Vol. 14, No. 4 Printed in U.S.A.
INFECTION AND IMMUNITY, Oct. 1976, p. 888-893 Copyright © 1976 American Society for Microbiology
Serum Bactericidal Activity in the Horseshoe Crab, Limulus polyphemus THOMAS G. PISTOLE* AND RITA M. FURMAN' Department of Microbiology, University of New Hampshire, Durham, New Hampshire 03824 Received for publication 26 April 1976
Serum from the horseshoe crab, Limulus polyphemus, was examined for bactericidal activity against five species of bacteria. Greatest activity was found against Pseudomonas putida and Flavobacterium sp.; with the former, serum dilutions as high as 1:20 were capable of reducing viable counts by 50% within 2 h. Bactericidal activity of a significantly lesser magnitude was demonstrated against Serratia marcescens and Salmonella minnesota. No killing was seen when the lobster pathogen Aerococcus viridans (formerly Gaffkya homari) was used. The cidal activity against Flavobacterium sp. remained relatively consistent for 6 months.
Extensive studies on bactericidal systems in dans, 6870 (formally Gaff7tya homari); and Salmoinvertebrates have been conducted in recent nella minnesota. Crabs. Male horseshoe crabs were collected and years. Notable among these are the inducible maintained as previously described (Furman and bactericidins in insects (see reference 4 for re- Pistole, in press). For each experiment, animals view) and in the lobster Homarus americanus were selected from a maintenance population of ap(1), the spiny lobster, Panulirus argus (9), and proximately 200, using random sampling techniques the sipunculid worm, Dendrostomum zosteri- (14). colum (10). Serum. Hemolymph was collected aseptically Serum, but not plasma, obtained from the from the hemocoel, using an 18-gauge hypodermic horseshoe crab, Limulus polyphemus, has been needle. After clotting, it was centrifuged for 20 min shown to possess native bactericidal activity at 675 x g and the clear serum was removed. Samagainst a variety of bacterial species (R. M. ples of serum were stored at 4°C and used within 10 Furman and T. G. Pistole, J. Invertebr. Pa- days. Bactericidal assays. A modification of the method thol., in press). Shirodkar et al. (22) reported of Schwab Reeves (21) was used for determining inhibitory activity against a number of bactericidaland activity (Furman and Pistole, in press). uncharacterized marine bacteria by using Lim- In brief, overnight cultures of the appropriate miulus hemolymph containing intact amebocytes; croorganism were diluted 1:1,000 in Trypticase soy duplicate samples with partially degranulated broth (BBL) and incubated for 4 h at 28°C with cells were considerably less antibacterial. Re- shaking. A 0. 1-ml amount of this culture was added cently, Johanssen et al. (11) described a bacteri- to L. polyphemus serum. Unless otherwise noted, cidal activity against gram-negative but not these serum samples were initially diluted 1:2 in gram-positive bacteria using cell-free hemo- 0.513 M NaCl + 0.05% peptone (Bacto-peptone, Difco). At 30-min intervals for 2 h, 0.02-ml aliquots lymph preparations. plated in duplicate onto Trypticase soy agar The present study was undertaken to charac- were (BBL). After incubation at temperatures appropriterize more fully the bactericidal activity of ate for the various organisms tested (37°C for S. serum from the horseshoe crab. Studies on this minnesota; 28°C for the others), colony counts were and other invertebrate species may provide in- performed. For each experiment, control suspensight into the defense mechanisms of mammals sions of the microorganisms plus diluent but without added serum were examined. On occasion, the including man. number of viable bacteria in the control system either increased or decreased markedly, presumably due in the former instance to growth in the diluent and in the latter to aggregation or nonspecific dieoff. To exclude as much as possible such extraneous influences on the viable cell count, arbitrary limits were applied to the control system: only those experimental systems in which the control plate counts varied within 50 to 200% of the initial count (t = 0) were included in the results.
MATERIALS AND METHODS Bacteria. The bacteria used were obtained from the New Hampshire stock culture collection and included Pseudomonas putida; Flavobacterium sp., ATCC 21048; Serratia marcescens; Aerococcus viriI
Present address: Carney Hospital, Boston, MA 02124.
888
VOL. 14, 1976
BACTERICIDAL ACTIVITY IN THE HORSESHOE CRAB
Analysis of data. The data were expressed as percentage of bacteria surviving at each given time interval and were examined by analysis of variance (2) and, where appropriate, ScheffM's test (14). Graphically, the data are represented as the mean ± standard error of the mean.
889
significant difference in the cidal activity of the various serum dilutions against each microorganism (P < 0.01) as well as over the 2-h assay period (P < 0.01). Furthermore, these differences were constant through the period of the assay (P > 0.10 for the interaction in each case). These results are illustrated in Fig. 6 and 7. It is noteworthy that in the case of cidal activity against Flavobacterium sp. there was a diminution in activity, whereas in the P. putida system the killing effect of L. polyphemus serum reached an apparent plateau at the 1:10 dilution (Fig. 6c) which remained at the 1:20 dilution (Fig. 6d). Bactericidal activity over time. Six consecutive monthly serum samples were collected from the same five crabs from November 1974 to April 1975 and tested for cidal activity against Flavobacterium sp. Statistical analysis
RESULTS Activity against gram-negative bacilli. In this experiment, sera from nine crabs were examined for cidal activity against four bacterial species. Statistical analysis of the results indicated a significant bactericidal activity over the time of the assay, which was 2 h (P < 0.01), and a significant difference in the activity directed at the various microorganisms tested (P < 0.01). The interaction between these two variables was not significant (P > 0.10). Further analyses were performed using Scheff6's test, in which all possible pairs of variable a (bacterial species) at a given level of TABLE 1. Paired analysis of the bactericidal effect of variable b (time) were tested for significant L. polyphemus serum against six bacterial species at differences (Table 1). The cidal response various times using Scheffe's test against P. putida and Flavobacterium sp. was Paired contrasts similar but quantitatively different from that directed against S. marcescens and S. minneS. p pUt.a p. pUt. P. put. Flavob. Flavob. marc. (ime) sota. This difference became greater with time. Fla- vs S. vs S. vs S. vs S. (i)vs The results are depicted graphically in Fig. 1 vob. marc. minn. marc. minn. minn. through 4. In Fig. 1 the greatest activity 0 0 0 0 0 0 30 against P. putida occurred between 30 and 60 0 0 0 * 0 * 60 min. As evidenced by the standard error val0 * * 0 * * 90 ues, the variability in cidal activity among the * * ** ** 0 120 0 sera tested decreased with time. a Abbreviations: P. put., P. putida; Flavob., FlaThe activity of L. polyphemus sera against Flavobacterium sp. (Fig. 2) and P. putida at 2 h vobacterium sp.; S. marc., S. marcescens; S. minn., in each case resulted in approximately a 90% S. bminnesota. 0, Difference not statistically significant (P > reduction in viable organisms. Similar to the * and **, difference significant at P < 0.05 and response seen with P. putida are the diminish- P0.05); < 0.01 levels, respectively. ing standard error values over time for activity against Flavobacterium sp. 100 The cidal activity against both S. marcescens (Fig. 3) and S. minnesota (Fig. 4) was significantly less than that against the first two or80 ganisms, with greater than 50% of the organisms surviving exposure to L. polyphemus serum for 2 h. Activity against A. viridans. In this experi- C,60 G ment, the bactericidal activity of Limulus se- .z rum against the lobster pathogen A. viridans was examined. There was no cidal activity di- i?40 rected toward this organism (Fig. 5); in fact, the .0 large values for the standard error depicted in t 20 the figure reflect the fact that some serum sam- 80 ples promoted the growth of this organism. Bactericidal activity of diluted sera. The 90 120 60 -o 30r two most active systems were selected for furTime (min) ther study. Serum samples diluted 1:2, 1:5, 1:10, FIG. 1. Bactericidal activity of Limulus sera and 1:20 were tested for cidal activity against P. putida and Flavobacterium sp. There was a against Pseudomonas putida.
890
INFECT. IMMUN.
PISTOLE AND FURMAN
.Y a)
(-
m 0140
Time FIG. 2. Bactericidal activity of Limulus against Flavobacterium sp.
sera
lysozyme has been demonstrated in a wide variety of extracts from invertebrates, both marine and terrestrial (25), and may be an important component of the humoral defense mechanism in these animals. Additionally, activities resembling those of terminal complement components have been found in the sera of several marine invertebrates, including L. polyphemus (8). Bactericidal mechanisms have been reported in a wide variety of marine invertebrates including the American lobster, Homarus americanus (1, 5, 19), sipunculid worms (10, 12, 15, 16, 20), the tunicate, Ciona intestinalis (13), and spiny lobsters, Panulirus sp. (9). Although it is generally agreed that structures comparable to vertebrate immunoglobulins are absent in invertebrate sera (3), apart from lysozyme the effector molecules have not been well de-
100 S
80, om60
L Cf
40
at0 a) 0
M 20
a0 30
60
Time (min)
90
FIG. 3. Bactericidal activity of Limulus against Serratia marcescens.
120 sera
FIG. 4. Bactericidal activity of Limulus against Salmonella minnesota.
sera
FIG. 5. Bactericidal activity of Limulus against Gafflkya homari.
sera
of the results indicated that there were significant differences as a function of time (30, 60, 90, and 120 min). No significant difference was found, however, in the repeated monthly experiments, indicating a reasonably consistent response over the 6-month period. These data are shown in Fig. 8. Standard error bars have been omitted for clarity. No significant interaction (P > 0.10) was found between the two variables, time and repeated measures, in this study.
DISCUSSION Internal defense in invertebrate hemolymph may be accomplished by (i) serum bactericidal activity, (ii) agglutinin activity, or (iii) phagocytic activity (24), or a combination of these, e.g., opsonization (18). The mucolytic enzyme
VOL. 14, 1976
BACTERICIDAL ACTIVITY IN THE HORSESHOE CRAB
891
a
Time (nin)
Time (min) FIG. 6. Bactericidal activity of diluted Limulus sera against Pseudomonas putida. Serum dilutions: (a) 1:2; (b) 1:5; (c) 1:10; (d) 1:20.
fined. In fact, there is good reason to believe that quite distinct molecular species may be involved in antimicrobial defense among various invertebrate hosts (25). The results of the present study confirm previous reports of antibacterial activity in L. polyphemus hemolymph or serum (11, 22; Furman and Pistole, in press). Of the five bacterial species examined in this study, all four gramnegative bacilli were inhibited to some extent by one or more serum components. However, the activity directed against P. putida and against Flavobacterium sp. was significantly greater than that against two other gram-negative species, S. marcescens and S. minnesota. Complement-mediated bacteriolysis using vertebrate sera is directed only against gram-negative bacteria (7). Serum from L. polyphemus has been shown to contain complement-like ac-
tivity (8), and this may be involved in the bactericidal activity here reported. A naturally occurring agglutinin for S. minnesota has been found in L. polyphemus serum (Pistole, Abstr. Annu. Meet. Am. Soc. Microbiol. 1974, M61, p. 76; Pistole, J. Invertebr. Pathol., in press). The relationship of this agglutinin to the serum bactericidal activity is not known. The only gram-positive organism tested, A. viridans, is a known pathogen for lobsters (23). No inhibitory activity was found in Limulus serum against this microorganism. Cornick and Stewart have reported similar results in the American lobster, H. americanus (5), and the Atlantic crab, Cancer irroratus (6), where, in each case, the growth of A. viridans was actually stimulated in the presence of serum. It would be of interest to determine whether this
F_.
INFECT. IMMUN.
PISTOLE AND FURMAN
892
a
Time (min)
Time (min)
FIG. 7. Bactericidal activity of diluted Limulus sera against Flavobacterium sp. Serum dilutions: (a) 1:2; (b) 1:5; (c) 1:10; (d) 1:20.
100
organism is pathogenic for L. polyphemus. The cidal activity of Limulus serum is probably independent of the hemocyanin since
plasma (fluid portion of hemolymph with intact cells removed) has no demonstrable activity (Furman and Pistole, in press). The differential cidal activity of serum versus plasma suggests
x 80"
,n60 (6
40 40-
m20 O
%lo
that, like the clotting components, the bactericidal substance or substances are derived from the amebocyte. _ ~~-\ K 4> \ \, _ The data presented indicate that the serum of L. polyphemus possesses bactericidal activity for four species of gram-negative bacilli but not for. the coccus A. viridans, that
INFECTION AND IMMUNITY, Oct. 1976, p. 888-893 Copyright © 1976 American Society for Microbiology
Serum Bactericidal Activity in the Horseshoe Crab, Limulus polyphemus THOMAS G. PISTOLE* AND RITA M. FURMAN' Department of Microbiology, University of New Hampshire, Durham, New Hampshire 03824 Received for publication 26 April 1976
Serum from the horseshoe crab, Limulus polyphemus, was examined for bactericidal activity against five species of bacteria. Greatest activity was found against Pseudomonas putida and Flavobacterium sp.; with the former, serum dilutions as high as 1:20 were capable of reducing viable counts by 50% within 2 h. Bactericidal activity of a significantly lesser magnitude was demonstrated against Serratia marcescens and Salmonella minnesota. No killing was seen when the lobster pathogen Aerococcus viridans (formerly Gaffkya homari) was used. The cidal activity against Flavobacterium sp. remained relatively consistent for 6 months.
Extensive studies on bactericidal systems in dans, 6870 (formally Gaff7tya homari); and Salmoinvertebrates have been conducted in recent nella minnesota. Crabs. Male horseshoe crabs were collected and years. Notable among these are the inducible maintained as previously described (Furman and bactericidins in insects (see reference 4 for re- Pistole, in press). For each experiment, animals view) and in the lobster Homarus americanus were selected from a maintenance population of ap(1), the spiny lobster, Panulirus argus (9), and proximately 200, using random sampling techniques the sipunculid worm, Dendrostomum zosteri- (14). colum (10). Serum. Hemolymph was collected aseptically Serum, but not plasma, obtained from the from the hemocoel, using an 18-gauge hypodermic horseshoe crab, Limulus polyphemus, has been needle. After clotting, it was centrifuged for 20 min shown to possess native bactericidal activity at 675 x g and the clear serum was removed. Samagainst a variety of bacterial species (R. M. ples of serum were stored at 4°C and used within 10 Furman and T. G. Pistole, J. Invertebr. Pa- days. Bactericidal assays. A modification of the method thol., in press). Shirodkar et al. (22) reported of Schwab Reeves (21) was used for determining inhibitory activity against a number of bactericidaland activity (Furman and Pistole, in press). uncharacterized marine bacteria by using Lim- In brief, overnight cultures of the appropriate miulus hemolymph containing intact amebocytes; croorganism were diluted 1:1,000 in Trypticase soy duplicate samples with partially degranulated broth (BBL) and incubated for 4 h at 28°C with cells were considerably less antibacterial. Re- shaking. A 0. 1-ml amount of this culture was added cently, Johanssen et al. (11) described a bacteri- to L. polyphemus serum. Unless otherwise noted, cidal activity against gram-negative but not these serum samples were initially diluted 1:2 in gram-positive bacteria using cell-free hemo- 0.513 M NaCl + 0.05% peptone (Bacto-peptone, Difco). At 30-min intervals for 2 h, 0.02-ml aliquots lymph preparations. plated in duplicate onto Trypticase soy agar The present study was undertaken to charac- were (BBL). After incubation at temperatures appropriterize more fully the bactericidal activity of ate for the various organisms tested (37°C for S. serum from the horseshoe crab. Studies on this minnesota; 28°C for the others), colony counts were and other invertebrate species may provide in- performed. For each experiment, control suspensight into the defense mechanisms of mammals sions of the microorganisms plus diluent but without added serum were examined. On occasion, the including man. number of viable bacteria in the control system either increased or decreased markedly, presumably due in the former instance to growth in the diluent and in the latter to aggregation or nonspecific dieoff. To exclude as much as possible such extraneous influences on the viable cell count, arbitrary limits were applied to the control system: only those experimental systems in which the control plate counts varied within 50 to 200% of the initial count (t = 0) were included in the results.
MATERIALS AND METHODS Bacteria. The bacteria used were obtained from the New Hampshire stock culture collection and included Pseudomonas putida; Flavobacterium sp., ATCC 21048; Serratia marcescens; Aerococcus viriI
Present address: Carney Hospital, Boston, MA 02124.
888
VOL. 14, 1976
BACTERICIDAL ACTIVITY IN THE HORSESHOE CRAB
Analysis of data. The data were expressed as percentage of bacteria surviving at each given time interval and were examined by analysis of variance (2) and, where appropriate, ScheffM's test (14). Graphically, the data are represented as the mean ± standard error of the mean.
889
significant difference in the cidal activity of the various serum dilutions against each microorganism (P < 0.01) as well as over the 2-h assay period (P < 0.01). Furthermore, these differences were constant through the period of the assay (P > 0.10 for the interaction in each case). These results are illustrated in Fig. 6 and 7. It is noteworthy that in the case of cidal activity against Flavobacterium sp. there was a diminution in activity, whereas in the P. putida system the killing effect of L. polyphemus serum reached an apparent plateau at the 1:10 dilution (Fig. 6c) which remained at the 1:20 dilution (Fig. 6d). Bactericidal activity over time. Six consecutive monthly serum samples were collected from the same five crabs from November 1974 to April 1975 and tested for cidal activity against Flavobacterium sp. Statistical analysis
RESULTS Activity against gram-negative bacilli. In this experiment, sera from nine crabs were examined for cidal activity against four bacterial species. Statistical analysis of the results indicated a significant bactericidal activity over the time of the assay, which was 2 h (P < 0.01), and a significant difference in the activity directed at the various microorganisms tested (P < 0.01). The interaction between these two variables was not significant (P > 0.10). Further analyses were performed using Scheff6's test, in which all possible pairs of variable a (bacterial species) at a given level of TABLE 1. Paired analysis of the bactericidal effect of variable b (time) were tested for significant L. polyphemus serum against six bacterial species at differences (Table 1). The cidal response various times using Scheffe's test against P. putida and Flavobacterium sp. was Paired contrasts similar but quantitatively different from that directed against S. marcescens and S. minneS. p pUt.a p. pUt. P. put. Flavob. Flavob. marc. (ime) sota. This difference became greater with time. Fla- vs S. vs S. vs S. vs S. (i)vs The results are depicted graphically in Fig. 1 vob. marc. minn. marc. minn. minn. through 4. In Fig. 1 the greatest activity 0 0 0 0 0 0 30 against P. putida occurred between 30 and 60 0 0 0 * 0 * 60 min. As evidenced by the standard error val0 * * 0 * * 90 ues, the variability in cidal activity among the * * ** ** 0 120 0 sera tested decreased with time. a Abbreviations: P. put., P. putida; Flavob., FlaThe activity of L. polyphemus sera against Flavobacterium sp. (Fig. 2) and P. putida at 2 h vobacterium sp.; S. marc., S. marcescens; S. minn., in each case resulted in approximately a 90% S. bminnesota. 0, Difference not statistically significant (P > reduction in viable organisms. Similar to the * and **, difference significant at P < 0.05 and response seen with P. putida are the diminish- P0.05); < 0.01 levels, respectively. ing standard error values over time for activity against Flavobacterium sp. 100 The cidal activity against both S. marcescens (Fig. 3) and S. minnesota (Fig. 4) was significantly less than that against the first two or80 ganisms, with greater than 50% of the organisms surviving exposure to L. polyphemus serum for 2 h. Activity against A. viridans. In this experi- C,60 G ment, the bactericidal activity of Limulus se- .z rum against the lobster pathogen A. viridans was examined. There was no cidal activity di- i?40 rected toward this organism (Fig. 5); in fact, the .0 large values for the standard error depicted in t 20 the figure reflect the fact that some serum sam- 80 ples promoted the growth of this organism. Bactericidal activity of diluted sera. The 90 120 60 -o 30r two most active systems were selected for furTime (min) ther study. Serum samples diluted 1:2, 1:5, 1:10, FIG. 1. Bactericidal activity of Limulus sera and 1:20 were tested for cidal activity against P. putida and Flavobacterium sp. There was a against Pseudomonas putida.
890
INFECT. IMMUN.
PISTOLE AND FURMAN
.Y a)
(-
m 0140
Time FIG. 2. Bactericidal activity of Limulus against Flavobacterium sp.
sera
lysozyme has been demonstrated in a wide variety of extracts from invertebrates, both marine and terrestrial (25), and may be an important component of the humoral defense mechanism in these animals. Additionally, activities resembling those of terminal complement components have been found in the sera of several marine invertebrates, including L. polyphemus (8). Bactericidal mechanisms have been reported in a wide variety of marine invertebrates including the American lobster, Homarus americanus (1, 5, 19), sipunculid worms (10, 12, 15, 16, 20), the tunicate, Ciona intestinalis (13), and spiny lobsters, Panulirus sp. (9). Although it is generally agreed that structures comparable to vertebrate immunoglobulins are absent in invertebrate sera (3), apart from lysozyme the effector molecules have not been well de-
100 S
80, om60
L Cf
40
at0 a) 0
M 20
a0 30
60
Time (min)
90
FIG. 3. Bactericidal activity of Limulus against Serratia marcescens.
120 sera
FIG. 4. Bactericidal activity of Limulus against Salmonella minnesota.
sera
FIG. 5. Bactericidal activity of Limulus against Gafflkya homari.
sera
of the results indicated that there were significant differences as a function of time (30, 60, 90, and 120 min). No significant difference was found, however, in the repeated monthly experiments, indicating a reasonably consistent response over the 6-month period. These data are shown in Fig. 8. Standard error bars have been omitted for clarity. No significant interaction (P > 0.10) was found between the two variables, time and repeated measures, in this study.
DISCUSSION Internal defense in invertebrate hemolymph may be accomplished by (i) serum bactericidal activity, (ii) agglutinin activity, or (iii) phagocytic activity (24), or a combination of these, e.g., opsonization (18). The mucolytic enzyme
VOL. 14, 1976
BACTERICIDAL ACTIVITY IN THE HORSESHOE CRAB
891
a
Time (nin)
Time (min) FIG. 6. Bactericidal activity of diluted Limulus sera against Pseudomonas putida. Serum dilutions: (a) 1:2; (b) 1:5; (c) 1:10; (d) 1:20.
fined. In fact, there is good reason to believe that quite distinct molecular species may be involved in antimicrobial defense among various invertebrate hosts (25). The results of the present study confirm previous reports of antibacterial activity in L. polyphemus hemolymph or serum (11, 22; Furman and Pistole, in press). Of the five bacterial species examined in this study, all four gramnegative bacilli were inhibited to some extent by one or more serum components. However, the activity directed against P. putida and against Flavobacterium sp. was significantly greater than that against two other gram-negative species, S. marcescens and S. minnesota. Complement-mediated bacteriolysis using vertebrate sera is directed only against gram-negative bacteria (7). Serum from L. polyphemus has been shown to contain complement-like ac-
tivity (8), and this may be involved in the bactericidal activity here reported. A naturally occurring agglutinin for S. minnesota has been found in L. polyphemus serum (Pistole, Abstr. Annu. Meet. Am. Soc. Microbiol. 1974, M61, p. 76; Pistole, J. Invertebr. Pathol., in press). The relationship of this agglutinin to the serum bactericidal activity is not known. The only gram-positive organism tested, A. viridans, is a known pathogen for lobsters (23). No inhibitory activity was found in Limulus serum against this microorganism. Cornick and Stewart have reported similar results in the American lobster, H. americanus (5), and the Atlantic crab, Cancer irroratus (6), where, in each case, the growth of A. viridans was actually stimulated in the presence of serum. It would be of interest to determine whether this
F_.
INFECT. IMMUN.
PISTOLE AND FURMAN
892
a
Time (min)
Time (min)
FIG. 7. Bactericidal activity of diluted Limulus sera against Flavobacterium sp. Serum dilutions: (a) 1:2; (b) 1:5; (c) 1:10; (d) 1:20.
100
organism is pathogenic for L. polyphemus. The cidal activity of Limulus serum is probably independent of the hemocyanin since
plasma (fluid portion of hemolymph with intact cells removed) has no demonstrable activity (Furman and Pistole, in press). The differential cidal activity of serum versus plasma suggests
x 80"
,n60 (6
40 40-
m20 O
%lo
that, like the clotting components, the bactericidal substance or substances are derived from the amebocyte. _ ~~-\ K 4> \ \, _ The data presented indicate that the serum of L. polyphemus possesses bactericidal activity for four species of gram-negative bacilli but not for. the coccus A. viridans, that
