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Agglutinins in the hemolymph of the hard clam, Mercenaria mercenaria
JOURNAL
OF INVERTEBRATE
PATHOLOGY
59,228-234
m lutinins
(1992)
in the Hemolymph of the Hard Clam, Mercenaria mercenaria M. R. TRIPP
School
of Life
and Health
Sciences,
University
of Delaware,
Newark,
Delaware,
19716
Received December 26, 1990; accepted August 28, 1991
MATERIALS
Lectins in the serum of the clam Mercenaria mercenaria agglutinate some red blood cells, bacteria, and yeast. The interaction of these substanceswith particles is affected by sugars, ions, temperature, and alteration of particle surfaces. Lectins are not needed for phagocytosis of foreign particles in vitro. In M. mercenaria these recognition molecules do not enhance defense mechanisms. CO1992 Academic Press, Inc.
KEY WORDS: Mercenaria mercenaria; phagocytosis; agglutination; opsonization.
Clams M. mercenaria were collected from Rehoboth Bay, Delaware, by a commercial dealer (C. Copp, Lewes, DE). They were maintained in the laboratory in artificial seawater (ASW; Forty Fathoms, Marine Enterprises, Baltimore, MD) at 20%0 and 12°C. Hemolymph Agglutination
INTRODUCTION
It is well known that the hemolymph of many mollusts contains soluble factors that react with a variety of biological substances (Fries, 1984; Chu, 1988; Olafsen, 1988). Considerable evidence has been presented that these substances are important for the identification of foreign materials by hemocytes (Sminia et al., 1979; van der Knaap et al., 1983; Renwrantz and Stahmer, 1983; Dikkeboom et al., 1985; Tuan and Yoshino, 1987; Suzuki and Mori, 1990; Yang and Yoshino, 1990a), but in other instances such substances are not needed for phagocytosis (Anderson and Good, 1976). It has been reported that hemolymph of the hard clam, Mercenaria mercenaria, contains a lectin that agglutinates a bacterium and enhances the phagocytosis of that bacterium (Arimoto and Tripp, 1977). However, hemocytes of M. mercenaria are capable of avid phagocytosis of a variety of particles in the absence of hemolymph (Tripp, 1991). These observations raise questions concerning the importance of humoral factors for defense mechanisms of this mollusc and the common assumption that agglutinins serve a defensive function (Sharon, 1984; Ofek and Sharon, 1988). This report explores further the agglutinating ability of M. mercenuria hemolymph, some of the characteristics of these agglutinins, and their relationship to phagocytosis by clam hemocytes.
0022-2011/92 Copyright All rights
$4.00 0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
AND METHODS
Assays
Hemolymph was obtained by filing the valves adjacent to the anterior adductor muscle and inserting a 20-gauge needle attached to a 3-ml syringe into the muscle sinus. The hemolymph was centrifuged for 10 min at about 400g at room temperature (22-2&C) and the supernatant fluid (=serum) was removed. Volumes of 25 ~1 of serum were added to the first two U-shaped wells of a row in 96-well plastic assay plates (Falcon brand, Becton-Dickinson, Oxnard, CA) and a doubling dilution series was prepared using phosphate-buffered saline (PBS) or ASW as the diluent. The 12th well was a PBS or ASW control. Suspensions of particles at appropriate concentrations were added to each well (25 ~1 per well) to give final concentrations of hemolymph diluted l/2 through l/2048 (diluted 2-l through 2-11). Trays were sealed, incubated for l-2 hr at 5”C, noted for a preliminary titer, and then incubated at 5°C overnight before the final titer was recorded. Negative reactions were dense buttons of unagglutinated particles in the center of the well; a positive reaction consisted of an even layer of particles spread over the surface of the U-shaped well. In some cases reactions were neither typically positive nor negative (due to treatment of the particles) and no reaction was recorded. Titers are reported as the exponential of the base 2 of the reciprocal of the last dilution showing positive agglutination (i.e., l/l6 = l/2*, reported as “4”).
Mercenaria
HEMOLYMPH
with PBS, and stored at 5°C. Before use yeast were washed twice with PBS and suspended in PBS or ASW at a concentration of 1%. Bacteria (Escherichia coli and Staphylococcus aureus from the University of Delaware teaching collection, courtesy of L. Clouser and T. della Volpe) were grown on trypticase soy (TS) agar plates or in TS nutrient broth at 37°C for 24 hr before harvesting. Bacteria were used live after washing 4x in PBS, or formalized (as RBC), or heat-killed (1OO’C for 30 min), or autoclaved (121°C for 15 min). Stock suspensions were stored at 5°C in PBS and washed 3~ with PBS before use. Yeast and bacteria agglutination titers were determined as for RBC.
TABLE 1 Typical Agglutination Titers of Pooled Clam (M. mercenuria) Serum with Fresh Vertebrate Red Blood Cells RBC species
Titer (2?
Range
Horse Burro Rabbit Sheep Chicken cow Human (A + )
9 3 4 1 0 0 0
7-12 3-5 3-6 O-4 -
Agglutination
Inhibition
229
AGGLUTININS
Tests
Solutions of 10 saccharides (glucose, glucosamine, galactose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine, trehalose, and sucrose; Sigma Chemical Co., St. Louis, MO) in 50 or 200 InM concentrations were prepared in PBS. These solutions were used to dilute pooled hemolymph as described above. Test RBC were prepared, agglutination tests performed, and titers reported as described.
Adsorption Procedures
Adsorption was done by adding 2 vol of serum to 1 vol of packed fresh or fixed RBC or fresh yeast at room temperature (22-24°C) with mild agitation for 3-4 hr. After centrifugation, the hemolymph was removed and added to fresh cells, mixed thoroughly, and allowed to stand at 5°C overnight. The adsorbed serum was used immediately or stored frozen ( - 20°C). Three separate pools of clam serum were tested.
Particle Preparation
Fresh red blood cells in Alsever’s solution (Cleveland Scientific, Bath, OH) were washed 3~ with PBS and resuspended at a concentration of 2% in PBS or ASW. RBC formalin fixation was carried out according to the procedure of Abdul-Salam and Michelson (1980) and glutaraldehyde fixation was according to Bing et al. (1967). Cells were stored at 5°C in PBS. Dried baker’s yeast (Saccharomyces cerevisiae; Fleishmann brand) were washed 3~ with PBS and stored at 5°C or treated with formalin (as RBC), washed with PBS or ASW, and stored at 5°C. Heatkilled yeast were autoclaved (121°C for 15 min) or heated in a water bath at 100°C for 30 min, washed 3 x
Dialysis and Heat Treatment
Pooled serum (10 ml) in dialysis tubing (12,00014,000 MW retention; Thomas Scientific, Swedesboro, NJ) was placed in 1 liter of water, 2% NaCl, Trisbuffered saline (TBS), TBS plus CaClz, or TBS + EDTA (Tuan and Yoshino, 1987) and stirred at 5°C. The fluid was replaced after the first 3 hr and dialysis continued for 24 hr. Dialyzed serum was used immediately or stored frozen ( - ZOOC). Hemocytes and Phagocytosis Assays
Up to 3 ml of hemolymph was obtained from a typical clam. Aliquots of 50 ~1 were placed in each of eight
TABLE 2
Agglutination
of Fresh or Fixed RBC after Adsorption of Pooled Clam (M. mercenaria)
Serum with Fresh (Unfixed)
RBC
or Yeast Agglutination
titer (2”) vs RBC of
Fresh
Formalinfixed
Hemolymph
Horse
Burro
Rabbit
Sheep
Horse
Burro
Rabbit
Sheep
Unadsorbed Adsorbed with Fresh RBC Horse Burro Rabbit Sheep Yeast (S. cerevisiuel
9
3
5
1
8
4
4
1
0 9 9 9 9
0 0 0 0 3
4 4 0 4 5
0 0 0 0 1
1 7 6 8 8
1 0 1 3 4
3 3 1 4 4
0 0 0 0 1
Note. Adsorption time.
with fresh RBC was repeated three times and with fixed RBC twice. Comparable titers (+I dilution)
were obtained each
230
M. R. TRIPP
wells (7 mm diameter, printed slides from Cel-Line Associates, Newfield, NJ) per slide. Slides were placed in moist incubation chambers for 30 min at room temperature (21-24°C). Serum was removed and the hemocyte layer was washed twice with 75 r~.l of ASW before test particles were added. For phagocytosis assays test particles were premixed in appropriate fluid and added to washed hemocytes in 50-~1 aliquots. Slides were incubated in moist chambers for 30 min at room temperature to allow phagocytosis to proceed. Hemocyte preparations were washed by dipping slides in large volumes (250 ml) of ASW to remove extracellular particles, fixed in Zenker’s solution, stained with Giemsa, and mounted under coverslips. Individual wells were examined under oil immersion and a minimum of 200 cells per well scored as positive (i.e., containing one or more particles) or negative. The scores of four wells for each treatment were averaged and are presented as percentage phagocytosis. RESULTS
M. mercenariu serum agglutinated red blood cells of several species of vertebrates (Table 1). Serum pooled from 4 to 6 individual clams consistently gave values as indicated although the reactivity of individual clams varied (e.g., hemolymph from 66 individual clams tested with fresh rabbit RBC had an average agglutination titer of 23.75 and ranged from 23 to 2?. Cross-adsorption experiments showed that RBC of some species removed only homologous agglutinins (e.g., “anti-horse” and “anti-rabbit”) but did not affect heterologous agglutinins (Table 2). An exception was the “anti-burro” material which was removed not only by burro RBC but also by horse, rabbit, and sheep RBC. After adsorption with formalin-fixed horse and rabbit RBC a trace of activity against the homologous fixed RBC remained. Yeast cells did not react with any antiRBC lectins since yeast-adsorbed hemolymph reacts with fresh and formalinized RBC just as it does with unadsorbed hemolymph (Table 2). Agglutination of RBC was affected only slightly by fixation with formaldehyde, but glutaraldehyde fixation had a greater effect on agglutinability (Table 3). The reactivity of burro and sheep cells was enhanced, while rabbit cells
TABLE 4 Agglutination Titers (2”) of Bacteria and Yeast in Clam (M. mercenaria) Serum Treatment Organism
Live
Formalin
St. aureus E. coli
2 AR 5
6 AR
S. cerevisiue
100°C
Auto
4 6
3 6
Note. AR, atypical reaction (i.e., neither typical positive nor negative reaction); lOo”C!, boiling water bath for 30 min; Auto, autoclaved (121W15 min).
no longer gave a visible reaction (Table 3) and horse RBC reactivity was decreased. Microbes treated in various ways to modify their surfaces showed different agglutination characteristics. St. aureus was agglutinated by clam hemolymph whether alive or killed by formalin or heating (1OOC or autoclaved) (Table 4). In contrast, E. coli was not agglutinated when alive or when killed by formalin but was agglutinated after boiling and autoclaving (Table 4). Yeast (5’. cereuisiae) were agglutinated by clam hemolymph when alive but not atter being killed (Table 4). When clam serum was heated it began to lose activity at 40-50°C and was inactive against horse, rabbit, and burro RBC at 70°C (Fig. 1). When dialyzed vs 2% NaCl horse RBC agglutinin is partially inactivated at room temperature (25°C) and totally inactivated at 35°C (Fig. 2). Activity against rabbit and burro RBC was lost completely after NaCl dialysis. Attempts were made to determine the ionic conditions needed for agglutination of horse and rabbit RBC by clam serum. Serum dialyzed against NaCl lost some of its ability to agglutinate horse RBC and addition of Ca2+ did not restore the lost activity (Table 5). Serum dialyzed against distilled water was inactive and activity was partially restored by addition of Ca2+; this is an artifact, however, since Ca2+ alone will agglutinate
TABLE 3 Titers (2”) of Red Blood Cells Agglutinated by Clam (M. mercenaria) Hemolymph Fixed RBC source Horse Burro Rabbit Sheep
Untreated 10 3 4 1
Formalin 8 4 4 1
Glutaraldehyde 7 7
20
30
40
50
60
70
60
OC
FIG. 1. Effect of temperature on agglutination of unfixed horse (X1, rabbit (01, and burro (0) red blood cells by M. mercenaria serum. Similar curves differing by no more than one dilution were obtained in four replicate experiments.
Mercenariu
HEMOLYMPH
231
AGGLUTININS
TABLE 5
‘.
$8 5 ;6 c N Ir4 F F .2 d a 40
Agglutination
t
of Unfixed RBC by Native and Dialyzed Clam Serum Titers (2”) of RBC
Hemolymph
25
35
45 "C
55
65
75
FIG. 2. Effect of temperature on the ability of undialyzed (0) and NaCl-dialyzed (0) M. mrcenaria serum to agglutinate unfixed horse red blood cells. Identical results were obtained in three replicate experiments.
horse RBC (titer = 23; Table 5). Dialysis against Trisbuffered saline partially inactivated anti-horse RBC agglutinin and when EDTA was added only minimal agglutination was detected (Table 5). Similar tests of dialyzed serum with rabbit RBC suggested a need for Ca 2+ for agglutination (Table 5). Results of experiments designed to demonstrate the inhibition of horse, burro, sheep, and rabbit RBC agglutination by saccharides are summarized in Table 6. Concentrations of 50 mM were not effective. At a concentration of 200 mM, glucosamine completely inhibited sheep RBC agglutination and horse RBC agglutination was partially inhibited by N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine. Otherwise there was no apparent inhibition. Serum dialyzed against TBS + EDTA lost its ability to agglutinate untreated RBC of burro, sheep, and rabbit; horse cells reacted minimally (Table 7). Sheep and rabbit cells reacted atypically after trypsinization as did glutaraldehyde-fixed rabbit RBC. Horse RBC agglutinability by normal serum is affected slightly by
Horse
Rabbit
Undialyzed Dialyzed vs NaCl NaCK + Ca’ + ) Distilled Hz0 Distilled H,O( + Ca2+ 1 Caz+ TBS TBS + EDTA Note. NaCl, 20%0; NaCl (+ Ca’+l, serum dialyzed vs 2% NaCl (then 0.09% CaClz added); distilled Hz0 (+ Ca2+), serum dialyzed vs distilled water (then 0.09% CaCl, added); Ca’+, 0.09% CaCl,; TBS, Tris-buffered saline; TBS + EDTA, TBS + EDTA (2 mM).
fixation or trypsinization, but trypsinization and glutaraldehyde fixation of burro and sheep RBC enhances their agglutinability (Table 7). Horse RBC are avidly phagocytosed by M. mercenaria hemocytes whether or not horse RBC agglutinin is present and whether fresh or fixed RBC are used (Fig. 3). Similarly, if hemagglutinin is present or absent, or if it is inactivated by heating or not, there is no appreciable effect on phagocytosis of horse or sheep RBC (Fig. 4). Finally, there is no correlation between the ability of clam hemolymph to agglutinate chemically altered horse RBC and the ability of clam hemocytes to phagocytose those particles (Fig. 5). Yeast, either living or dead, were as readily phagocytosed in ASW as in hemolymph (Fig. 6). St. aureus was phagocytosed when alive or heat-killed, but after formalin treatment very little phagocytosis occurred. E. coli, in contrast, was extensively phagocytosed when
TABLE 6 Effects of Saccharides
on Agglutination
of Red Blood Cells by Clam (M. mercenaria) Agglutination
Horse
Glucose Gala&se Mannose Glucosamine Fucose N-Acetylglucosamine N-Acetylgalactosamine N-Acetylmannosamine Trehalose Sucrose Note. -,
not performed.
Sheep 3
8 8 8 8 8 7 7 7 8 8
titer (2”) vs RBC
Burro
Control sugar (mM) 6 7 6 6 4 5 4 7 6
4 4 4 4 4 4 4 4 4 4
Serum
cm3)
4 (50)
3 3 3 2 3 3 3 4 3
4 4 4 3 4 4 4 4 4 4
Rabbit
3 3 3 0 3 3 3 3 2
4 4 4 4 4 5 5 5 5 5
4 3 3 3 4 4 3 4 3
232
M. R. TRIPP TABLE
7
Agglutination of Red Blood Cells (Fresh, Fixed, or Trypsinized) by Clam (M. mercenariu) Hemolymph (Normal or Dialyzed) Agglutination titer (2”) vs Serum treatment
Horse RBC
Burro RBC
Sheep RBC
Rabbit RBC
Un
Glut
Tryp
Un
Glut
Tryp
Un
Glut
Tryp
Un
Glut
‘Jh
Normal
9
7
8
TBS TBS + EDTA
6 1
0 0
0 0
3 0 0
7 2 0
7 3 0
1 0 0
6 2 2
AR AR
4 4
AR AR
AR AR
AR
0
AR
AR
Note. Serum treatment:
Normal, normal serum; TBS, serum dialyzed vs Tris-buffered saline; and TBS + EDTA, serum dialyzed vs TBS Un, unfixed; Glut, glutaraldehyde fixed, and Tryp, tr-ypsinized RBC fixed with glutaraldehyde. AR, atypical reaction, neither typical positive nor negative.
with 2 mu EDTA. RBC treatment:
alive, formalin-killed, or heat-killed (100°C) but after autoclaving it was very resistant to phagocytosis (Fig. 6). In no case does hemolymph appear to affect phagocytosis. DISCUSSION
Many invertebrates have substances in their tissues that react with various biological materials (Fries, 1984). These lectins (i.e., proteins or glycoproteins that react with specific sugars; Sharon, 1984) may be found in the hemolymph (Bayne, 1983; Ratcliffe et al., 1985) or associated with hemocyte membranes (Vasta et al., 1984; Sminia et al., 1979; Renwrantz and Stahmer, 1983) or both (Yang and Yoshino, 1990a). The membrane-bound lectins facilitate hemocyte attachment and phagocytosis (i.e., opsonization), as do some, but not all, of the soluble lectins (e.g., Anderson and Good, 1976). It is clear that the serum lectins of M. mercenaria do not serve as recognition molecules that promote the phagocytosis of foreign particles. Several experiments have shown that avid phagocytosis of a variety of particles occurs in the absence of soluble serum aggluti-
FRESH
I
nins. It is still not clear what role lectins play in normal M. mercenaria, but they are not necessary for cellular defense. Clam hemolymph agglutinates red blood cells of several species but the range of reactivity is more limited than that reported for C. virginica (Vasta et al., 1982). The amount of agglutinating substance present in clam hemolymph (as measured by titration assay) varies with the species of red cell tested. Thus the reaction with horse red cells is consistently strong (titer = 7-12), with rabbit and burro cells moderate (titer = 3-61, and with cow, sheep, chicken, and human cells weak or negative. This suggests a family of specific lectins, and cross-adsorption tests support this notion. Horse and rabbit cells specifically remove “anti-horse” and “anti-rabbit” activities. In contrast, “anti-burro” activity is removed by horse, rabbit, and sheep cells as well as burro cells, clearly a less specific reaction. The interrelationships among these molecules are unclear at present. 100 80
4 cc 6 8 I
111
FIXED
FIG. 3. Phagocytosis of fresh or formalin-fixed horse red blood cells by M. mercenaria hemocytes in ASW (O), serum (ml, or serum absorbed with fresh horse red blood cells (a). Agglutination titer is the greatest dilution (2? of serum that agglutinates fresh or fixed horse red blood cells.
FIG. 4. Phagocytosis of unfixed horse and sheep red blood cells by M. mercenariu hemocytes in homologous (home.) serum (i.e., from same clam that donated phagocytes), pooled (pool) serum, or pooled serum heated at 56 or 100°C for 30 min. Artificial seawater (ASW) was the control medium. Agglutination titer (aggl. titer) is the greatest dilution (2”) of M. mercenariu serum that agglutinates fresh horse or sheep red blood cells.
Mercenuriu
HEMOLYMPH
FIG. 5. Phagocytosis of formalin (form.)or glutaraldehyde (glut.)-fixed or trypsinized (tryp.) horse red blood cells in artificial seawater (0) or M. mercenuriu serum cm). Agglutination titer (aggl. titer) is the greatest dilution (2”) of M. mercenuria serum that agglutinates treated horse red blood cells.
agglutinins seem to be similar to lecdescribed from other molluscan species. They are inactivated by heat (70°C) and ion deprivation. Agglutination of some species of RBC is blocked by specific sugars as has been reported in other mollusks (McDade and Tripp, 1967; Arimoto and Tripp, 1977; van der Knaap et al., 1982; Pauley et al., 1971). Partial species specificity is demonstrable by cross-adsorption tests. All of these observations suggest that M. mercenaria hemolymph contains a family of naturally occurring lectins with different specificities whose normal function is not known. Physical or chemical alteration of bacterial surfaces affects their ability to be phagocytosed by clam hemocytes. St. aureus treated with formaldehyde and autoclaved E. coli are very poorly phagocytosed while living cells or those killed in other ways are readily taken in M. mercenaria
tins
:I i
by hemocytes. In contrast, yeast are phagocytosed readily whether alive or killed. There is no correlation between the agglutination reactions of these particles and their ability to be phagocytosed. This is consistent with the idea that agglutinins are not involved in phagocytosis. Two biologically active substances in the hemolymph of M. mercenaria have been reported previously. Agglutination of four gram-negative isolates and a marine alga was reported by Arimoto and Tripp (1977). In that study formalinized E. coli were not agglutinated by M. mercenaria serum but in the present study such cells were agglutinated. The discrepancy may be due to differences in the strain of bacteria used and/or the formalinization procedure used. The second hemolymph factor, a naturally occurring hemolysin, was reported by Anderson (1981). This material was most active against rabbit red blood cells and required divalent cations. In the present study hemolytic activity was not detected either in agglutination assays or by the spectrophotometric assay of Anderson (1981). Different genetic backgrounds of clams, different sources of erythrocytes, or variation in laboratory procedures may account for these contrasting observations. Red blood cell agglutinins with similar heat sensitivity and Ca2+ requirements were reported by Vasta et al. (1980). The hemolymph of M. mercenaria contains lectins that agglutinate red blood cells, bacteria, and yeast. Despite the consistent presence of these materials in hemolymph, they do not facilitate phagocytosis of these particles. This represents one end of a spectrum of molluscan lectin interactions that ranges from total dependence (Prowse and Tait, 1969) through various degrees of enhanced phagocytosis (e.g., Anderson and Good, 1976; Tripp and Kent, 1967; Tripp, 1966; Sminia et al., 1979; Noda and Loker, 1989) to lectin independence for Mercenaria hemocytes. It is becoming clear that molluscan defense mechanisms are complex and that the roles played by phagocytic cells and hemolymph factors vary from species to species. Renwrantz (1983) has reviewed the immunobiological importance of lectins and their possible interactions with hemocytes and other cells (Renwrantz, 1986). As more is learned about molluscan lectins their evolutionary history becomes more complex (Yang and Yoshino, 1990b) and understanding the details of their interactions with phagocytic cells becomes more important. It is clear that “simple” molluscan defense mechanisms are more complex and diverse than has been thought.
-
FIG. 6. Phagocytosis of yeast (S. cerevisiue) and bacteria reus and E. coli) after no treatment (live), formalinization heating at 100°C for 30 min (loo”), or autoclaving (auto.). for phagocytosis was artificial seawater (0) or serum (W).
(St. uu-
(form.), Medium
233
AGGLUTININS
ACKNOWLEDGMENT I thank
L. Fawcett
for technical
assistance.
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234
M. R. TRIPP
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currence origin and roles in immunity. Symp. 2001. Sot. London 56, 81-93. Renwrantz, L., Schancke, W., Harm, H., Erl, H., Liebsch, H., and Gercken, J. 1981. Discriminative ability and function of the immunobiological recognition system of the snail Helix pomatia. J. Comp. Physiol. 141, 477-488. Renwrantz, L., and Stahmer, A. 1983. Opsonizing properties of an isolated hemolymph agglutinin and demonstration of lectin-like recognition molecules at the surface of hemocytes from Mytilus edulis. J. Comp. Physiol. 149, 535-546. Sharon, N. 1984. Carbohydrates as recognition determinants in phagocytosis and in lectin-mediated killing of target cells. Biol. Cell 51, 239-246. Sminia, T., and van der Knaap, W. P. W. 1986. Immunorecognition in invertebrates with special reference to molluscs. In “Immunity in Invertebrates” (M. Brehelin, Ed.), pp. 112-124. SpringerVerlag, Berlin. Sminia, T., and van der Knaap, W. P. W. 1987. Cells and molecules in molluscan immunology. Dev. Comp. Immunol. 11, 17-28. Sminia, T., van der Knaap, W. P. W., and Edelenbosch, P. 1979. The role of serum factors in phagocytosis of foreign particles by blood cells of the fresh water snail Lymnaea stagnulis. Dev. Comp. Immunol. 3, 37-44. Suzuki, T., and Mori, K. 1990. Hemolymph lectin of the pearl oyster, Pinctada fucuta murtensii: A possible non-self recognition system. Dev. Comp. Immunol. 14, 161-173. Tripp, M. R. 1966. Hemagglutinin in the blood of the oyster Crassostrea virginica. J. Invertebr. Pathol. 8, 478484. Tripp, M. R. 1992. Phagocytosis by hemocytes of the hard clam, Mercenaria mercenuria. J. Invertebr. Pathol., 59, 222-227. Tripp, M. R., and Kent, V. E. 1967. Studies on oyster cellular immunity. In vitro 3, 129-135. Tuan, T-L., and Yoshino, T. P. 1987. Role of divalent cations in plasma opsonin-dependent and -independent erythrophagocytosis by hemocytes of the Asian clam, Corbiczdu fluminea. J. Invertebr. Pathol. 50, 310319. Van der Knaap, W. P. W., Doderer, A., Boerrigter-Barendsen, L. H., and Sminia, T. 1982. Some properties of an agglutinin in the hemolymph of the pond snail Lymnnea stagnalis. Biol. Bull. 162, 404-412. Van der Knaap, W. P. W., Sminia, T., Schutte, R., and BoerrigterBarendsen, L. H. 1983. Cytophilic receptors for foreignness and some factors which influence phagocytosis by invertebrate leucocytes: In vitro phagocytosis by amoebocytes of the snail Lymnaecz stagnalis. Immunology 48, 377383. Vasta, G. R., Cheng, T. C., and Marchalonis, J. J. 1984. A lectin on the hemocyte membrane of the oyster (Crassostrea virginica). Cell. Immunol. 88, 475-488. Vasta, G. R., Cohen, E., and Bhargawa, A. K. 1980. Separation and characterization of hemagglutinins and hemolysins from Mercenaria mercenaria (Venus clam) hemolymph. Am. Zool. 20, 805. Vasta, G. R., Sullivan, J. T., Cheng, T. C., Marchalonis, J. J., and Ware, G. W. 1982. A cell membrane-associated lectin of the oyster hemocyte. J. Invertebr. Pathol. 40, 367377. Yang, R., and Yoshino, T. P. 1990a. Immunorecognition in the freshwater bivalve, Corbicula fluminea. I. Electrophoretic and immunologic analysis of opsonic plasma components. Dev. Comp. Zmmunol. 14, 385-395. Yang, R., and Yoshino, T. P. 199Ob. Immunorecognition in the freshwater bivalve, Corbiculu fluminea. II. Isolation and characterization of a plasma opsonin with hemagglutinating activity. Dev. Comp. Immunol. 14, 97-404.
OF INVERTEBRATE
PATHOLOGY
59,228-234
m lutinins
(1992)
in the Hemolymph of the Hard Clam, Mercenaria mercenaria M. R. TRIPP
School
of Life
and Health
Sciences,
University
of Delaware,
Newark,
Delaware,
19716
Received December 26, 1990; accepted August 28, 1991
MATERIALS
Lectins in the serum of the clam Mercenaria mercenaria agglutinate some red blood cells, bacteria, and yeast. The interaction of these substanceswith particles is affected by sugars, ions, temperature, and alteration of particle surfaces. Lectins are not needed for phagocytosis of foreign particles in vitro. In M. mercenaria these recognition molecules do not enhance defense mechanisms. CO1992 Academic Press, Inc.
KEY WORDS: Mercenaria mercenaria; phagocytosis; agglutination; opsonization.
Clams M. mercenaria were collected from Rehoboth Bay, Delaware, by a commercial dealer (C. Copp, Lewes, DE). They were maintained in the laboratory in artificial seawater (ASW; Forty Fathoms, Marine Enterprises, Baltimore, MD) at 20%0 and 12°C. Hemolymph Agglutination
INTRODUCTION
It is well known that the hemolymph of many mollusts contains soluble factors that react with a variety of biological substances (Fries, 1984; Chu, 1988; Olafsen, 1988). Considerable evidence has been presented that these substances are important for the identification of foreign materials by hemocytes (Sminia et al., 1979; van der Knaap et al., 1983; Renwrantz and Stahmer, 1983; Dikkeboom et al., 1985; Tuan and Yoshino, 1987; Suzuki and Mori, 1990; Yang and Yoshino, 1990a), but in other instances such substances are not needed for phagocytosis (Anderson and Good, 1976). It has been reported that hemolymph of the hard clam, Mercenaria mercenaria, contains a lectin that agglutinates a bacterium and enhances the phagocytosis of that bacterium (Arimoto and Tripp, 1977). However, hemocytes of M. mercenaria are capable of avid phagocytosis of a variety of particles in the absence of hemolymph (Tripp, 1991). These observations raise questions concerning the importance of humoral factors for defense mechanisms of this mollusc and the common assumption that agglutinins serve a defensive function (Sharon, 1984; Ofek and Sharon, 1988). This report explores further the agglutinating ability of M. mercenuria hemolymph, some of the characteristics of these agglutinins, and their relationship to phagocytosis by clam hemocytes.
0022-2011/92 Copyright All rights
$4.00 0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
AND METHODS
Assays
Hemolymph was obtained by filing the valves adjacent to the anterior adductor muscle and inserting a 20-gauge needle attached to a 3-ml syringe into the muscle sinus. The hemolymph was centrifuged for 10 min at about 400g at room temperature (22-2&C) and the supernatant fluid (=serum) was removed. Volumes of 25 ~1 of serum were added to the first two U-shaped wells of a row in 96-well plastic assay plates (Falcon brand, Becton-Dickinson, Oxnard, CA) and a doubling dilution series was prepared using phosphate-buffered saline (PBS) or ASW as the diluent. The 12th well was a PBS or ASW control. Suspensions of particles at appropriate concentrations were added to each well (25 ~1 per well) to give final concentrations of hemolymph diluted l/2 through l/2048 (diluted 2-l through 2-11). Trays were sealed, incubated for l-2 hr at 5”C, noted for a preliminary titer, and then incubated at 5°C overnight before the final titer was recorded. Negative reactions were dense buttons of unagglutinated particles in the center of the well; a positive reaction consisted of an even layer of particles spread over the surface of the U-shaped well. In some cases reactions were neither typically positive nor negative (due to treatment of the particles) and no reaction was recorded. Titers are reported as the exponential of the base 2 of the reciprocal of the last dilution showing positive agglutination (i.e., l/l6 = l/2*, reported as “4”).
Mercenaria
HEMOLYMPH
with PBS, and stored at 5°C. Before use yeast were washed twice with PBS and suspended in PBS or ASW at a concentration of 1%. Bacteria (Escherichia coli and Staphylococcus aureus from the University of Delaware teaching collection, courtesy of L. Clouser and T. della Volpe) were grown on trypticase soy (TS) agar plates or in TS nutrient broth at 37°C for 24 hr before harvesting. Bacteria were used live after washing 4x in PBS, or formalized (as RBC), or heat-killed (1OO’C for 30 min), or autoclaved (121°C for 15 min). Stock suspensions were stored at 5°C in PBS and washed 3~ with PBS before use. Yeast and bacteria agglutination titers were determined as for RBC.
TABLE 1 Typical Agglutination Titers of Pooled Clam (M. mercenuria) Serum with Fresh Vertebrate Red Blood Cells RBC species
Titer (2?
Range
Horse Burro Rabbit Sheep Chicken cow Human (A + )
9 3 4 1 0 0 0
7-12 3-5 3-6 O-4 -
Agglutination
Inhibition
229
AGGLUTININS
Tests
Solutions of 10 saccharides (glucose, glucosamine, galactose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine, trehalose, and sucrose; Sigma Chemical Co., St. Louis, MO) in 50 or 200 InM concentrations were prepared in PBS. These solutions were used to dilute pooled hemolymph as described above. Test RBC were prepared, agglutination tests performed, and titers reported as described.
Adsorption Procedures
Adsorption was done by adding 2 vol of serum to 1 vol of packed fresh or fixed RBC or fresh yeast at room temperature (22-24°C) with mild agitation for 3-4 hr. After centrifugation, the hemolymph was removed and added to fresh cells, mixed thoroughly, and allowed to stand at 5°C overnight. The adsorbed serum was used immediately or stored frozen ( - 20°C). Three separate pools of clam serum were tested.
Particle Preparation
Fresh red blood cells in Alsever’s solution (Cleveland Scientific, Bath, OH) were washed 3~ with PBS and resuspended at a concentration of 2% in PBS or ASW. RBC formalin fixation was carried out according to the procedure of Abdul-Salam and Michelson (1980) and glutaraldehyde fixation was according to Bing et al. (1967). Cells were stored at 5°C in PBS. Dried baker’s yeast (Saccharomyces cerevisiae; Fleishmann brand) were washed 3~ with PBS and stored at 5°C or treated with formalin (as RBC), washed with PBS or ASW, and stored at 5°C. Heatkilled yeast were autoclaved (121°C for 15 min) or heated in a water bath at 100°C for 30 min, washed 3 x
Dialysis and Heat Treatment
Pooled serum (10 ml) in dialysis tubing (12,00014,000 MW retention; Thomas Scientific, Swedesboro, NJ) was placed in 1 liter of water, 2% NaCl, Trisbuffered saline (TBS), TBS plus CaClz, or TBS + EDTA (Tuan and Yoshino, 1987) and stirred at 5°C. The fluid was replaced after the first 3 hr and dialysis continued for 24 hr. Dialyzed serum was used immediately or stored frozen ( - ZOOC). Hemocytes and Phagocytosis Assays
Up to 3 ml of hemolymph was obtained from a typical clam. Aliquots of 50 ~1 were placed in each of eight
TABLE 2
Agglutination
of Fresh or Fixed RBC after Adsorption of Pooled Clam (M. mercenaria)
Serum with Fresh (Unfixed)
RBC
or Yeast Agglutination
titer (2”) vs RBC of
Fresh
Formalinfixed
Hemolymph
Horse
Burro
Rabbit
Sheep
Horse
Burro
Rabbit
Sheep
Unadsorbed Adsorbed with Fresh RBC Horse Burro Rabbit Sheep Yeast (S. cerevisiuel
9
3
5
1
8
4
4
1
0 9 9 9 9
0 0 0 0 3
4 4 0 4 5
0 0 0 0 1
1 7 6 8 8
1 0 1 3 4
3 3 1 4 4
0 0 0 0 1
Note. Adsorption time.
with fresh RBC was repeated three times and with fixed RBC twice. Comparable titers (+I dilution)
were obtained each
230
M. R. TRIPP
wells (7 mm diameter, printed slides from Cel-Line Associates, Newfield, NJ) per slide. Slides were placed in moist incubation chambers for 30 min at room temperature (21-24°C). Serum was removed and the hemocyte layer was washed twice with 75 r~.l of ASW before test particles were added. For phagocytosis assays test particles were premixed in appropriate fluid and added to washed hemocytes in 50-~1 aliquots. Slides were incubated in moist chambers for 30 min at room temperature to allow phagocytosis to proceed. Hemocyte preparations were washed by dipping slides in large volumes (250 ml) of ASW to remove extracellular particles, fixed in Zenker’s solution, stained with Giemsa, and mounted under coverslips. Individual wells were examined under oil immersion and a minimum of 200 cells per well scored as positive (i.e., containing one or more particles) or negative. The scores of four wells for each treatment were averaged and are presented as percentage phagocytosis. RESULTS
M. mercenariu serum agglutinated red blood cells of several species of vertebrates (Table 1). Serum pooled from 4 to 6 individual clams consistently gave values as indicated although the reactivity of individual clams varied (e.g., hemolymph from 66 individual clams tested with fresh rabbit RBC had an average agglutination titer of 23.75 and ranged from 23 to 2?. Cross-adsorption experiments showed that RBC of some species removed only homologous agglutinins (e.g., “anti-horse” and “anti-rabbit”) but did not affect heterologous agglutinins (Table 2). An exception was the “anti-burro” material which was removed not only by burro RBC but also by horse, rabbit, and sheep RBC. After adsorption with formalin-fixed horse and rabbit RBC a trace of activity against the homologous fixed RBC remained. Yeast cells did not react with any antiRBC lectins since yeast-adsorbed hemolymph reacts with fresh and formalinized RBC just as it does with unadsorbed hemolymph (Table 2). Agglutination of RBC was affected only slightly by fixation with formaldehyde, but glutaraldehyde fixation had a greater effect on agglutinability (Table 3). The reactivity of burro and sheep cells was enhanced, while rabbit cells
TABLE 4 Agglutination Titers (2”) of Bacteria and Yeast in Clam (M. mercenaria) Serum Treatment Organism
Live
Formalin
St. aureus E. coli
2 AR 5
6 AR
S. cerevisiue
100°C
Auto
4 6
3 6
Note. AR, atypical reaction (i.e., neither typical positive nor negative reaction); lOo”C!, boiling water bath for 30 min; Auto, autoclaved (121W15 min).
no longer gave a visible reaction (Table 3) and horse RBC reactivity was decreased. Microbes treated in various ways to modify their surfaces showed different agglutination characteristics. St. aureus was agglutinated by clam hemolymph whether alive or killed by formalin or heating (1OOC or autoclaved) (Table 4). In contrast, E. coli was not agglutinated when alive or when killed by formalin but was agglutinated after boiling and autoclaving (Table 4). Yeast (5’. cereuisiae) were agglutinated by clam hemolymph when alive but not atter being killed (Table 4). When clam serum was heated it began to lose activity at 40-50°C and was inactive against horse, rabbit, and burro RBC at 70°C (Fig. 1). When dialyzed vs 2% NaCl horse RBC agglutinin is partially inactivated at room temperature (25°C) and totally inactivated at 35°C (Fig. 2). Activity against rabbit and burro RBC was lost completely after NaCl dialysis. Attempts were made to determine the ionic conditions needed for agglutination of horse and rabbit RBC by clam serum. Serum dialyzed against NaCl lost some of its ability to agglutinate horse RBC and addition of Ca2+ did not restore the lost activity (Table 5). Serum dialyzed against distilled water was inactive and activity was partially restored by addition of Ca2+; this is an artifact, however, since Ca2+ alone will agglutinate
TABLE 3 Titers (2”) of Red Blood Cells Agglutinated by Clam (M. mercenaria) Hemolymph Fixed RBC source Horse Burro Rabbit Sheep
Untreated 10 3 4 1
Formalin 8 4 4 1
Glutaraldehyde 7 7
20
30
40
50
60
70
60
OC
FIG. 1. Effect of temperature on agglutination of unfixed horse (X1, rabbit (01, and burro (0) red blood cells by M. mercenaria serum. Similar curves differing by no more than one dilution were obtained in four replicate experiments.
Mercenariu
HEMOLYMPH
231
AGGLUTININS
TABLE 5
‘.
$8 5 ;6 c N Ir4 F F .2 d a 40
Agglutination
t
of Unfixed RBC by Native and Dialyzed Clam Serum Titers (2”) of RBC
Hemolymph
25
35
45 "C
55
65
75
FIG. 2. Effect of temperature on the ability of undialyzed (0) and NaCl-dialyzed (0) M. mrcenaria serum to agglutinate unfixed horse red blood cells. Identical results were obtained in three replicate experiments.
horse RBC (titer = 23; Table 5). Dialysis against Trisbuffered saline partially inactivated anti-horse RBC agglutinin and when EDTA was added only minimal agglutination was detected (Table 5). Similar tests of dialyzed serum with rabbit RBC suggested a need for Ca 2+ for agglutination (Table 5). Results of experiments designed to demonstrate the inhibition of horse, burro, sheep, and rabbit RBC agglutination by saccharides are summarized in Table 6. Concentrations of 50 mM were not effective. At a concentration of 200 mM, glucosamine completely inhibited sheep RBC agglutination and horse RBC agglutination was partially inhibited by N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine. Otherwise there was no apparent inhibition. Serum dialyzed against TBS + EDTA lost its ability to agglutinate untreated RBC of burro, sheep, and rabbit; horse cells reacted minimally (Table 7). Sheep and rabbit cells reacted atypically after trypsinization as did glutaraldehyde-fixed rabbit RBC. Horse RBC agglutinability by normal serum is affected slightly by
Horse
Rabbit
Undialyzed Dialyzed vs NaCl NaCK + Ca’ + ) Distilled Hz0 Distilled H,O( + Ca2+ 1 Caz+ TBS TBS + EDTA Note. NaCl, 20%0; NaCl (+ Ca’+l, serum dialyzed vs 2% NaCl (then 0.09% CaClz added); distilled Hz0 (+ Ca2+), serum dialyzed vs distilled water (then 0.09% CaCl, added); Ca’+, 0.09% CaCl,; TBS, Tris-buffered saline; TBS + EDTA, TBS + EDTA (2 mM).
fixation or trypsinization, but trypsinization and glutaraldehyde fixation of burro and sheep RBC enhances their agglutinability (Table 7). Horse RBC are avidly phagocytosed by M. mercenaria hemocytes whether or not horse RBC agglutinin is present and whether fresh or fixed RBC are used (Fig. 3). Similarly, if hemagglutinin is present or absent, or if it is inactivated by heating or not, there is no appreciable effect on phagocytosis of horse or sheep RBC (Fig. 4). Finally, there is no correlation between the ability of clam hemolymph to agglutinate chemically altered horse RBC and the ability of clam hemocytes to phagocytose those particles (Fig. 5). Yeast, either living or dead, were as readily phagocytosed in ASW as in hemolymph (Fig. 6). St. aureus was phagocytosed when alive or heat-killed, but after formalin treatment very little phagocytosis occurred. E. coli, in contrast, was extensively phagocytosed when
TABLE 6 Effects of Saccharides
on Agglutination
of Red Blood Cells by Clam (M. mercenaria) Agglutination
Horse
Glucose Gala&se Mannose Glucosamine Fucose N-Acetylglucosamine N-Acetylgalactosamine N-Acetylmannosamine Trehalose Sucrose Note. -,
not performed.
Sheep 3
8 8 8 8 8 7 7 7 8 8
titer (2”) vs RBC
Burro
Control sugar (mM) 6 7 6 6 4 5 4 7 6
4 4 4 4 4 4 4 4 4 4
Serum
cm3)
4 (50)
3 3 3 2 3 3 3 4 3
4 4 4 3 4 4 4 4 4 4
Rabbit
3 3 3 0 3 3 3 3 2
4 4 4 4 4 5 5 5 5 5
4 3 3 3 4 4 3 4 3
232
M. R. TRIPP TABLE
7
Agglutination of Red Blood Cells (Fresh, Fixed, or Trypsinized) by Clam (M. mercenariu) Hemolymph (Normal or Dialyzed) Agglutination titer (2”) vs Serum treatment
Horse RBC
Burro RBC
Sheep RBC
Rabbit RBC
Un
Glut
Tryp
Un
Glut
Tryp
Un
Glut
Tryp
Un
Glut
‘Jh
Normal
9
7
8
TBS TBS + EDTA
6 1
0 0
0 0
3 0 0
7 2 0
7 3 0
1 0 0
6 2 2
AR AR
4 4
AR AR
AR AR
AR
0
AR
AR
Note. Serum treatment:
Normal, normal serum; TBS, serum dialyzed vs Tris-buffered saline; and TBS + EDTA, serum dialyzed vs TBS Un, unfixed; Glut, glutaraldehyde fixed, and Tryp, tr-ypsinized RBC fixed with glutaraldehyde. AR, atypical reaction, neither typical positive nor negative.
with 2 mu EDTA. RBC treatment:
alive, formalin-killed, or heat-killed (100°C) but after autoclaving it was very resistant to phagocytosis (Fig. 6). In no case does hemolymph appear to affect phagocytosis. DISCUSSION
Many invertebrates have substances in their tissues that react with various biological materials (Fries, 1984). These lectins (i.e., proteins or glycoproteins that react with specific sugars; Sharon, 1984) may be found in the hemolymph (Bayne, 1983; Ratcliffe et al., 1985) or associated with hemocyte membranes (Vasta et al., 1984; Sminia et al., 1979; Renwrantz and Stahmer, 1983) or both (Yang and Yoshino, 1990a). The membrane-bound lectins facilitate hemocyte attachment and phagocytosis (i.e., opsonization), as do some, but not all, of the soluble lectins (e.g., Anderson and Good, 1976). It is clear that the serum lectins of M. mercenaria do not serve as recognition molecules that promote the phagocytosis of foreign particles. Several experiments have shown that avid phagocytosis of a variety of particles occurs in the absence of soluble serum aggluti-
FRESH
I
nins. It is still not clear what role lectins play in normal M. mercenaria, but they are not necessary for cellular defense. Clam hemolymph agglutinates red blood cells of several species but the range of reactivity is more limited than that reported for C. virginica (Vasta et al., 1982). The amount of agglutinating substance present in clam hemolymph (as measured by titration assay) varies with the species of red cell tested. Thus the reaction with horse red cells is consistently strong (titer = 7-12), with rabbit and burro cells moderate (titer = 3-61, and with cow, sheep, chicken, and human cells weak or negative. This suggests a family of specific lectins, and cross-adsorption tests support this notion. Horse and rabbit cells specifically remove “anti-horse” and “anti-rabbit” activities. In contrast, “anti-burro” activity is removed by horse, rabbit, and sheep cells as well as burro cells, clearly a less specific reaction. The interrelationships among these molecules are unclear at present. 100 80
4 cc 6 8 I
111
FIXED
FIG. 3. Phagocytosis of fresh or formalin-fixed horse red blood cells by M. mercenaria hemocytes in ASW (O), serum (ml, or serum absorbed with fresh horse red blood cells (a). Agglutination titer is the greatest dilution (2? of serum that agglutinates fresh or fixed horse red blood cells.
FIG. 4. Phagocytosis of unfixed horse and sheep red blood cells by M. mercenariu hemocytes in homologous (home.) serum (i.e., from same clam that donated phagocytes), pooled (pool) serum, or pooled serum heated at 56 or 100°C for 30 min. Artificial seawater (ASW) was the control medium. Agglutination titer (aggl. titer) is the greatest dilution (2”) of M. mercenariu serum that agglutinates fresh horse or sheep red blood cells.
Mercenuriu
HEMOLYMPH
FIG. 5. Phagocytosis of formalin (form.)or glutaraldehyde (glut.)-fixed or trypsinized (tryp.) horse red blood cells in artificial seawater (0) or M. mercenuriu serum cm). Agglutination titer (aggl. titer) is the greatest dilution (2”) of M. mercenuria serum that agglutinates treated horse red blood cells.
agglutinins seem to be similar to lecdescribed from other molluscan species. They are inactivated by heat (70°C) and ion deprivation. Agglutination of some species of RBC is blocked by specific sugars as has been reported in other mollusks (McDade and Tripp, 1967; Arimoto and Tripp, 1977; van der Knaap et al., 1982; Pauley et al., 1971). Partial species specificity is demonstrable by cross-adsorption tests. All of these observations suggest that M. mercenaria hemolymph contains a family of naturally occurring lectins with different specificities whose normal function is not known. Physical or chemical alteration of bacterial surfaces affects their ability to be phagocytosed by clam hemocytes. St. aureus treated with formaldehyde and autoclaved E. coli are very poorly phagocytosed while living cells or those killed in other ways are readily taken in M. mercenaria
tins
:I i
by hemocytes. In contrast, yeast are phagocytosed readily whether alive or killed. There is no correlation between the agglutination reactions of these particles and their ability to be phagocytosed. This is consistent with the idea that agglutinins are not involved in phagocytosis. Two biologically active substances in the hemolymph of M. mercenaria have been reported previously. Agglutination of four gram-negative isolates and a marine alga was reported by Arimoto and Tripp (1977). In that study formalinized E. coli were not agglutinated by M. mercenaria serum but in the present study such cells were agglutinated. The discrepancy may be due to differences in the strain of bacteria used and/or the formalinization procedure used. The second hemolymph factor, a naturally occurring hemolysin, was reported by Anderson (1981). This material was most active against rabbit red blood cells and required divalent cations. In the present study hemolytic activity was not detected either in agglutination assays or by the spectrophotometric assay of Anderson (1981). Different genetic backgrounds of clams, different sources of erythrocytes, or variation in laboratory procedures may account for these contrasting observations. Red blood cell agglutinins with similar heat sensitivity and Ca2+ requirements were reported by Vasta et al. (1980). The hemolymph of M. mercenaria contains lectins that agglutinate red blood cells, bacteria, and yeast. Despite the consistent presence of these materials in hemolymph, they do not facilitate phagocytosis of these particles. This represents one end of a spectrum of molluscan lectin interactions that ranges from total dependence (Prowse and Tait, 1969) through various degrees of enhanced phagocytosis (e.g., Anderson and Good, 1976; Tripp and Kent, 1967; Tripp, 1966; Sminia et al., 1979; Noda and Loker, 1989) to lectin independence for Mercenaria hemocytes. It is becoming clear that molluscan defense mechanisms are complex and that the roles played by phagocytic cells and hemolymph factors vary from species to species. Renwrantz (1983) has reviewed the immunobiological importance of lectins and their possible interactions with hemocytes and other cells (Renwrantz, 1986). As more is learned about molluscan lectins their evolutionary history becomes more complex (Yang and Yoshino, 1990b) and understanding the details of their interactions with phagocytic cells becomes more important. It is clear that “simple” molluscan defense mechanisms are more complex and diverse than has been thought.
-
FIG. 6. Phagocytosis of yeast (S. cerevisiue) and bacteria reus and E. coli) after no treatment (live), formalinization heating at 100°C for 30 min (loo”), or autoclaving (auto.). for phagocytosis was artificial seawater (0) or serum (W).
(St. uu-
(form.), Medium
233
AGGLUTININS
ACKNOWLEDGMENT I thank
L. Fawcett
for technical
assistance.
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