The role of opsonin in phagocytosis by coelomocytes of the earthworm
Yeflim Kalaç*, Ayten Kimiran, Gülruh Ulako¤lu and Ayfl›n Çotuk
University of ‹stanbul, Faculty of Science, Department of Biology, 34459 Vezneciler, ‹stanbul, Turkey
(* author for correspondence)
Received 9 July 2001; Accepted 25 October 2001
The phagocytes which reside in the coelomic cavity of the earthworms have roles in cellular immunity. The
phagocytic activity of coelomic cells of the earthworm Dendrobaena veneta against the pathogenic and
non-pathogenic bacteria was investigated in vitro. The coelomic cells of D. veneta having phagocytic activity was
separated by density gradient centrifugation method. It was observed that these cells have neutrophil and basophil
structure. Among the pathogenic bacteria, B. megaterium and A. hydrophila, and non-pathogenic bacteria
P. maltophilia and B. subtilis, to various earthworms, B. megaterium was significantly ingested by phagocytes
8 Yeflim Kalaç et al.
(Cameron, 1932; Millar and Ratcliffe, 1994; Rittig et
al., 1996). The free coelomocytes of the coelomic
cavity of annelids play a key role in defence
mechanisms (Dales, 1978; Dales and Kalaç, 1992).
The coelomocytes of annelids vary widely in
morphology, not only between classes but also
between species of the same family (Stein and
Cooper, 1983). The coelomocytes are generally
divided in two main groups by light microscopy:
ameobocytes (phagocytes) and eleocytes
(chloragogen cells), which are not phagocytic
(Ratcliffe and Rowley, 1981). Eleocytes, which can
be found in almost all annelids, are generally big cells
and have granules (chloragosomes) that contain lipid,
lipid-like and protein substances. Although such cells
have no phagocytic features, they are responsible for
synthesizing humoral factors such as bacteriostatic
substances, hemolysins and hemagglutinins in
Oligochaeta (Ratcliffe et al., 1985; Tripp, 1992;
Valembois et al., 1982; Valembois et al., 1986).
Ameobocytic coelomocytes are extremely efficient
removing foreign particles, such as bacteria, fungi
and nematodes from the coelomic cavity, by either
phagocytosis, nodule formation or encapsulation
(Millar and Ratcliffe, 1994) and can reach all tissues
and all parts of the earthworm body (Bilej et al.,
1990a; Cameron, 1932; Cooper et al., 1974; Cooper,
1986; Dales and Kalaç, 1992).
Earthworms Lumbricus terrestris and Eisenia
foetida phagocytes generally have four cell types such
as basophils, neutrophils, acidophils and granulocytes
according to their morphological and cytochemical
properties (Anderson, 1975; Dales, 1978; Millar and
Ratcliffe, 1994; Ratcliffe and Rowley, 1981; Ratcliffe
et al., 1985; Stein and Cooper, 1978).
Earthworm coelomic cavity very often can contain
bacteria, fungi, protozoa and nematodes in spite of
interaction between outer environment and coelomic
cavity via dorsal pores and nephridiopores (Cameron,
As we know that annelids have both cellular and
humoral response to those infective agents, although
earthworms have both cellular and humoral defence
mechanisms, some researchers have demonstrated
that bacteria such as Aeromonas hydrophila, Bacillus
megaterium, Serratia marcescens (Çotuk and Dales,
1984a), Bacillus thuringiensis (Heimpel, 1966;
Smirnoff and Heimpel, 1961) and Yersinia ruckeri
(Çotuk and Kalaç, 1990) are pathogenic to some
species of earthworms. The primary cellular response
against invading microorganisms starts with
recognition step and is followed by ingestion and
killing. Following contact of the foreign particles with
the phagocytic cell, the next step is attachment.
Recognition and attachment may be mediated, in part,
in a non-specific way by physiochemical properties of
the foreign material such as surface change and
hydrophobicity (Anderson, 1975; Bilej et al., 1990b;
Millar and Ratcliffe, 1994). On the other hand
humoral factors may facilitate recognition and
subsequent ingestion of foreign material by
phagocytic cells. These factor, which facilitate or
enhance phagocytosis, are generally referred to as
opsonins (Cooper et al., 1974; Kelly et al., 1993;
Laulan et al., 1988).
In vertebrates opsonins are mainly
immunoglobulins and the third component of
complement and specific recognition may also take
place through carbohydrate-binding proteins called
lectins. In invertebrates the body fluids and some
tissues may have lectins and may act as recognition
molecules. It has been suggested that earthworm
coelomic fluid acts as an opsonin against some
microbial agents (Bilej et al., 1990a; Laulan et al.,
1988; Stein et al., 1977). In vitro investigations by
Stein and Cooper (1981) underlined the importance of
time and temperature in the phagocytosis of yeast by
L. terrestris phagocytes.
In this study cellular response to pathogenic and
non-pathogenic bacteria in earthworm Dendrobaena
veneta was investigated in vitro. We worked on
recognizing the cell type of this species of
earthworms by light microscopy. The phagocytosis
experiments were run for different incubation periods
and temperatures. The opsonin effect of the coelomic
fluid on phagocytosis was studied using pathogenic
and saprophytic bacteria to understand probable
Materials and Methods
Dendrobaena veneta was derived from a single
source in Istanbul University garden, and has been
maintained in the laboratory for a number of years.
The bacteria Aeromonas hydrophila 9926 and
Bacillus megaterium that are pathogenic to D. veneta
and Pseudomonas maltophilia KUEN 1297 and
Bacillus subtilis ATCC 6633 that are saprophytic
were obtained from different reference laboratories.
Aeromonas hydrophila 9926 was grown at 22°C, the
other bacteria were grown at 37°C for 24 h then
centrifuged and the pellet was washed three times
with saline solution. Bacteria were adjusted to 3x10 8
cells/ml by Nephelometry method (Mc Farland,
1907), before the experiment.
The preparetion of coelemic cells
Coelomic cells were liberated via the dorsal pores by
5 V stimulation (Roch, 1979). Cell free fluid was
obtained by centrifugation at 20000 g for 15 min.
Coelomic cells were prevented from clumping by
liberation into ice-cold Ca 2+ /Mg 2+ free Holtfreter
saline, containing 5 mM ethylen glycol-bis (ßaminoethyl
ether) N,N,N',N'- tetraacetic acid (EGTA)
(Sigma) adjusted to pH 7.02 and 200 mOsm.
Coelomic fluid was filtered through a 0.2 µm pore
size membrane filter and then used.
Separation of phagocytes and short term cell cultures
Phagocytes were separated from eleocytes by
density gradient centrifugation at 2500 g. Following
the centrifugation, eleocytes were carefully removed
from the interface, and the lower phase containing the
phagocytes and non-adherent cells were diluted two
times with Ca 2+ /Mg 2+ free Holtfreter containing 5 mM
EGTA, recentrifuged and resuspended according to
Dales and Kalaç (1992) methods. Cell suspensions
were handled in silicone-coated glass tubes (2%
dimethyldichlorosilane in 1,1,1-trichloroethane)
(Sigma) to prevent probable clumping then they were
counted by haemocytometer before using.
Phagocytes (2x10 6 cells/ml) were allowed to
attach to sterile glass coverslips for 30 min. and then
immersed in 5 cm Petri dishes containing 5 ml sterile
medium. Medium was based on Eagles minimum
essential medium with Earl's salts (Sigma) adjusted
by addition of 0.42 g CaCl2·2H2O, 0.09 g KCl, 0.88 g
MgSO4·7H2O, 1.27 g NaCl, 5.0 mg adenosine, 0.5 mg
sodium pyruvate, 1.0 mg peptone per liter and
adjusted to 200 mOsm and pH 7.2. An antibiotic
cocktail was added to keep the culture for long-term
(Kalaç, 1997). After the separation, attached cells
were stained with fluorescein isothiocyanate (FITC)
(Sigma) (Bellinati-Peres, 1989) and acridine orange
(Sigma) (Oda and Maeda, 1986) for 45-60 seconds
and modified Wright's stain (Sigma) for 2 min (Stein
and Cooper, 1981) to distinguish microscopically
different types of D. veneta coelomocytes.
For the measurement of phagocytosis rate the
suspension of bacteria were added into the cell culture
in such a way that cells and the bacteria ratio was
1:50. Opsonin effect was investigated by using
coelomic fluid collected by electrical stimulation
(Roch, 1979) and sterilized by filtration as described
above. 500 µl steril coelomic fluid was added into the
cell cultures contained 5 ml medium. The cells and
bacteria were incubated for 30 min and 60 min both at
22°C and 37°C. Bacteria were not added to controls.
After the incubation, the coverslips were gently
washed in Ca 2+ /Mg 2+ free Holtfreter and coverslips
were stained with a modified Wright's stain (Sigma)
for 2 min. All experimental groups also were tested
for viability by using 0,1 % nigrosin (w/v) (Sigma)
(Dales and Kalaç, 1992). Then air dried coverslips
were stuck on clean slides by entellan (Sigma). The
cells were counted under the light microscope and
both phagocytic index (PI) and percentage of
phagocytosis (%P) were calculated for every slide
using equations below respectively (Çotuk et al.,
Role of opsonin in phagocytosis 9
Number of cell phagocytosed bacteria
Number of total cells
Number of cell phagocytosed bacteria
% P= X 100
Number of total cells
Data were evaluated by Student's t test and variant
analysis. All values are expressed as the mean ±
10 Yeflim Kalaç et al.
standard error of the mean. Number of replicates is
given as N.
Coelomocytes of D. veneta
The coelomocytes of D. veneta have been
distinguished, based on morphological properties as
seen by light and U.V. microscope stained by
flouroscent dyes. According to our observations
D. veneta has two groups of cells, phagocytes and
eleocytes (chloragocytes). Eleocytes were easily
separated by gradient centrifuge method and
distinguished by their yellow color. Eleocytes are not
responsible for phagocytosis. Also they clump to each
other more than other cells. Following Wright's stain,
our observations show that phagocytic cells can be
grouped in two different types. The first one is likely
to be basophils, as L. terrestris cells, with
distinguishable nucleus. The other group is likely to
be neutrophils, has a nucleus and a large cytoplasm
containing vacuoles with bacteria (Figures 1, 2, 3, 4).
Furthermore while we counted the cells we saw that
the number of basophils were more than neutrophils.
In vitro phagocytosis experiments
Our results have shown that D. veneta phagocytes had
a phagocytosis activity for all kinds of bacteria used
in the experiments (Table 1 and Figure 5). Percentage
of phagocytosis rate at 22°C after 30 and 60 min.
incubation without coelomic fluid (opsonin) was
6-9% for B. megaterium, 7-16% for A. hydrophila,
2-3% for P. maltophilia and B. subtilis (Figure 6).
With respect to our evaluation the phagocytosis rates
were found to be insignificant at 22°C after 30 and 60
min. incubation both with and without opsonin.
Among the bacteria we tested at 37°C, percentage of
phagocytosis rates for A. hydrophila and B. subtilis
showed that the increase of phagocytosis rates were
significant (p0.05) was observed
between percentages of phagocytosis rates at 22°C for
Figure 1: Basophilic (A) and neutrophilic (B) cells stained
by Wright dye.
Figure 2: Eleocyte (el) and phagocytic (ph) cells stained by
fluorecein isothiocyanate dye (FITC).
all bacteria with and without opsonin. As it is seen
from Figure 5, B. megaterium was the most ingested
one among the tested bacteria without opsonin, for 30
Figure 3: Phagocytic cells stained by acridine orange dye.
and 60 min. incubation (p
12 Yeflim Kalaç et al.
Table 1: Phagocytic index and percentage of phagocytosis of D. veneta phagocytes against certain bacteria at different
temperatures and times.
B. subtilis 22
* Values expressed as means ± S.E.
** N = Number of replicates.
Phagocytic Index* Percentage of Phagocytosis*
Without Opsonin With Opsonin Without Opsonin With Opsonin
Figure 5: Percentage of phagocytosis of D. veneta
phagocytes against different bacteria in 30 and 60 minutes
with and without opsonin at 22˚C (A) and 37˚C (B).
had no significant effect on phagocytosis ability of L.
terrestris coelomocytes and also L. terrestris showed
that 60 min. of phagocytosis time was better than 30
Various annelid species possess agglutinins and/or
lysins. They are structurally unrelated to vertebrate
immunoglobulins and vary from species to species in
their molecular structure and biochemical
composition (Cooper et al., 1974; Millar and
Ratcliffe, 1994; Ohta et al., 2000). These substances
might have recognition molecules. Many authors
have also shown that the body fluid of invertebrates
acts as an opsonin (Lackie, 1980; Olafsen, 1988;
Ratcliffe et al., 1985; Renwrantz, 1983; Yeaton,
1981). The aim of some of our experiments was to
investigate whether the coelomic fluid acted as an
opsonin or not. We have observed that phagocytosis
activity of the D. veneta coelomocytes in both
saprophytic (P. maltophilia, B. subtilis) and
pathogenic (B. megaterium, A. hydrophila) bacteria
was suppressed by coelomic fluid (opsonin). This
suppression was found to be statistically significant at
Figure 6: Percentage of D. veneta phagocytes in 60
minutes against different bacteria with and without
opsonin at 37°C.
differences on phagocytosis rate of Halocynthic
roretzi ameobocytes when they phagocytosed sheep
red blood cells and latex beads in vitro. Another in
vitro study has shown that opsonin (hemolymph) did
not increase phagocytosis activity of mollusc
Mercenaria mercenaria phagocytic cells for
erythrocytes, bacteria and yeast (Tripp, 1992). Kelly
et al. (1993) declared that they saw a stimulating
effect of body fluid at 30 min. incubation in
Urochordate, Styela deva, but at 60 min. incubation
they did not find any differences in conditions with or
without opsonin. Stein and Cooper (1981) reported
that opsonins in the coelomic fluid of L. terrestris
facilitated phagocytosis of yeast by neutrophils but
not by basophils or granular ameobocytes. According
to Stein et al. (1977), the overall phagocytic capacity
of L. terrestris is not greatly effected by coelomic
fluid factors, since neutrophils constitute less than
20% of the coelomocyte population. In the cells
population the number of basophiles is higher (64%),
so that they may not be effected by opsonin. With
respect to the above results, a similar condition may
also fit for D. veneta coelomocytes. Because our
observations shows that the number of basophiles is
higher than neutrophils which seems to be more
Our results showed that no significant difference
was observed in the phagocytosis of bacteria treated
with and without opsonin, except B. megaterium.
Though this explains why phagocytosis in many
basophils is not effected by opsonin, the whole
concept will be better understood by knowing the
ratio between the coelomic cells in D. veneta. An in
vitro study with cockroachs, Anderson (1975) shows
that the effect of the opsonin on phagocytosis of
erythrocytes coated by opsonin was under the control
values, similar to the results obtained in our study. In
some studies, another phagocytosis called “coiling
phagocytosis” has been reported both in vertebrate
and invertebrate during which the bacteria attract the
phagocytic cells without opsonin (Akbafl, 1996;
Rittig, 1996). Opsonins in invertebrates do not have
the ability to facilitate the phagocytic activity of cells
against foreign particles everytime. It appears that
there is a different response to foreign particles
among the earthworms species.
Role of opsonin in phagocytosis 13
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