had a
dose-dependent negative effect on phagocytosis (Fig. 4a).
Such functional interference by xenobiotics can be
observed at the morphological level. For example, expo-
sure to 3 µg/cm2 Arochlor, the cytoplasm of earthworm
macrophages appeared condensed in a round, smooth,
cellular body (Fig. 4c). Exposed cells had few short
pseudopodia attaching the cell to the substratum com-
pared with the relatively numerous and longer pseudopo-
dia observed in unexposed macrophages (Fig. 4b).

Phagocytosis is one of the most ancestral and essential
functions driven by the cytoskeleton. Any compound that
inhibits phagocytosis will have a major impact on sur-
vival. More than 50 % of macrophages derived from un-
exposed earthworms were able to phagocytose at least 2
General enhancement of plasma cytolysis
The four tested compounds significantly increased cy-
tolytic capacity of coelomic fluid (Fig. 5). The most ef-
ficient was carbaryl > T2 toxin > 2,4 D > Arochlor. It

Pedobiologia (2003) 47, 676–688

682 Edwin L. Cooper and Philippe Roch
must be noted that the lytic capacity of non-exposed
earthworms varied considerably from one batch of
coelomic fluid to another (Fig. 5). Such variability can
be only partially explained by variations in the num-
bers of erythrocytes used as targets.
PO activity in Lumbricus terrestris
Evidence
Putative DOPA-chrome reaction, revealing the pres-
ence of a phenoloxidase cascade, was tested at different
temperatures (10–60 °C), different pHs (pH 6–9) and
various incubation durations (0–6 h). Optimal condi-
tions, allowing clear L-DOPA-chrome staining without
interference of DOPA spontaneous oxidation, were de-
fined as 25 °C, pH 8.0 and records of absorption every
hour during 4 h. Using these conditions, the coelomic
fluid underwent no noticeable change in absorption.
The supernatant of various sonicated L. terrestris
tissues were incubated with L-DOPA. There is essen-
tially no difference between reactions developed by
normal coelomic fluid, coelomic fluid collected from
Pedobiologia (2003) 47, 676–688
Fig. 4. Effect of Arochlor, 2,4 D, carbaryl
and T2 toxin on phagocytosis of earth-
worm macrophages. Coelomic cells from
single earthworms were collected in
buffer containing 5 mM EDTA. Chlor-
agogue cells were pelleted by 5 min cen-
trifugation at 50 g. Supernatant contain-
ing leukocytes and macrophages was in-
cubated on microscope slides for 30 min
at 20 °C in presence of calcium. Non-ad-
hering cells were eliminated by gentle
washing. Phagocytosis of heat-killed
yeasts was performed using the remain-
ing monolayers of adherent macro-
phages in a proportion of 1 ma-crophage
for 50 yeasts (Toupin et al. 1977). After
45 min of incubation at 20 °C, non-
phagocytosed yeasts were washed away
and a minimum of 200 macrophages ex-
amined randomly using a phase contrast
microscope to determine numbers of
macrophages that had phagocytosed two
or more yeasts (a). Scanning electron mi-
croscopy observation of E. f. andrei unex-
posed (b) and Arochlor exposed (c)
macrophages. Bar, 1 µm (partially from
Ville et al. 1995). * indicates values sta-
tistically significantly different from unex-
posed earthworms (p < 0.01)
earthworms starved for 17 days and coelomic cell
lysate (Fig. 6). Chloragogue stem cells produced
lower activity, whereas extracts from macrophage/
leukocyte stem cells, gonads, body wall muscles and
gut contents, failed to exhibit any activity. We tested
the coelomic fluid and the coelomic cell lysate of 67
individual L. terrestris; all were able to develop a
black coloration indicative of phenoloxidase activ-
ity. Reaction kinetics showed some minor variability,
but there was no correlation between speed of reac-
tion and earthworm size, sexual maturity and volume
of coelomic fluids collected from experimental
worms.
Chloragogue cells originate from splanchnopleure
(Valembois et al. 1985) and they gave a positive
DOPA-chrome reaction. Chloragogue cells are in-
volved in diverse nutrition-related activities (Valem-
bois & Cazaux 1970) and immunodefense (Valembois
et al. 1982). Macrophages and several sub-populations
of leukocytes originate from the somatopleure (Valem-
bois et al. 1985) where we found no significant DOPA-
chrome reaction. In addition, digestive enzymes or in-
testinal flora that can contaminate coelomic fluid sam-
ples does not mediate the DOPA-chrome reaction.
 Earthworm immunity: a model of immune competence 683

Fig. 5. Effect of Arochlor, 2,4 D, carbaryl

and T2 toxin on cytolytic capacity of E.
fetida coelomic fluid. Cytolytic activity
was evaluated by mixing different dilu-
tions of cell-free coelomic fluid with a
suspension of sheep red blood cells
(SRBC). After 1 h incubation at 37 °C, the
remaining non-lysed SRBC were pelleted
and hemoglobin content of supernatants
was evaluated at 541 nm (Roch et al.
1989). Results are presented as percent-
ages of hemolysis. Partially from Ville et
al. 1997. * indicates values statistically
significantly different from unexposed
earthworms (p < 0.01)

Fig. 6. Evidence for phenoloxidase activity in various L. terrestris

tissues and extracts as inferred from oxidation of added DOPA. L-
DOPA-chrome staining was recorded at 492 nm after 4 h incuba-
tion at 25 °C, pH 8.0

In vivo stimulation In vitro activation

Figure 7a indicates that only the injection of M.
lysodeikticus into the coelomic cavity induced a signif-
icant increase of DOPA-chrome reaction; the injection
of 100 µl of saline or of 5 % BSA were not stimulatory.
One day after being injected with 5 % M. lysodeikticus
suspension, earthworms were sick with a low level of
viscous, grayish coelomic fluid, exhibiting higher
DOPA-chrome reaction. Chlorogogue cells are known
to be involved in elimination of foreign debris and
senescent tissues by the formation of brown bodies
(Valembois et al. 1992), a process that includes
melanization.
Incubation of coelomic fluid with fresh SRBC or
Escherichia coli (E. coli), and with glutaraldehyde sta-
bilized SRBC (SRBC glu) or E. coli (E. coli glu) prior
to add L-DOPA, did not significantly modify the
DOPA-chrome reaction (Fig. 7b). In contrast, the same
incubation with M. lysodeikticus cell wall resulted in a
significantly higher activity. Contact between
coelomic fluid and M. lysodeikticus cell walls stimu-
lated melanization suggesting that the coelomic fluid
contains factors that can be activated. We controlled
that, in absence of coelomic fluid, M. lysodeikticus cell
wall did not oxidize L-DOPA.

Pedobiologia (2003) 47, 676–688

684 Edwin L. Cooper and Philippe Roch
Activation of DOPA-chrome reaction by both β-glu-
cans and bacterial cell wall has been reported in insects
(Vargas-Albores et al. 1996). In arthropods, oxidation of
catecholamines represents an essential step in the bio-
chemical pathway leading to the synthesis of quinine pre-
cursors of melanin that participates in the encapsulation
of parasites and senescent self-tissues. Recent evidence of
these compounds in brown bodies of earthworms, along
with the present data, argues in favor of a metabolic path-
way related to the so-called phenol-oxidase system in
arthropods (Söderhäll & Cerenius 1998; Nappi & Vass
2001). However, the in vitro DOPA-chrome reaction of
earthworms is extremely slow, and necessitates several
hours compared to minutes in insects.
Relationships between the pro-PO
system and CCF in Eisenia fetida
The 42-kDa protein named CCF (coelomic cytolytic
factor) is localized in chloragogue cells near the gut
wall and in cells with macrophage-like function (Bilej
et al. 1998). CCF shows homology with the saccha-
ride-binding motif of bacterial and animal β-1,3-glu-
canases, with Gram-negative bacteria binding protein
and β-1,3-glucan-recognition proteins of arthropods,
and with glucan-sensitive factor G from the horseshoe
crab Limulus polyphemus (see Bilej et al. 2000, for re-
view). Although these proteins show high homology in
the putative polysaccharide-binding domain and the
catalytic sites of the bacterial glucanases, neither CCF
nor its invertebrate homologues exhibit glucanase ac-
tivity. Glucan-binding proteins may have appeared
from a primitive glucanase that evolved to proteins
without enzymatic activity, but then were able to bind
glucans and operate as elicitors of immune reactions
by recognizing non-self (Söderhäll & Cerenius 1998).
Pedobiologia (2003) 47, 676–688
Fig. 7. Phenoloxidase activity in L. terrestris coelomic
fluid measured after 4 h of incubation at 25 °C and pH
8.0. a: Effect of various injections on DOPA-chrome reac-
tion. 100 µl of saline, of 5 % BSA or of 5 % of a cell wall
suspension from M. lysodeikticus were injected into the
coelomic cavity of three groups of earthworms (n = 36).
After 24 h, coelomic fluids were collected and tested for
DOPA-chrome reaction. b: Effect of 1 h in vitro incuba-
tion at 4 °C of coelomic fluid with fresh E. coli, SRBC, glu-
taraldehyde stabilized E. coli (E. coli gluta) or SRBC
(SRBC gluta) and M. lysodeikticus cell walls, on the
DOPA-chrome reaction. *indicates a result significantly
different from untreated coelomic fluid (p < 0.01)
CCF binds efficiently the O-antigen of LPS, muramyl
dipeptide of peptidoglycan, β-1,3-glucans and N, N’-
diacetylchitobiose (β-1,4-N-acetylglucosidic link)
(Bilej et al. 2001). After recognizing cell wall compo-
nents of bacteria or yeast, CCF can trigger the activa-
tion of the pro-PO cascade.
After recognizing polysaccharides of microbial cell
walls, serine proteinases cleave by limited proteolysis
inactive fetidins to active cytolytic proteins (Fig. 8).
Similarly, inactive pro-PO is cleaved to active phe-
noloxidase (PO). Then, PO catalyses the O-hydroxyla-
tion of monophenols and oxidation of diphenols to
quinones, which in turn polymerize to melanin that can
then effect cytotoxic and antibacterial activities (Jo-
hansson & Söderhäll 1996). CCF is important in medi-
ating several immune-related reactions, including pro-
PO in E. fetida. This has been confirmed by showing
that when CCF is removed from the coelomic fluid, the
activation cascade is blocked. Exogenous supply of re-
combinant CCF can restore the L-DOPA oxidation in
CCF-depleted coelomic fluid (Beschin et al. 1998).
The precursors of melanin involved in the pro-PO acti-
vating system stimulate, besides antimicrobial properties,
a wide range of other biological activities including
phagocytosis and opsonization, capsule/nodule formation,
and wound healing. In earthworm, cytotoxic and antimi-
crobial activities are intimately associated with agglutinat-
ing, hemolytic and opsonizing activities (Roch 1996). For
example, cytotoxicity of leukocytes from E. fetida against
leukocytes of L. terrestris is blocked by anti-CCF mono-
clonal antibody. With respect to antimicrobial properties,
CCF agglutinates smooth but not rough Gram-negative
and Gram-positive bacteria (Beschin et al. 1998). Phago-
cytosis is enhanced by the opsonizing properties of CCF
that also potentiates lytic activity against various erythro-
cytes. CCF may now be considered as a pattern recogni-
tion molecule that exerts a prominent role in innate im-
mune mechanisms of earthworms.

Fig. 8. Putative activation cascade of immune reactions in earthworms going from external stimuli to immune capacities through lysozyme,
CCF, protease equilibrium and PO activation (adapted from Beschin et al. 2002)
Prospective
Most of our knowledge on immune defense mecha-
nisms is mainly based on data obtained in vertebrates,
particularly mice and humans, while about 95 % of the
two million animal species belong to invertebrate
phyla. Moreover, invertebrates have evolved for hun-
dreds of millions of years often surviving in hostile en-
vironments. One survival strategy includes the produc-
tion of numerous offsprings. In addition, invertebrates
have invented a variety of active innate immune mech-
anisms based on pattern recognition receptors. Inverte-
brate innate immune mechanisms include both unique
pathways unknown in vertebrates and more universal
mechanisms present throughout the entire animal king-
dom. In this respect, knowledge of the less complex in-
vertebrate immune strategies may contribute to the un-
derstanding of the vertebrate immune system, as well
as lead to the identification of new factors with possi-
ble therapeutic use. Though the earthworm has been
used as a drug in China and the Far East for millennia,
more detailed scientific studies have been performed
Earthworm immunity: a model of immune competence 685
only recently (Mihara et al. 1991, 1992; Nakajima et
al. 1996). One example is Lumbrokinase, (earthworm
powder enzymes or EPE, e-PPA, Boluoke®) a fibri-
nolytic enzyme isolated from the earthworm L. rubel-
lus commercially available as an orally administered
drug for the prevention and treatment of cardiac and
cerebrovascular diseases (Canada RNA Biochemical
Inc.).
Invertebrates, like vertebrates, possess factors regu-
lating responses to infection or wounding. Lytic mole-
cules mediate one mechanism in earthworms. The se-
quences of fetidin, lysenin and CCF derived from E.
fetida are known, but we need more precisions on eise-
niapore and lumbricin I. Evidently, within the
oligochaete annelid group, there are several families of
lytic proteins, making these function more compli-
cated than expected.
Despite being a model for comparative immunolo-
gists since the early sixties, such review aims to em-
phasize the absolute importance of furthering our un-
derstanding of the immune competences of earth-
worms. They occupy a broad ecological and ecotoxi-
Pedobiologia (2003) 47, 676–688
686 Edwin L. Cooper and Philippe Roch
cological status. Earthworms benefit in environmental
monitoring and in the acquisition of novel molecules
for human therapeutic purposes.
Acknowledgements. The Alexander von Humboldt Founda-
tion supports Edwin L. Cooper, a GAAC grant from the Fed-
eral Republic of Germany and a NATO Cooperative Re-
search Grant (971128). Philippe Roch is partially supported
by CNRS, IFREMER and the University of Montpellier 2.
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