Download The Role of Lysosomes in Molluscan Inflammation1

AMER. ZOOL., 23:129-144 (1983)
The Role of Lysosomes in Molluscan Inflammation1
THOMAS C. CHENG
Marine Biomedical Research Program and Department of Anatomy (Cell Biology),
Medical University of South Carolina, Charleston, South Carolina 29412
SYNOPSIS. Phagocytosis and related phenomena represent integral features of inflammation in all metazoans. Reviewed herein are the results of studies directed at understanding the role(s) of lysosomal enzymes synthesized and released from circulating hemocytes, especially granulocytes, of gastropods and bivalves as a result of challenge with
exogenous, nonself materials. From what is known, most of the mechanisms underlying
this inflammation-associated process parallel those of mammalian macrophages; however,
immunoglobulins and most probably components of complement are not involved. The
required energy for phagocytosis in molluscs appears to be derived from glycolysis alone.
Furthermore, nitroblue tetrazolium reduction and the myeloperoxidase-H2O2-halide antimicrobial system, both characteristic of mammalian phagocytes, appear to be absent in
molluscs. It is concluded that by studying phagocytosis by molluscan hemocytes, a great
deal can be learned about the evolution of inflammatory response and its constitutent
elements.
rewarding. Specifically, studies involving
Inflammation, as generally defined, is the molluscan hemocytes have been advantareaction of tissues to injury. The process, geous for elucidating the role of lysosomal
regardless of the causative agent, is char- enzymes in inflammation.
Prior to delving into what is currently
acterized by local heat, swelling, redness,
known
about hemocytic lysosomal enzymes
and pain. Furthermore, in mammals the
pathologic picture involves primary vaso- in molluscs, it bears restating that during
constriction followed by vasodilatation, acute inflammation in mammals, local veswhich result in the slowing down of cir- sel dilation allows influx of plasma proteins
culation, accompanied by the accumula- and phagocytic cells into the tissue spaces,
tion and emigration of leucocytes, exuda- which leads to swelling. Also, if the acute
tion of fluid, and deposition of fibrin. As inflammatory response does not rid the host
those interested in the comparative aspects of the causative agent, then phagocytic cells
of inflammatory response will recognize, continue to be attracted to the inflamed
these features of vertebrate, especially site, followed in some instances by formamammalian, inflammation are not all tion of an abcess.
Continuing acute inflammation may
applicable to invertebrates. Nevertheless,
nonspecific cellular response does occur in become chronic. Chronic inflammatory
these animals. At this point, a seldom responses are characterized by an infiltraremembered fact relative to inflammation tion of lymphocytes and cells of the monoin molluscs bears recalling. Specifically, cyte-macrophage lineage, which are
Hurst and Walker (1933, 1935) have actively phagocytic. It is thus apparent that,
reported that the body temperature of the as Metchnikoff emphasized in 1892,
gastropod Lymnaea stagnalis is 2.7 times phagocytosis and associated phenomena
higher in specimens infected with larval represent an integral part of inflammation.
trematodes than in nonparasitized ones.
In our laboratory we have been employThe intent of this contribution, hopefully, ing molluscan hemocytes as models in the
is to point out that studying aspects of study of phagocytosis and related phenominflammation in invertebrates can be ena. Hopefully, it will become apparent why
we have elected this system. Briefly, (1)
molluscan hemocytes have been known to
be
phagocytic since the report by Haeckel
1
From the Symposium on Comparative Aspects of (1862); (2) molluscs, and all invertebrates
Inflammation presented at the Annual Meeting of the
for that matter, do not synthesize immuAmerican Society of Zoologists, 27-30 December
noglobulins and all available evidences
1981, at Dallas, Texas.
INTRODUCTION
129
130
THOMAS C. CHENG
INTERNALIZATION
CHEMOTAXIS
RECEPTOR
RESIDUAL BODY
Fic. 1. Schematic, simplified diagrams showing sequence of events during the phagocytosis and intracellular
degradation of a foreign particle.
indicate that they do not have a complement system; therefore, one can study the
phenomenon of phagocytosis without having to decipher if it is enhanced by antibodies or some component of complement;
(3) from the standpoint of elucidating the
evolution of cellular immunity, including
phagocytosis, a great deal can be learned
about the basic mechanisms involved along
the protostomate schizocoelous line of
invertebrates by studying molluscs.
In that area of comparative immunology
pertaining to the internal defense mechanisms of invertebrates, the greatest advances during the past decade have been
made in understanding the molluscs and
insects. Salt (1970) and Ratcliffe and Rowley (1979) have contributed authoritative
reviews of the mechanisms operative in
insects, especially what has been commonly
designated as cellular immunity. Cheng
(1967), Cheng and Rifkin (1970), and others have presented reviews of what was
known about phagocytosis, encapsulation,
and nacrezation in molluscs at the time
those reviews were written. In this presentation, as indicated by the title, it is my
intent to offer a synthesis of what has been
learned during the past decade relative to
one aspect of cellular immunity in molluscs, i.e., the role of lysosomes in inflammation.
In his classic study, Stauber (1950) has
demonstrated that foreign particles experimentally introduced into a mollusc, the
American oyster, Crassostrea virginica, are
phagocytosed and subsequently are eliminated from the animal's body by the exomigration of particle-laden phagocytes
across epithelial borders. Following
Stauber, Tripp (1958, 1960) and Feng
(1959, 1965) have extended their mentor's
study to show that digestible foreign materials may also be degraded intracellularly
within phagocytes.
The reports by Tripp and Feng may be
ROLE OF LYSOSOMES IN MOLLUSCAN INFLAMMATION
131
TABLE 1. Comparison of combined differential analyses of
hemolymph cells of Crassostrea virginica in two experiments carried out in vitro at 22°C*
Experiment
Granulo- Hyalinocytes
cytes
%
%
Staphylococcus aureus
x = 87.28 12.31
SD = 34.55 5.37
Escherichia coli
x = 83.48 16.80
SD = 36.01
9.17
x = 87.49 12.85
Control (millipore filtered
sea water)
SD = 38.73 6.90
* The results are expressed as percent of total differential counts for each experiment. (After Foley
and Cheng, 1975; with permission of Academic Press.)
FIG. 2. Electron micrograph showing the presence
of concentrically positioned digestive lamellae within
phagosome of Crassostrea virginica that had phago-
eated vesicles occur in the cytoplasm and
these commonly enclose arrays of concentric lamellae (Fig. 2). Earlier, Cheng and
considered the spring boards for studies Cali (1974) had demonstrated by electron
aimed at understanding the cell biology of microscopy, and subsequently, Cheng and
what is involved in intracellular degrada- Rudo (1976), by employing isotopic tracers,
have confirmed, that glycogen is synthetion and related phenomena.
sized in granulocytes that had phagocyCELL TYPES
tosed carbohydrates. Apparently the comIt is well known that in mammalian mac- plex carbohydrates are first depolymerized
rophages the degradation of phagocytosed to glucose and resynthesized as stored glynonself materials is dependent on the com- cogen. Also, Cheng and Cali (1974) had
patible fusion of lysosomes with the pri- shown that foreign materials in the process
mary phagosome and the release of lyso- of being degraded intracellularly are sursomal hydrolases into the phagosome (Fig. rounded by concentric digestive lamellae
1). Surprisingly, even as recently as 1974, within phagosomes (Fig. 2). Thus, it became
and still true to some extent, there was no apparent that the occurrence of glycogen
agreement as to how many types of he- granules and digestive lamellae are charmocytes occur in molluscs, let alone as to acteristic of granulocytes at a later phase
which category of hemocytes is the most of the endocytosis-intracellular degradaactive from the standpoint of phagocytosis. tion process when the typical cytoplasmic
In that year, Foley and Cheng (1974), who granules had been discharged via degranstudied the morphology and in vitro behav- ulation. Cheng (1981) has presented a
ior of the circulating hemocytes of Merce- detailed account of molluscan cell types
naria mercenaria, came to the conclusion based on functional attributes.
that there are three categories of cells:
granulocytes, hyalinocytes, and fibrocytes.
PHAGOCYTIC CELLS
This conclusion, however, was later
Having ascertained that there are two
emended by Cheng and Foley (1975) who general categories of circulating molluscan
studied the fine structure of these cells. hemocytes, the next question that needed
The basis of their drawing the conclusion to be answered was: Which type is the most
that only two general types of cells occur, active in phagocytosis? Although most
granulocytes and hyalinocytes, was the authors believed that granulocytes are most
finding that what had been designated as active, it was not until Foley and Cheng
fibrocytes consistently include large aggre- (1975) performed a quantitative study on
gates of glycogen granules. Furthermore, the hemocytes of C. virginica and M. merlarge, electron-lucid, membrane-delin- cenaria that it was ascertained that all of
cytosed Bacillus megaterium.
132
THOMAS C. CHENG
TABLE 2. Mean numbers of Bacillus megaterium associated with hemolymph cells of Mercenaria mercenaria
in vitro at three different temperatures (n = 50).*
Experiment
Granulocytes
28-1
Hyalinocyles
<1
<1
x=
—
—
SD =
2.56
x=
22°C
12.82
SD =
0.96
2.19
3.08
x=
37°C
17.88
SD =
1.60
8.57
<1
<1
x=
Control (22°)
—
SD =
—
* After Foley and Cheng, 1975; with permission of
Academic Press.
4°C
50
75
3
Saltconc.(mMxlO- )
FIG. 3. Effect of various salt concentrations on the
lytic activity of Crassostrea virginica whole hemolymph
lysozyme using NaCl ( • - • ) , KC1 (o-o), and MgCl2
(A—A). Lysozyme activity is expressed as AOD M0 /
min X 10-2 at 25°C and pH 5.5. (After Rodrick and
Cheng, 1974a; with permission of Academic Press.)
the cells are phagocytic; however, the
granulocytes are considerably more active.
As indicated in Table 1, when C. virginica
hemocytes were exposed to Staphylococcus
aureus, 87.28% of the granulocytes as compared to 12.32% of the hyalinocytes had
phagocytosed bacteria. Similarly, when sumption by M. mercenaria hemocytes
exposed to Escherichia coli, 83.48% of oys- actively phagocytosing Bacillus megaterium.
ter granulocytes as compared to 16.80% of The utilization of glucose and glycogen,
hyalinocytes were associated with bacteria. coupled with the production of lactate and
In the case of M. mercenaria, 12.82% of no increase in oxygen utilization indicate
granulocytes as compared to 2.56% of that glycolysis is the energy providing pathhyalinocytes were associated with Bacillus way. This conclusion is strengthened by
megaterium in in vitro exposure tests (Table the fact that KCN does not inhibit phagocytosis. It was also reported that nitroblue
2).
tetrazolium reduction, characteristic of
ENERGY REQUIREMENT
mammalian phagocytes, is absent in M.
Another question related to phagocy- mercenaria hemocytes. Also, the myelopertosis in molluscs that required answering oxidase-H2O2-halide antimicrobial system
was: Since phagocytosis and related phe- of mammalian phagocytes is absent in Mernomena are energy expensive processes, cenaria phagocytes.
what is the source of the energy? It is noted
LYSOSOMES
that biochemical studies on mammalian
cells during the past two decades (Iyer et
Since the major distinguishing characal., 1961; Cagan and Karnovsky, 1964; teristic between molluscan granulocytes
Rossi and Zatti, 1964, 1966; Selvaraj and and hyalinocytes is the occurrence of large
Sbarra, 1966; Sbarra et al., 1971; and oth- numbers of cytoplasmic granules in the
ers) have contributed considerably to this former, the nature of these organdies and
aspect of phagocytosis. Anderson et al. their possible association with intracellular
(1973) were the only ones who had studied degradation following endocytosis had to
energy production and utilization by inver- be ascertained. Consequently, Yoshino and
tebrate phagocytes, in the roach Blaberus Cheng (1976), by employing acid phoscraniifer, prior to the report by Cheng phatase as a marker as has been advocated
(1976r) on this topic as related to the bivalve by deDuve (1969), deDuve et al. (1955),
Mercenaria mercenaria. It was reported that, and Novikoff (1963), have demonstrated
unlike the situation in mammalian phago- at both the light and electron microscope
cytes, there is no increase in oxygen con- levels that these prominent, cytoplasmic,
ROLE OF LYSOSOMES IN MOLLUSCAN INFLAMMATION
133
3P-
205-
8-
o
4
1.00.5-
2-
10
.20
40
80
160
320
Ionic cone. [mM|
FIG. 4. Effect of ionic concentration on the lytic
activity of Crassostrea virginica whole hemolymph at
pH
pH 8.0. Lysozyme activity is expressed as AOD510/
min X 10~2 at 25°C. • - • , ethylene diamine; o-o, Mg2+
FIG. 5. Effect of pH on the lytic activity ofCrassostrea
as MgCl2; A—A, 1,4-diaminobutane. (After Rodrick
and Cheng, 1974a; with permission of Academic virginica whole hemolymph lysozyme using 0,7 M acetate ( • - • ) , 0.1 M glycylglycine (o-o), and 0.1 Af phosPress.)
phate (A—A). Lysozyme activity is expressed as
AODM0/min X 10"2 at 25°C. (After Rodrick and
Cheng, 1974a; with permission of Academic Press.)
electron-opaque vesicles act as true lysosomes. It is noted that earlier, Cheng and
Foley (1972) had demonstrated by use of
scanning electron microscopy of ruptured
C. virginica granulocytes that the cytoplasmic vesicles are membrane bound.
LYSOSOMAL ENZYMES
Concurrent with the cytochemical studies mentioned above, Rodrick and Cheng
(1974a) carried out biochemical studies on
the lysozyme from the hemolymph of C.
virginica. It is noted that earlier McDade
and Tripp (1967a, b) had reported the
occurrence of this lysosomal hydrolase in
the hemolymph of this oyster. However,
Rodrick and Cheng (1974a) advanced our
knowledge by reporting that lysozyme
activity occurs in both the serum and hemocytes of C. virginica. When whole hemolymph was subjected to centrifugation at
4,000 and 10,000 X g, the enzyme activity
was greater in both instances in the serum
than in the cells. Furthermore, it was determined that the lytic activity of the mollus-
centration (Fig. 4). The optimal pH of the
molluscan enzyme, however, ranges from
5.0 to 5.5 (Fig. 5), depending on the buffer
employed.
When tested against a number of bacteria, the oyster lysozyme was found to be
active not only against M. lysodeikticus but
also against Bacillus subtilis, B. megaterium,
Escherichia coli, Gaffkya tetragena, Salmonella
pullorum, and Shigella sonnei, although it is
less active against the last four mentioned.
It was not active against Staphylococcus
aureus. Rodrick and Cheng (1974a) have
postulated that the serum lysozyme had its
origin in the lysosomes of granulocytes and
was released when these organelles ruptured. Subsequently, Foley and Cheng
(1977) have demonstrated quantitatively
that the lysosomes of M. mercenaria are
released from granulocytes by degranulation as do those of mammalian macrophages. Degranulation occurs rapidly in
actively phagocytosing cells and represents
can lysozyme on Micrococcus lysodeikticus, like the morphological basis for the release of
that of egg-white lysozyme, is salt depen- lysosomal enzymes.
dent (Fig. 3), is relatively heat stable, and
Along similar lines, Cheng and Rodrick
is very sensitive to changes in ionic con- (1974) identified and characterized the
134
THOMAS C. CHENG
6-
o
x
V
o
<]
25
75
100
125
150
Salt cone. <mMxlO"3)
'
20C
FIG. 6. Effect of various salt concentrations on the
lytic activity of the lysozyme in the whole hemolymph
of Mya arenaria using NaCl (A), KCI (•), and MgCI2
(o). The lysozyme activity is expressed as AOD540/ Fic. 7. Effect of pH on the activity of the lysozyme
min X 10"2 at 25°C and pH 5.5. (After Cheng and in whole hemolymph of Mya arenaria using 0.1 M
Rodrick, 1974; with permission of Biological Bulletin.) glycylglycine (•), 0.1M Tris-HCl (o), 0.1 M imidazole
(D), and 0.1 M phosphate (A) as buffers. The lysozyme
activity is expressed as AODM0/min X 10~2 at 25°C.
(After Cheng and Rodrick, 1974; with permission of
lysozyme from the hemolymph of Mya Biological Bulletin.)
arenaria. As is the case in C. virginica, they
reported that the lysozyme activity is
greater in serum than in cells. Further- allel studies on the lysozyme of Mercenaria
more, the enzyme from M. arenaria is also mercenaria and found its characteristics to
salt dependent (Fig. 6), relatively heat sta- be essentially identical to those from the
ble, very sensitive to alterations in ionic two other marine bivalves.
concentration and the presence of heavy
Further studies from our laboratory on
metals (Table 3), and has an optimal pH molluscan lysosomal enzymes have revealed
of 5.0 when 0.1 M glycylglycine, 0.1 M the presence of lysozyme, alkaline phosimidazole, or 0.1 M phosphate buffers are phatase, acid phosphatase, /3-glucuroniemployed, but an optimal pH of 4.5 when dase, amylase, and lipase in the hemo0.1 M Tris-HCl buffer is used (Fig. 7). Also, lymph of the freshwater gastropod
a Hill plot of the data resulting from salt Biomphalaria glabrata (Rodrick and Cheng,
reactivation studies indicated that the lyso- 19746) and in the serum and hemocytes of
zyme in M. arenaria hemolymph includes C. virginica and M. mercenaria (Cheng and
at least 2.0 interacting binding sites for Rodrick, 1975). Some characterization of
NaCl and KCI (Fig. 8). Like the oyster lyso- the /3-glucuronidase from the serum and
zyme, that of Mya is active against several cells of C. virginica and M. mercenaria has
species of bacteria, including Micrococcus been carried out by Cheng (1976a). It has
lysodeikticus, Bacillus megaterium, Proteus vulbeen reported that the optimal pH of this
garis, Salmonella pullorum, Shigella sonnei, enzyme is 4.5 when 0.2 M phosphate, 0.2
Bacillus subtilis, and Escherichia coli, althoughM glycylglycine, and 0.2 M acetate are
it is most active against the first two species employed as buffers (Fig. 9). In the same
listed. It is not active against Staphylococcus report, Cheng (1976a) has speculated that
aureus. Since the reports by Rodrick and since /3-glucuronidase can hydrolyze acid
Cheng (1974a) and Cheng and Rodrick mucopolysaccharides, which are constitu(1974) on the identification and character- ents of bacterial walls, it may play a role in
ization of the lysozymes from C. virginim internal defense by acting on susceptible
and M. arenaria, we have performed par- invading microorganisms. Similarly, since
135
ROLE OF LYSOSOMES IN MOLLUSCAN INFLAMMATION
8000
SD356O-
•St»573
•NaCI n 2 3
• KCI n 2 8
g 7000
5
I 6000
8> 5000
| 4000
SIX268
"SD2S1-0
•S0455O
S3000
| 2000
* 1
.2
.3
A .!> .6.7.8.91D
W
VI)
Salt cone. ( m * x 1O'3 )
Fic. 8. Hill plot analysis of the effect of various concentrations of NaCI (A) and KCI (•) on the lytic activpH
ity of the lysozyme in the whole hemolymph of Mya
arenaria. The slope (n) is equal to the minimum num- Fic. 9. Specific activities of/3-glucuronidase in Merber of interacting binding sites. (After Cheng and cenaria mercenaria serum with 0.2 M phosphate
Rodrick, 1974; with permission of Biological Bulletin.) (D-D), 0.2 M acetate ( • - • ) , and 0.2 M glycylglycine
(o—o) buffers at several pHs. SD = standard deviations. The /3-glucuronidase activities are reported as
modified Sigma units/100 ml. n = 25. (After Cheng,
it is known that helminth teguments include 1976a; with permission of Academic Press.)
acid mucopolysaccharides (Lee, 1966),
/3-glucuronidase may play a role in the
destruction of incompatible helminth parasites. In addition, this lysosomal hydrolase
in cells may be correlated with cell proliferation. The details and evidences supporting this hypothesis have been presented by Cheng (1978, 1979).
Relative to the possible role of lysosomal
enzymes acting as defensive molecules
against incompatible helminths, it is noted
that Cheng el al. (19786) have reported
that the levels of aminopeptidase activity
in the hemocytes and serum of B. glabrata
at 20 and 30 days post-exposure to irradiated miracidia of the trematode Echinostoma lindoense is significantly elevated at
both time intervals when compared to the
levels in control snails (Table 4). Furthermore, there is a significantly higher level
of aminopeptidase activity in the serum of
snails at 30 days than at 20 days post-exposure (Table 4). Earlier, Lie el al. (1975) had
reported that£. glabrata (NIH albino strain)
is capable of developing acquired resistance to E. lindoense larvae. Specifically,
when susceptible snails are exposed to irradiated miracidia, the resulting sporocysts
are destroyed in the ventricle by amoebocytic capsules and the snails become
resistant to subsequent challenge with nonirradiated E. lindoense miracidia. Further-
TABLE 3. The effects of sodium tartrate, zinc acetate, and lead nitrate on lysozyme activity in the hemolymph of Mya
arenaria.*
Sample
Fresh
Fresh
Fresh
Fresh
whole
whole
whole
whole
hemolymph
hemolymph + 0.1 mM sodium tartrate
hemolymph + 5 iuM zinc acetate
hemolymph + 0.6 fiM lead nitrate
AODM0/min
Specific activity
% inhibition
0.025
0.008
0.011
0.005
0.020
0.006
0.008
0.003
68.0
56.0
80.0
* After Cheng and Rodrick, 1974; with permission of Biological Bulletin.
0
136
THOMAS C. CHENG
TABLE 4. Levels of aminopeptidase activity in serum and hemocytes of Biomphalaria glabrata collected at 20 and SO
days postexposure to irradiated Echinostoma lindoense miraddia.'
Aminopeptidase activity
(sigma units/ml ± SD)
Snail group
20-day
20-day
30-day
30-day
controls (n = 19)
postexposure (n = 6)b
controls (n = 20)
postexposure (n = 6)b
Serum
0.0424
0.0955
0.0550
0.9850
±
±
±
±
0.0457
0.0467
0.0565
0.9890
Cells
0.0035
0.0111
0.0051
0.0370
±
±
±
±
0.0070
0.0167
0.0056
0.0479
" After Cheng et at., 19786; with permission of Academic Press.
b
The sample size of six represent two determinations on each of three pooled samples.
more, these sensitized snails may be resensitized by exposing them to a large number
of normal miracidia, resulting in development of a higher degree of resistance than
in snails sensitized only once (Lie and
Heyneman, 1976). Since it is generally
accepted that molluscs are incapable of
synthesizing immunoglobulins (Tripp,
1974; Cheng, 19766; and others), and there
is some evidence that soluble molecules, in
addition to cellular immunity, may play a
role in acquired resistance, Cheng et al.
(19786) have suggested that the elevated
level of serum aminopeptidase may alter
the surface proteins of secondarily introduced parasites and thus act as a form of
acquired humoral immunity. It is noted that
Cushing et al. (1971), Bayne (1980), Jeong
etal. (1980), andSminiaand van der Knaap
(1981) have also provided evidence of
enhanced cellular and humoral reactions
as a result of previous challenge with foreign materials. It is of interest to note that
Ratanarat-Brockelman (1977) has identified a nematode inhibitor in the hemolymph of Helix aspersa; however, its chemical nature remains to be elucidated.
It needs to be noted that enhancement
of host reactions does not appear to be the
case always. Exposure to nonself materials
may enhance, suppress, or not effect molluscan internal defense mechanisms,
depending perhaps on the nature and fate
of the foreign material. Examples of
enhancement have been cited. To the contrary, Lie et al. (1977) have reported that
infection of B. glabrata by sporocysts of
Echinostoma paraensei suppresses innate
resistance to infection with Schistosoma
mansoni. Furthermore, both Dondero et al.
(1977), who challenged Lymnaea rubiginosa
with irradiated miracidia of either Echinostoma audyi or Hypoderaeum dingeri, and
Locker (1978), who initially challenged
Lymnaea catascopium with irradiated miracidia of Schistosomatium douthitti, reported
that none of these treatments altered the
snails' resistance to subsequent infection
with homologous, normal miracidia.
In order to at least partially reveal the
mechanism(s) underlying instances where
suppression of host cellular reactions occur,
Cheng et al. (1981) have demonstrated that
hemocytes collected from C. virginica from
2 hr on post-challenge with Bacillus megaterium are significantly less chemotactic to
this bacterium (Fig. 10). Since chemotaxis
between molluscan hemocytes and nonself
materials appears to be a prerequisite to
effective phagocytosis (Cheng and Howland, 1979), the reported reduction could
be an explanation of reduced cellular reaction.
The following are three possible explanations for the reduction of chemotaxis
between B. megaterium and oyster hemocytes as related to surface recognition: (1)
There are qualitative differences in recognition sites or hemocyte surfaces which
are capable of recognizing chemotactic signals. In oysters that had been prechallenged with bacteria, such sites on certain
subpopulations of hemocytes may have
been preempted by the chemotactic agent
emitted by B. megaterium comprising the in
vivo challenge and consequently are
unavailable for subsequent recognition. As
a consequence, these cells do not demonstrate chemotaxis. (2) There are quantitative differences in surface recognition
ROLE OF LYSOSOMES IN MOLLUSCAN INFLAMMATION
137
80-,
H|I
70-
supernatant
without
B. megaterium
pellet
supernatant
10-
hjn
Z1 30-
1——1
1
pellet
I.
1—|—1
>>60
with
B megaterium
Fic. 11. Distributions of lysozyme activity in the
supernatant (serum) and pellet (cell) fractions of Mercenaria mercenaria hemolymph that had not and had
TIME(hr)
been incubated with Bacillus megaterium. The decrease
Fic. 10. Mean number of hemocytes per oil-immer- of enzyme activity in the pellet fraction of hemolymph
sion field from Crassostrea virginica preinjected with that had been incubated with B. megaterium is statislive Bacillus megaterium (A), preinjected with saline (•), tically significant (P < 0.01) as is the increase in the
and left untampered (•). n = 60 in each instance. Ver- supernatant fraction (P < 0.01). (After Cheng et al.,
tical bars represent one standard deviation. (After
1975; with permission of Academic Press.)
Cheng et al., 1981; with permission of Academic Press.)
release of lysozyme from cells into serum
(Figs. 11, 12). Subsequently, Cheng and
Yoshino (1976a) have demonstrated that
when Mya arenaria is challenged in vivo with
B. megaterium, there is an elevation of lipase
activity in both the hemocytes and serum
(Table 5). The elevated level in serum is
believed to have been released from cells.
Similarly, Cheng and Yoshino (19766) have
demonstrated that when whole hemolymph of B. glabrata is exposed to live and
sonicated B. megaterium in vitro, there is an
elevation in the lipase activity levels in both
the cells and serum (Tables 6, 7). On the
other hand, exposure to sonicated vegetative bacterial cells did not induce elevated lipase activity levels in either cells or
serum. Thus, it would appear that the
mechanism of induction of increased depoSOURCES OF SERUM ENZYMES
Since elevated serum lysosomal enzymes sition of lipase in lysosomes and subsequent
are believed to play a role in molluscan release of this enzyme into serum appears
immunity, the question that needed to be to rest with whole bacteria.
asked was: What is the source(s) of the
Further evidence for the hypersynthesis
serum enzymes? It is now known as a result of lysozyme and its subsequent release into
of studies by Cheng et al. (1975) that when serum of gastropod molluscs was contribM. mercenaria hemocytes are induced to uted by Cheng et al. (1977) who demonengage in phagocytosis by exposure to strated that the in vivo challenge of B. glaBacillus megaterium, there is enhanced brata with B. megaterium results in an
sites on subpopulations of molluscan hemocytes. According to this hypothesis, on
some cells there are more of the specific
recognition sites, and these are preempted
by the bacterial chemotactic agent introduced by the in vivo challenge, and consequently, such cells are less responsive, if
at all, to that agent during a subsequent
challenge. (3) The introduction of bacteria
may have in some qualitative and/or quantitative manner altered the composition of
oyster serum and such changes, in turn,
had caused a decrease in response on the
part of hemocytes to the chemoattractant.
Known instances of alterations in molluscan serum composition due to infection
have been cited by Cheng et al. (1981).
138
THOMAS C. CHENG
80-|
70-
£•»
•fl
so
|
1001
2:1
20:1
Bacteria hemolymph cell ratio
200:1
Fic. 12. Distribution of lysozyme activity in the
supernatant (serum) and pellet (cell) fractions of Mercenaria mercenaria hemolymph that had not (0:1) and
had been incubated with Bacillus megaterium at three
different bacteria: hemolymph cell ratios. As indicated, there is the tendency for the enzyme activity
to decrease in the pellet fraction and increase in the
supernatant fraction as the number of bacteria was
increased. The differences between the levels of lysozyme activity in both fractions of hemolymph that
had been incubated with bacteria and in nonexposed
hemolymph are significant (P < 0.01). (After Cheng
et al., 1975; with permission of Academic Press.)
fact suggests that the phenomenon is not
purely biologic, e.g., associated with bacterial cells; it may have a biophysical basis,
for example, alteration in cell surface
charges. Additional studies demonstrating
hypersynthesis of lysosomal enzymes and
their subsequent release during phagocytosis by molluscan hemocytes have been
contributed by Cheng et al. (1978a) and
Cheng and Butler (1979). These studies
have revealed that there is hypersynthesis
of aminopeptidase, lysozyme, and acid
phosphatase in actively phagocytosing
hemocyte of B. glabrata and these lysosomal enzymes are released into serum by
degranulation.
As a result of the studies reviewed up to
this point, the pattern involving lysosomal
hydrolases in hemocytes, particularly granulocytes, has been established. However,
Yoshino and Cheng (1977) posed the question as to whether there may be other
sources for serum lysosomal enzymes. As
a consequence, it was ascertained that both
the headfoot and the visceral mass of B.
glabrata include subequal levels of aminopeptidase. Similarly, Cheng et al. (19806)
have demonstrated that the tissues of the
headfoot of the terrestrial gastropod Theba
pisana also contains acid and alkaline phosphatases, lysozyme, and /3-glucuronidase.
Since gastropods and pelecypods have open
circulatory systems, it is possible that the
serum aminopeptidase could have originated in the headfoot and visceral mass in
addition to hemocytes.
elevation of the activity of this lysosomal
hydrolase in hemocytes at 1 hr post-challenge and in serum at 2 and 4 hr postchallenge (Table 8). These data, again,
support our contention that during phagocytosis of foreign materials, there is the
hypersynthesis of lysosomal enzymes and
their subsequent release into serum. Of
particular interest in the study by Cheng
RECOGNITION SITES
et al. (1977) is the fact that challenging B.
glabrata with sterile distilled water also
In light of our present knowledge of
results in an elevation of serum lysozyme induction phenomena at the cellular level,
activity at 1 hr post-injection (Table 8). This what has been stated thus far suggests that
T A B L E 5. Specific lipase activities in hemolymph cells and sera of Mya arenaria that had not (controls) and had
(experimentals) been injected with 30 X 10* heat-killed Bacillus megaterium."
Control cells
Experimental
cells
0.16 ± SD 0.11
( n = 19)
0.29 ± SD 0.16
(n=17)
P < 0.01"
Control serum
Experimental
serum
0.26 ± SD0.19
(n =
0.40 ± SD 0.24
(n =
0.05b
' All enzyme activities are reported as Sigma-Tietz units/milliliter of sample. The discrepancies between
the sample sizes are due to the discarding of a few inadequate cell samples. (After Cheng and Yoshino, 1976a;
with permission of Academic Press.)
h
Statistically significant.
139
ROLF, OF LVSOSOMES IN MOLLUSCAN INFLAMMATION
TABLE 6. Lipase activities in the sera of Biomphalaria glabrata that had been challenged with sonicated and live
Bacillus megaterium and incubated at 22° and 37°C compared with the activity in unchallenged control serum.'
Treatment of hemolymph
Lipase activity
(Sigma-Tietz units/ml ± SD)
Challenged with heat-killed, sonicated B. megaterium
0.037 ±
and incubated at 22°C
Challenged with heat-killed, sonicated B. megaterium
0.042 ±
and incubated at 37°C
Challenged with live B. megaterium and incubated
0.036 ±
at 37°C.
0.021 +
Unchallenged controls
' After Cheng and Yoshino, 1976i; with permission of Academic Press.
b
Statistically significant.
Kest
0.038
P > 0. 05
0.048
P > 0 . 05
0.023
0.013
P > 0. 05"
at least three categories of binding (or rec- cifically, Renwrantzand Cheng(1977a), by
ognition) sites must occur. Specifically, (1) studying the interaction of Helix pomatia
there must be one category on the surface hemocytes with various agglutinins (or lecof granulocytes that receives the signal tins) with known saccharide specificities
generated by the challenge; (2) there must (anti-A and Anti-B antisera, phytohemagbe another category of binding sites on the glutinin, M form (PHA), concanavalin A
nuclear surface which initiates the signal- (Con A), wheat germ agglutinin (WGA),
ling of intranuclear events, e.g., the acti- Limulus polyphemus agglutinin, Anguilla
vation or enhanced activity of the genomes anguilla agglutinin (Anti-Heel), Dolichos
responsible for the hypersynthesis of lyso- biflorus seed agglutinin (anti-ADB), Labsomal enzymes; and (3) there must be a urnum alpinum seed agglutinin (antithird category of signal-receiving sites on HLa), Cepaea hortensis albumin gland aggluthe surface of lysosomes the activation of tinin (anti-ACH), Helix pomatia albumin
which induces degranulation, i.e., the gland agglutinin (anti-AHP), Soja sp. seed
mechanism for lysosomal enzyme released agglutinin, Ricinus communis seed aggluti(Fig. 13).
nin, Ulex europaeus seed agglutinin, EnvonA limited number of studies have been ymus europaeus seed coat agglutinin, Aaptos
conducted to ascertain whether binding papillata agglutinin, Axinella polypoides
sites occur on molluscan hemocytes. Fur- agglutinin, and Cerianthus sp. agglutinin),
thermore, these have been carried out pri- came to the conclusion that binding sites
marily in relation to understanding phago- exist and that the carbohydrate compocytosis. Nevertheless, such studies, as nents of these sites of Helix cells include
indicated below, have revealed the occur- galactose, fucose, mannose or glucose or
rence of binding sites on cell surfaces. Spe- both, and N-acetylneuraminic acid or poly-
TABLE 7. Lipase activities in hemolymph cells o/Biomphalaria glabrata that had been challenged with sonicated and
live Bacillus megaterium and incubated at 22? and 37°C compared with the activity in unchallenged control cells.'
Treatment of hemolymph
Lipase activity
(Sigma-Tietz units/ml ± SD)
Challenged with heat-killed, sonicated B. megaterium
0.016 ±
and incubated at 22°C
Challenged with heat-killed, sonicated B. megaterium
0.009 ±
and incubated at 37°C
Challenged with live B. megaterium and incubated
0.030 ±
at 37°C
0.014 +
Unchallenged controls
' After Cheng and Yoshino, 1976A; with permission of Academic Press.
b
Statistically significant.
(-lest
0.025
P > 0.20
0.011
P > 0.10
0.021
0.013
P > 0.05
140
THOMAS C. CHENG
TABLE 8. Levels of lysozyme activity in the serum and cell fractions of the hemolymph of challenged and nonchallenged
Biomphalaria glabrata."
Time postinjection
Treatment of snail
Lysozyme activity in serum
Bacteria-injected
Water-injected
Sham-injected
Noninjected
Lysozyme activity in cells'"
Bacteria-injected
I hr
2hr
4 hr
0.997 ± 0.387(n = 10)0.948 ± 0.493
(n = 14)0.426 ± 0.312
(n = 10)-
0.291 ± 0.102c
(n= 10)0.908 ± 0.405
(n = 5)0.844 ± 0.507
(n = 5)-
b
0.860 ± 0.519
(n = 8)«
1.133 ± O.515c
(n= 12)0.399 ± 0.156
(n= 10)d
0.624 ± 0.363
(n = 14)-
0.351 ± 0.483
1.936 ± 1.302'
1.147 ± 0.676
(n= 10)(n = 10)(n = 8)"
Water-injected
0.092 ± 0.053
1.128 ± 1.583
1.208 ± 1.078
(n = 14)(n = 5)(n = 12)Sham-injected
0.296 ± 0.229
0.492 ± 0.580
0.334 ± 0.263
(n = 5)(n=10)(n= 10)d
Noninjected
0.786 ± 0.948
(n = 14)' After Cheng el al, 1977; with permission of Academic Press.
b
The enzyme activities are expressed as micrograms of hen egg white lysozyme equivalents per milliliter
of serum or per milliliter of cell homogenate.
c
Values significantly different from that of noninjected controls.
- Each sample consisted of the pooled hemolymph fraction from five snails.
galactose. Furthermore, in order to ascertain whether these binding sites serve the
function of linking Helix hemocytes to
exogenous cells, Renwrantz and Cheng
(19776) performed rosette-formation tests
to determine whether agglutinins known
to bind molluscan cells will also bind to
mammalian erythrocytes. These tests
involved pretreatment of Helix hemocytes
with each of 15 non-native agglutinins (lectins) and incubating them with human
erythrocytes. It was found that of the
agglutinins tested, WGA, as well as those
from Ricinus communis, Axinella polypoides,
Anguilla anguilla, Limulus polyphemus, and
Con A caused rosette formation with
erythrocytes (Fig. 14). In addition, it was
found that a small number of Helix hemocytes are capable of direct binding to
erythrocytes of mice, rabbits, rats, and
sheep.
In addition to Helix cells, Schoenberg and
Cheng (1980) have tested the effect of
eleven lectins on the attachment of human
erythrocytes to hemocytes of two strains of
Biomphalaria glabrata by employing micro-
hemadsorption assays. As a result, it was
determined that Con A, the type II-A
agglutinin of Ricinus communis (RCA-120),
and WGA strongly enhanced hemadsorption. Moreover, RCA-120 and Con A
enhancement were restricted to what
Schoenberg and Cheng (1980) have
referred to as "type A" hemocytes, which
have since been identified as granulocytes
(Schoenberg and Cheng, 1981a). It is noted
that the lectins from Helix pomatia (HPA),
Lens culinaris (LCA), Tetragonolobus purpureas (TPA), and Ulex europeus (UEA-60)
weakly enhanced hemadsorption. Also,
LCA more strongly enhanced hemadsorption with hemocytes from one strain of B.
glabrata (NIH albino) than with cells from
another strain (10R-2). These findings suggest that gastropod hemocytes of different
origins differ quantitatively in their arrays
of cell surface oligosaccharides capable of
lectin binding.
Subsequently, Schoenberg and Cheng
(1981ft) also have demonstrated the occur-
ROLE OF LYSOSOMES IN MOLLUSCAN INFLAMMATION
141
FIG. 14. Rosette formation of human erythrocytes
with agglutinin-pretreated Helix hemocytes.*
FIG. 13. Schematic diagram showing positions of at
least three recognition sites associated with lysosomal
enzyme hypersynthesis and release (degranulation) in
molluscan phagocyte.
rence of specific lectin-binding sites on
hemocytes of another pulmonate, Bulinus
truncatus. Also, Cheng et al. (1980a), by
employing several plant and animal lectins,
ascertained that three subpopulations (1,
3, and 4) of C. virginica hemocytes were
agglutinated with Con A and extracts of
the albumin glands of Helix pomatia and
Cepaea nemoralis while another subpopulation of hemocytes (subpopulation 2) was
agglutinated with the same three lectins as
well as wtih WGA. Furthermore, by applying the Con A-peroxidase cytochemical
technique, it was determined that approximately 20% of the granulocytes of subpopulations 1 and 3 do not possess Con Abinding sites and only 18% of the large cells
comprising subpopulation 5 possess such
sites. This study represents the first to demonstrate that different subpopulations of
molluscan granulocytes exist and in addition to differences in their dimensions and
densities, they may be further subdivided
by differences in specific surface binding
sites. The implications of this finding relative to the recognition of exogenous stimulation is presently not known, although it
is possible that different subpopulations of
granulocytes are qualitatively a n d / o r
quantitatively differentially stimulated by
exogenous factors. It is noted that Sminia
Agglutinins
Erylhrocytesb
Anti-ADb
Anti-ACH
Anti-ACN
Anti-AHP
Soja
PHA
WGA
Ricinus
Axinella
Cerianthus
Anti-H^,
Laburnum
Vlex
Con A
Limulus
A
A
A
A
O
O
Rosette
formation0
o
o
o
o
o
o
o
o
o
"After Renwrantz and Cheng, 19774; with permission of Academic Press.
b
The letters designate the type of human erythrocytes.
c
— = no rosettes, + = rosette formation.
et al. (1981), employing ultrahistochemical
tests, have also demonstrated the presence
of Con A-binding sites on the hemocyte
surface of Lymnaea stagnalis.
FUNCTIONAL ROLES OF
LYSOSOMAL ENZYMES
The question that remains to be completely elucidated is: What are the functional roles of the released lysosomal
enzymes in molluscan inflammation? As
stated thus far, there is some evidence that
hypersynthesized enzymes released into
serum play a protective role in destroying
susceptible infectious, biotic agents. Thus,
they may be important in the removal of
inflammation-provoking agents. Also, elevated levels of serum lysosomal enzymes
may alter the molecular configuration of
the surface of exogenous cells, which otherwise would be immunologically recognized as self, and as a consequence become
recognized as nonself and are attacked by
phagocytes.
Another possibility that must be considered is that elevated levels of serum lysosomal hydrolases may initiate autolysis in
inflamed areas and this, in turn, serves to
induce further inflammation as the
142
THOMAS C. CHENG
degraded tissues become recognized as foreign.
It is apparent from this review that lysosomes and their enzymes play a central role
in molluscan inflammation, although
numerous details and yet unknown aspects
of the total phenomenon have yet to be
elucidated. Nevertheless, we are beginning
to understand the roles of lysosomes in
molluscs which, in turn, have contributed
to our understanding of the phylogeny of
inflammatory response and possibly aid in
the understanding of the basic mechanisms
in higher animals.
on subsequent chemotactic response by its hemocytes. J. Invert. Pathol. 38:122-126.
Cheng, T. C. and M. S. Butler. 1979. Experimentally
induced elevations of acid phosphatase activity in
hemolymph of Biomphalaria glabrata (Mollusca).
J. Invert. Pathol. 34:119-124.
Cheng, T. C. and A. Cali. 1974. An electron microscope study of the fate of bacteria phagocytized
by granulocytes of Crassostrea virginica. Contemp.
Top. Immunobiol. 4:25-35.
Cheng, T. C, M. J. Chorney, and T. P. Yoshino.
1977. Lysozymelike activity in the hemolymph
of Biomphalaria glabrata challenged with bacteria.
J. Invert. Pathol. 29:170-174.
Cheng, T. C. and D. A. Foley. 1972. A scanning
electron microscope study of the cytoplasmic
granules of Crassostrea virginica granulocytes. J.
Invert. Pathol. 20:372-374.
ACKNOWLEDGMENTS
Cheng, T. C. and D. A. Foley. 1975. Hemolymph
cells of the bivalve mollusc Mercenaria mercenaria:
T h e original information presented
An electron microscopical study. J. Invert. Pathol.
herein has resulted from research sup26:341-351.
ported by grants from the National Sci- Cheng, T. C , V. G. Guida, and P. L. Gerhart. 1978a.
ence Foundation (PCM-8020884, PCMAminopeptidase and lysozyme activity levels and
serum protein concentrations in Biomphalaria gla8208016).
brata (Mollusca) challenged with bacteria. J.
REFERENCES
Invert. Pathol. 32:297-302.
Anderson, R. S., B. Holmes, and R. A. Good. 1973. Cheng, T.C. and K. H. Howland. 1979. Chemotactic
attraction between hemocytes of the oyster, CrasComparative biochemistry of phagocytizing insect
sostrea virginica, and bacteria. J. Invert. Pathol.
hemocytes. Comp. Biochem. Physiol. 46B:59533:204-210.
602.
Bayne, C. J. 1980. Molluscan immunity: Induction Cheng, T. C, J. W. Huang, H. Karadogan, L. R.
Renwrantz, and T. P. Yoshino. 1980a. Separaof elevated immunity in the land snail (Helix) by
tion of oyster hemocytes by density gradient ceninjection of bacteria (Pseudomonas aeruginosa). Dev.
trifugation and identification of their surface
Comp. Immunol. 4:43-54.
receptors. J. Invert. Pathol. 36:35-40.
Cagan, R. H. and M. C. Karnovsky. 1964. Enzymatic
basis of the respiratory stimulation during phago- Cheng, T. C , K. J. Lie, D. Heyneman, and C. S.
cytosis. Nature 204:255-257.
Richards. 1978i. Elevation of aminopeptidase
activity in Biomphalaria glabrata (Mollusca) parCheng, T. C. 1967. Marine molluscs as hosts for
asitized by Echinostoma indoense (Trematoda). J.
symbioeses: With a review of known parasites of
Invert. Pathol. 31:57-62.
commercially important species. Adv. Mar. Biol.
5: 1-424.
Cheng, T. C. and G. E. Rodrick. 1974. Identification
and characterization of lysozyme from the hemoCheng, T. C. 1976a. Beta-glucuronidase in the serum
lymph of the soft-shelled clam Mya arenaria. Biol.
and hemolymph cells of Mercenaria mercenaria and
Bull. 147:311-320.
Crassostrea virginica (Mollusca: Pelecypoda). J.
Invert. Pathol. 27:125-128.
Cheng, T. C. and G. E. Rodrick. 1975. Lysosomal
and other enzymes in the hemolymph of CrasCheng, T. C. 1976A. Humoral immunity in molluscs.
sostrea virginica and Mercenaria mercenaria. Comp.
Proc. 1st Int. Collo. Invert. Pathol., pp. 190-194.
Biochem. Physiol. 52B:443-447.
Queen Univ. Press, Kingston, Ontario, Canada.
Cheng, T. C. 1976c. Aspects of substrate utilization Cheng, T. C, G. E. Rodrick, D. A. Foley, and S. A.
Koehler. 1975. Release of lysozyme from hemoand energy requirement during molluscan
lymph cells of Mercenaria mercenaria during
phagocytosis. J. Invert. Pathol. 27:263-268.
phagocytosis. J. Invert. Pathol. 25:261-265.
Cheng, T. C. 1978. The role of lysosomal hydrolases
in molluscan cellular response to immunologic Cheng, T. C, G. E. Rodrick, S. Moran, and W. A.
Sodeman. 19806. Notes on Theba pisana and
challenge. Comp. Pathobiol. 4:59-71.
quantitative determinations of lysosomal enzymes
Cheng, T. C. 1979. The role of hemocytic hydrolases
and transaminases from the head-foot of this gasin the defense of molluscs against invading partropod. J. Invert. Pathol. 36:1-5.
asites. Haliotis 8:193-209.
Cheng, T. C. 1981. Bivalves. /iiN.A. Ratcliffe and Cheng, T. C. and B. M. Rudo. 1976. Distribution of
glycogen resulting from degradation of r e A. F. Rowley (eds.), Invertebrate blood cells, pp.
labelled bacteria in the American oyster, Cras233-300. Academic Press, London.
sostrea virginica.]. Invert. Pathol. 27:259-262.
Cheng, T. C , M. H. Bui, K. H. Howland, D. A.
Schoenberg, and J. T. Sullivan. 1981. Effect of Cheng, T.C. and T. P. Yoshino. 1976a. Lipase activpreinjection of Crassostrea virginica with bacteria
ity in the serum and hemolymph cells of the soft-
ROLE OF LYSOSOMES IN MOLLUSCAN INFLAMMATION
143
shelled clam, Mya arenaria, during phagocytosis.
induced by irradiated miracidia of Echinostoma
J. Invert. Pathol. 27:243-245.
lindoense in Biomphalaria glabrata snails. Int. J.
Parasitol. 5:627-631.
Cheng, T. C. andT. P. Yoshino. 1976*. Lipase activity in the hemolymph of Biomphalaria glabrata Lie, K. J., D. Heyneman, and C. S. Richards. 1977.
(Mollusca) challenged with bacterial lipids. J.
Studies on resistance in snails: Interference by
Invert. Pathol. 28:143-146.
nonirradiated echinostome larvae with natural
resistance to Schistosoma mansoni in Biomphalaria
Cushing,J. E., E. E. Evans, and M. L. Evans. 1971.
glabrata.]. Invert. Pathol. 29:118-125.
Induced bactericidal responses of abalones. J.
Invert. Pathol. 17:446-448.
Loker, E. S. 1978. Schistosomatium douthitti exposure
of Lymnaea catascopium to irradiated miracidia.
DeDuve, C. 1969. The lysosome in retrospect. In J.
Exp. Parasitol. 46:134-140.
T. Single and H. B. Bell (eds.), Lysosomes in biology
and pathology, Vol. 1, pp. 3-40. North-Holland, McDade.J. E. and M. R. Tripp. 1967a. Lysozyme in
Amsterdam.
the hemolymph of the oyster, Crassostrea virginica. J. Invert. Pathol. 9:531-535.
DeDuve, C, B. C. Pressman, R. Gianetto, R. Waltiaux, and F. Applemans. 1955. Tissue fractionMcDade, J. E. and M. R. Tripp. 1967A. Lysozyme in
ation studies—6. Intracellular distribution patoyster mantle mucus. J. Invert. Pathol. 9:581terns of enzymes in rat-liver tissue. Biochem. J.
582.
60:604-617.
Metchnikoff, E. 1892. Lectures on the comparative
pathology of inflammation. (Translated from French
Dondero, T. J., Jr., C. K. Ow-Yang, and K. J. Lie.
by F. A. Starling and E. H. Starling.) Dover Publ.,
1977. Failure of irradiated Echinostoma audyi and
New York.
Hypoderaeum dingeri to sensitize Lymnaea rubiginosa snails. Southeast Asian J. Trop. Med. Pub. Novikoff, A. B. 1963. Lysosomes in the physiology
Health 8:359-363.
and pathology of cells: Contributions of staining
methods. In A. V. S. de Reuck and M. P. Cameron
Feng, S. Y. 1959. Defense mechanism of the oyster.
(eds.), Lysosomes, pp. 36-73. Little Brown, Boston.
Bull. N.J. Acad. Sci. 4:17.
Ratanarat-Brockelman, C. 1977. Isolation of nemaFeng, S. Y. 1965. Pinocytosis of proteins by oyster
tode inhibitor from hemolymph of the snail, Helix
leucocytes. Biol. Bull. 128:95-105.
aspersa. Biol. Bull. 152:406-414.
Foley, D. A. and T. C. Cheng. 1974. Morphology,
Ratcliffe, N. A. and A. F. Rowley. 1979. Role of
hematologic parameters, and behavior of hemohemocytes in defense against biological agents.
lymph cells of the quahaug clam, Mercenaria merIn A. P. Gupta (ed.), Insect hemocytes, pp. 331cenaria. Biol. Bull. 146:343-356.
414. Cambridge Univ. Press, Cambridge,
Foley, D. A. and T. C. Cheng. 1975. A quantitative
England.
study of phagocytosis by hemolymph cells of the
pelecypods Crassostrea virginica and Mercenaria Renwrantz, L. R. and T. C. Cheng. 1977a. Identimercenaria.]. Invert. Pathol. 25:189-197.
fication of agglutinin receptors on hemocytes of
Helix pomatia.]. Invert. Pathol. 29:88-96.
Foley, D. A. and T. C. Cheng. 1977. Degranulation
and other changes of molluscan granulocytes
Renwrantz, L. R. and T. C. Cheng. 19776. Aggluassociated with phagocytosis. J. Invert. Pathol.
tinin-mediated attachment of erythrocytes to
29:321-325.
hemocytes of Helix pomatia. ]. Invert. Pathol. 29:
97-100.
Haeckel.E. 1862. Die Radiolarien, pp. 104-106. Geo.
Reimer, Berlin.
Rodrick, G. E. andT. C.Cheng. 1974a. Kinetic propHurst, C.T. and C.A.Walker. 1933. A temperature
erties of lysozyme from Crassostrea virginica
reaction in a cold-blooded animal. Anat. Rec. 57
hemolymph. J. Invert. Pathol. 24:41-48.
(Suppl. 4): 100.
Rodrick, G. E. and T. C. Cheng. 1974*. Activities
Hurst, C.T. and C. A. Walker. 1935. Increased heat
of selected hemolymph enzymes in Biomphalaria
production in a poikilothermus animal in paraglabrata (Mollusca). J. Invert. Pathol. 24:374-375.
sitism. Am. Nat. 69:461-466.
Rossi, F. and M. Zatti. 1964. Biochemical aspects of
Iyer, G. Y. N., M. F. Islam, and J. M. Quastel. 1961.
phagocytosis in polymorphonuclear leucocytes.
Biochemical aspects of phagocytosis. Nature 192:
NADA and NADPH oxidation by the granules
of resting and phagocytizing cells. Experientia
535-541.
20:21-23.
Jeong, K. H., K. G. Lie, and D. Heyneman. 1980.
Leucocytosis in Biomphalaria glabrata sensitized Salt, G. 1970. The cellular defense reactions oj insects.
and resensitized to Echinostoma lindoense. J. Invert.
Cambridge Univ. Press, Cambridge, England.
Pathol. 35:8-13.
Sbarra, A. J., A. A.Jacobs, R. R. Strauss, B. B. Paul,
Lee, D. L. 1966. The structure and composition of
and G. E. Mitchel. 1971. The biochemical and
the helminth cuticle. In B. Dawes (ed.), Advances
antimicrobial activities of phagocytizing cells. Am.
in parasitology, Vol. 5, pp. 187-254. Academic
J. Clin. Nutr. 24:272-281.
Press, London.
Schoenberg, D. A. and T. C. Cheng. 1980. Lectinbinding specificities of hemocytes from two strains
Lie, K. J. and D. Heyneman. 1976. Studies in resistance in snails. 3. Tissue reactions to Echinostoma
of Biomphalaria glabrata as determined by microlindoense sporocysts in sensitized and resensitized
hemadsorption assays. Dev. Comp. Immunol. 4:
Biomphalaria glabrata. J. Parasitol. 62:51-58.
617-628.
Lie, K. J., D. Heyneman, and H. K. Lim. 1975. Stud- Schoenberg, D. A. and T. C. Cheng. 1981a. The
ies on resistance in snails: Specific resistance
behavior of Biomphalaria glabrata (Gastropoda:
144
THOMAS C. CHENG
Pulmonata) hemocytes following exposure to lectins. Trans. Am. Microsc. Soc. 100:345-354.
Schoenberg, D. A. and T. C. Cheng. 1981ft. Lectinbinding specificities of Bulinus truncatus hemocytes as determined by microhemadsorption. Dev.
Comp. Immunol. 5:145-149.
Selvaraj, R. J. and A. J. Sbarra. 1966. Relationship
of glycolytic and oxidative metabolism to particle
entry and destruction in phagocytizing cells.
Nature 211:1272-1276.
Sminia, T. and W. P. W. van der Knaap. 1981. The
internal defense system of the freshwater snail
Lymnaea stagnalis. Dev. Comp. Immunol. 5:8797.
Sminia, T., A. A. Winsemius, and W. P. W. van der
Knaap. 1981. Recognition of foreignness by
blood cells of the freshwater snail Lymnaea stagnalis, with special reference to the role and structure of the cell coat. J. Invert. Pathol. 38:175183.
Stauber, L. A. 1950. The fate of midia ink injected
intracardially into the oyster, Ostrea virginica
Gmelin. Biol. Bull. 98:227-241.
Tripp, M. R. 1958. Disposal by the oyster of intracardially injected red blood cells of vertebrates.
Proc. Nat. Shellfish. Assoc. 48:143-147.
Tripp, M. R. 1960. Mechanisms of removal of injected
microorganisms from the American oyster, Crassostrea virginica (Gmelin). Biol. Bull. 119:210-223.
Tripp, M. R. 1974. Molluscan immunity. Ann. N.Y.
Acad. Sci. 234:23-27.
Yoshino, T. P. and T. C. Cheng. 1976. Fine structural localization of acid phosphatase in granulocytes of the pelecypod Mercenaria mercenaria.
Trans. Am. Microsc. Soc. 95:215-220.
Yoshino, T. P. and T. C. Cheng. 1977. Aminopeptidase activity in the hemolymph and body tissues
of the pulmonate gastropod Biomphalaria glabrata. J. Invert. Pathol. 30:76-79.