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Effect of zymosan on phagocytosis in larvae of the greater wax moth, Galleria mellonella
JOURNAL
OF INVERTEBRATE
Effect of Zymosan
PATHOLOGY
10,
(1968)
176-179
on Phagocytosis Galleria
in Larvae mellonella
of the Greater
Wax Moth,
JOHN C. HAR~HBARGER L 2 AND ARTHUR M. HEIMPEL Entomology Research Division, U. S. Department of Agriwlture, Received
Agricultural Reltsville,
February
Research Maryland
Service, 20705
2, 1967
Larvae of the greater wax moth, Galleria mellonella, were more susceptible to Although the infection by bacteria after intrahemocoelic injections of zymosan. zymosan particles were rapidly phagocytized, efficiency of the phagocytes was not impaired. Polysterene latex particles were also rapidly phagocytized but neither caused the serum to melanize (as did zymosan) or increased susceptibility. The possibility that zymosan may cause the removal of substances that inhibit bacteria is discussed.
However, because of its insoluble nature, zymosan itself is probably phagocytized by
INTRODUCTION
ZYmosan is an insoluble Carbohydrate from yeast that causes insect blood to melanize (Stephens, 1962). When it was injected into the hemocoel, larvae of the silkworm, Bombyx mori, became susceptible to infection by a normally avirulent strain of Escherichia COG (Briggs, 1958). Bullock ( 1963) electrophoretically compared blood of Galleria mellonella larvae darkened by injections of zymosan with blood from controls and found less protein in the melanized serum; he suggested that plasma proteins may either react directly with foreign substances or may act as opsonins by coating the foreign substance and thereby increasing the efficiency of the phagocytes. In either case, less protein would be available in the serum after melanization for defense against pathogens. 1 This research was partially supported by U.S. Public Health Service Grant No. 5358 (to E. A. Steinhaus ) from the National Institute of Allergy and Infectious Diseases, while the senior author was at the University of ‘California, Irvine, California 92664. s Present Address: Registry of Tumors in Lower Animals, Smithsonian Institution, Washington, D. C. 20560. 176
hemocytes.
The
question
is
then
whether
the hemocytes might become so packed with particles of zymosan that their effectiveness in engulfing and destroying bacteria would be significantly reduced. MATERIALS,
METHODS,
AND RESULTS
The experimental animals were last-instar larvae of the greater wax moth, G. mellonda, reared according to Dutky et al. ( 1962). The experimental materials were injected through the prolegs following CO, anesthesia by using a Dutky-Fest (1942) microinjector, a l-ml Beckton-Dickinson tuberculin syringe, and a 30-gauge needle. The injected volume was always 3.57 ~1. The zymosan suspension was prepared by mixing 100 mg in 1 ml of sterile distilled water in a test tube. Before use, the mixture was agitated 30 set by a vortex mixer; within 1 hr after the injection, the larvae became dark. At 1 hr after this injection, bacteria from 24-hr-old thioglycollate cultures were injected as suspensionsin sterile distilled water. To determine whether particles of zymosari were phagocytized by blood cells, we
EFFECT
OF
ZYMOSAN
ON
PHAGOCYTOSIS
Srst injected five larvae intrahemocoelically with zymosan. This dose rate (approx 1.785 mg/g of body wt ) was considerably higher than that employed by Briggs (approx 0.2457 mg/g) but fell within the range of doses reported by Bullock (0.25-125 mg/g ). One hour after the injection, a blood smear was made from each larva and stained with Wright’s Giemsa stain. Microscopic examination revealed that the 3 X 5~ particles had been phagocytized by 83.6% + 5.8% of the blood cells. The susceptibility of G. meZZoneZZa to infection by Pseudomonas aeruginosa and E. coli after treatment with zymosan was tested as follows: For each species of bacteria, two groups of larvae were injected intrahemocoelically, one with water and the other with zymosan. Then after 1 hr, as noted, each larva was injected with bacteria. Table 1 shows that susceptibility to both species of bacteria was increased after treatment with zymosan. In larvae challenged by P. aeruginosu, the time of death was accelerated at least 20%. In larvae challenged by E. coli, 50% of the larvae receiving injections of zymosan died within
‘TABLE SUSCEPTIBILITY AFTER
Treatment Zymosan + P. aerffginosa Zymosan + P. aerugifwsa Water + P. aeruginosa Water + P. aeruginosa Zymosan + E. coli Water + E. coli Zymosan + water Latex (5 X lo7 particles) + water Latex (5 X lo7 particles) + E. coli (2 X 108) a One died at 23 hr. b None died in 24 hr.
IN
AN
177
INSECT
24 hr compared with only 5% of those that received water injections. In contrast, initial injection of a particulate material that did not cause the blood to melanize had no effect on susceptibility. Twenty larvae were each injected with 50 million biological inert polysterene latex particles ( 1.305 p in diameter) obtained from the Dow Chemical Company. These particles were readily phagocytized, but when the larvae were challenged 1 hr later by E. coli, no deaths occurred in 24 hr (Table 1). To determine whether phagocytes could still function efficiently after gorging themselves with zymosan particles, we divided 48 larvae into two equal groups. One group was injected with the zymosan suspension and the other group with water. Each larva was reinjected 1 hr later with 5 million polysterene latex particles. After one more hour, blood smears were made and stained with Wright’s Giemsa. Under phase-contrast microscopy, the latex particles contrasted vividly with the blood cells (Fig. 1)) but actual counts of the number of latex particles in the first 100 cells in
1
OF G. mellonelk TO P. aeruginosa AND E. coli PREVIOUS TREATMENT WITH ZYMOSAN
Replication I
Bacterial antial
cells
No. of animals
LT50
(h)
I I I
100 100 100 100 2 x 108 2 x 10s -
10 10 10 10 20 20 20
12 10 15 14 24 -n -a
I
-
20
-b
I
-
20
-b
II I II
178
HARSHBARGER
AND
HEIMPEL
to exist. Berheimer et al. ( 1952), Briggs ( 1958), and Stephens ( 1959) (see also review article by Briggs, 1964) were unable to demonstrate the existence in insects of agglutinins, precipitins, and complement to a variety of antigens. An indirect relationship between removal of serum proteins by melanization after treatment with zymosan and susceptibility to bacterial challenge is therefore indicated. An attractive possibility is that nonprotein bacterial inhibitory materials are removed from the blood. EviFIG. 1. Phase-contrast photomicrograph showdence is accumulating that such materials ing oval zymosan (Z) granules and small spherical contribute significantly to immunity in latex (L) particles inside a phagocyte. insects. For example, Briggs (1958) each smear showed no difference between demonstrated a relatively nonspecific bacterial inhibitory principle in five species of the two groups. In insects treated with zymosan for 1 hr, the number of phago- lepidopteran larvae that increased after cytized latex particles was 4.8 -+ 2.3/tell. vaccination with any of several species of In the controls, the number was 4.8 -+ 1.9/ bacteria (in a sixth species, B. mori, baccell. The quantity of cells appeared the terial inhibition occurred only after vacThe increase corresponded to same for both treatments, based on their cination). proximity to one another in the smears. an increased tolerance for the bacteria. Stephens and Marshall (1962) described a DISCUSSION AND CONCLUSIONS nonprotein bactericidal principle in larvae Polysterene latex particles are easier to of G. mellonella immunized with P. am@use than bacteria in studying phagocytosis. nosa. P. aeruginosa also stimulated OncopAlso, they give more reliable quantitative teltus fasciatus to produce “nonprotein lytic data because they can be readily identified substances” first detectable at 4 hr (Ginginside the phagocyte and do not cause rich, 1964) ; these lytic materials were assoclumping of blood cells as frequently as do ciated with, and seemed to be attached to, bacteria. Because the phagocytosis of the several proteins in electrophoretically fraclatex particles was unaffected by previous tionated serum. treatment of the insects with zymosan, Although the insects used in our study blood cells are not inactivated by the level were not vaccinated before they were of zymosan used, and opsonins, if present, challenged with living bacteria, sufficient are not removed. bacterial inhibitory substances may have Since the biologically inert polysterene been present in the controls to retard latex particles had no effect on the sus- growth of the bacterial population. The ceptibility of larvae to bacterial infection treatment with zymosan and the resultant though zymosan, which removed serum melanization of the blood could have preproteins by stimulating the hemolymph cipitated proteins vital to the normal functo melanize, increased susceptibility, a re- tion of these inhibitory substances or delationship exists between proteins and im- pleted enzymes necessary for their synmunity as Bullock ( 1963) suggested. Howthesis. If either possibility pertains, inever, a direct relationship between serum sects would be more susceptible to bacterial proteins and foreign materials is not known infection after melanization.
EFFECT
OF
ZYMOSAN
ON
PHAGOCYTOSIS
REFERENCES BERNHEIMER, A. W., CASPERI, E., AND KAISER, A. D. 1952. Studies on antibody formation in caterpillars. .I. Exptl. Zool., 119, 23-25. BRIGGS, J. T. 1958. Humoral immunity in lepidopterous larvae. J. Exptl. ZooI., 138, 135-188. BRIMS, J. T. 1964. Immunological responses. In “The Physiology of Insecta,” (Morris Rockstein, ed.) Vol. III, pp. 259-283. Academic Press, New York. BULLOCK, H. R. 1963. A study of hemolymph proteins of insects in relation to melanization and natural defense against microorganisms. Ph. D. thesis, 160 pp. DUTKY, S. R., AND FEST, W. C. 1942. Microinjector. U. S. patent 2,270,804.
IN
AN
INSECT
179
DUTKY, S. R., THOMPSON, J. V., AND CANTWELL, G. E. 1962. A technique for mass rearing the greater wax moth ( Lepidoptera: Galleridae) . Proc. Entomol. Sot. Wash., 64, 56-58. GINGRICH, R. E. 1964. Acquired humoral immune response of the large milkweed bug, Oncopeltus fusciutus (Dallas), to injected materials. J. Insect Physiol., lo, 179-194. STEPHENS, J. M. 1959. Immune responses of some bacterial antigens. Can. J. Microbial., 5, 20% 228. STEPHENS, J. M. 1962. Bactericidal activity of the blood of actively immunized wax moth larvae. Can. J. Microobiol., 8, 491499. STEPHENS, J. M., AND MARSHALL, J. H. 1962. Some properties of an immune factor isolated from the blood of actively immunized wax moth larvae. Can. I. Microbial., 8, 719-725.
OF INVERTEBRATE
Effect of Zymosan
PATHOLOGY
10,
(1968)
176-179
on Phagocytosis Galleria
in Larvae mellonella
of the Greater
Wax Moth,
JOHN C. HAR~HBARGER L 2 AND ARTHUR M. HEIMPEL Entomology Research Division, U. S. Department of Agriwlture, Received
Agricultural Reltsville,
February
Research Maryland
Service, 20705
2, 1967
Larvae of the greater wax moth, Galleria mellonella, were more susceptible to Although the infection by bacteria after intrahemocoelic injections of zymosan. zymosan particles were rapidly phagocytized, efficiency of the phagocytes was not impaired. Polysterene latex particles were also rapidly phagocytized but neither caused the serum to melanize (as did zymosan) or increased susceptibility. The possibility that zymosan may cause the removal of substances that inhibit bacteria is discussed.
However, because of its insoluble nature, zymosan itself is probably phagocytized by
INTRODUCTION
ZYmosan is an insoluble Carbohydrate from yeast that causes insect blood to melanize (Stephens, 1962). When it was injected into the hemocoel, larvae of the silkworm, Bombyx mori, became susceptible to infection by a normally avirulent strain of Escherichia COG (Briggs, 1958). Bullock ( 1963) electrophoretically compared blood of Galleria mellonella larvae darkened by injections of zymosan with blood from controls and found less protein in the melanized serum; he suggested that plasma proteins may either react directly with foreign substances or may act as opsonins by coating the foreign substance and thereby increasing the efficiency of the phagocytes. In either case, less protein would be available in the serum after melanization for defense against pathogens. 1 This research was partially supported by U.S. Public Health Service Grant No. 5358 (to E. A. Steinhaus ) from the National Institute of Allergy and Infectious Diseases, while the senior author was at the University of ‘California, Irvine, California 92664. s Present Address: Registry of Tumors in Lower Animals, Smithsonian Institution, Washington, D. C. 20560. 176
hemocytes.
The
question
is
then
whether
the hemocytes might become so packed with particles of zymosan that their effectiveness in engulfing and destroying bacteria would be significantly reduced. MATERIALS,
METHODS,
AND RESULTS
The experimental animals were last-instar larvae of the greater wax moth, G. mellonda, reared according to Dutky et al. ( 1962). The experimental materials were injected through the prolegs following CO, anesthesia by using a Dutky-Fest (1942) microinjector, a l-ml Beckton-Dickinson tuberculin syringe, and a 30-gauge needle. The injected volume was always 3.57 ~1. The zymosan suspension was prepared by mixing 100 mg in 1 ml of sterile distilled water in a test tube. Before use, the mixture was agitated 30 set by a vortex mixer; within 1 hr after the injection, the larvae became dark. At 1 hr after this injection, bacteria from 24-hr-old thioglycollate cultures were injected as suspensionsin sterile distilled water. To determine whether particles of zymosari were phagocytized by blood cells, we
EFFECT
OF
ZYMOSAN
ON
PHAGOCYTOSIS
Srst injected five larvae intrahemocoelically with zymosan. This dose rate (approx 1.785 mg/g of body wt ) was considerably higher than that employed by Briggs (approx 0.2457 mg/g) but fell within the range of doses reported by Bullock (0.25-125 mg/g ). One hour after the injection, a blood smear was made from each larva and stained with Wright’s Giemsa stain. Microscopic examination revealed that the 3 X 5~ particles had been phagocytized by 83.6% + 5.8% of the blood cells. The susceptibility of G. meZZoneZZa to infection by Pseudomonas aeruginosa and E. coli after treatment with zymosan was tested as follows: For each species of bacteria, two groups of larvae were injected intrahemocoelically, one with water and the other with zymosan. Then after 1 hr, as noted, each larva was injected with bacteria. Table 1 shows that susceptibility to both species of bacteria was increased after treatment with zymosan. In larvae challenged by P. aeruginosu, the time of death was accelerated at least 20%. In larvae challenged by E. coli, 50% of the larvae receiving injections of zymosan died within
‘TABLE SUSCEPTIBILITY AFTER
Treatment Zymosan + P. aerffginosa Zymosan + P. aerugifwsa Water + P. aeruginosa Water + P. aeruginosa Zymosan + E. coli Water + E. coli Zymosan + water Latex (5 X lo7 particles) + water Latex (5 X lo7 particles) + E. coli (2 X 108) a One died at 23 hr. b None died in 24 hr.
IN
AN
177
INSECT
24 hr compared with only 5% of those that received water injections. In contrast, initial injection of a particulate material that did not cause the blood to melanize had no effect on susceptibility. Twenty larvae were each injected with 50 million biological inert polysterene latex particles ( 1.305 p in diameter) obtained from the Dow Chemical Company. These particles were readily phagocytized, but when the larvae were challenged 1 hr later by E. coli, no deaths occurred in 24 hr (Table 1). To determine whether phagocytes could still function efficiently after gorging themselves with zymosan particles, we divided 48 larvae into two equal groups. One group was injected with the zymosan suspension and the other group with water. Each larva was reinjected 1 hr later with 5 million polysterene latex particles. After one more hour, blood smears were made and stained with Wright’s Giemsa. Under phase-contrast microscopy, the latex particles contrasted vividly with the blood cells (Fig. 1)) but actual counts of the number of latex particles in the first 100 cells in
1
OF G. mellonelk TO P. aeruginosa AND E. coli PREVIOUS TREATMENT WITH ZYMOSAN
Replication I
Bacterial antial
cells
No. of animals
LT50
(h)
I I I
100 100 100 100 2 x 108 2 x 10s -
10 10 10 10 20 20 20
12 10 15 14 24 -n -a
I
-
20
-b
I
-
20
-b
II I II
178
HARSHBARGER
AND
HEIMPEL
to exist. Berheimer et al. ( 1952), Briggs ( 1958), and Stephens ( 1959) (see also review article by Briggs, 1964) were unable to demonstrate the existence in insects of agglutinins, precipitins, and complement to a variety of antigens. An indirect relationship between removal of serum proteins by melanization after treatment with zymosan and susceptibility to bacterial challenge is therefore indicated. An attractive possibility is that nonprotein bacterial inhibitory materials are removed from the blood. EviFIG. 1. Phase-contrast photomicrograph showdence is accumulating that such materials ing oval zymosan (Z) granules and small spherical contribute significantly to immunity in latex (L) particles inside a phagocyte. insects. For example, Briggs (1958) each smear showed no difference between demonstrated a relatively nonspecific bacterial inhibitory principle in five species of the two groups. In insects treated with zymosan for 1 hr, the number of phago- lepidopteran larvae that increased after cytized latex particles was 4.8 -+ 2.3/tell. vaccination with any of several species of In the controls, the number was 4.8 -+ 1.9/ bacteria (in a sixth species, B. mori, baccell. The quantity of cells appeared the terial inhibition occurred only after vacThe increase corresponded to same for both treatments, based on their cination). proximity to one another in the smears. an increased tolerance for the bacteria. Stephens and Marshall (1962) described a DISCUSSION AND CONCLUSIONS nonprotein bactericidal principle in larvae Polysterene latex particles are easier to of G. mellonella immunized with P. am@use than bacteria in studying phagocytosis. nosa. P. aeruginosa also stimulated OncopAlso, they give more reliable quantitative teltus fasciatus to produce “nonprotein lytic data because they can be readily identified substances” first detectable at 4 hr (Ginginside the phagocyte and do not cause rich, 1964) ; these lytic materials were assoclumping of blood cells as frequently as do ciated with, and seemed to be attached to, bacteria. Because the phagocytosis of the several proteins in electrophoretically fraclatex particles was unaffected by previous tionated serum. treatment of the insects with zymosan, Although the insects used in our study blood cells are not inactivated by the level were not vaccinated before they were of zymosan used, and opsonins, if present, challenged with living bacteria, sufficient are not removed. bacterial inhibitory substances may have Since the biologically inert polysterene been present in the controls to retard latex particles had no effect on the sus- growth of the bacterial population. The ceptibility of larvae to bacterial infection treatment with zymosan and the resultant though zymosan, which removed serum melanization of the blood could have preproteins by stimulating the hemolymph cipitated proteins vital to the normal functo melanize, increased susceptibility, a re- tion of these inhibitory substances or delationship exists between proteins and im- pleted enzymes necessary for their synmunity as Bullock ( 1963) suggested. Howthesis. If either possibility pertains, inever, a direct relationship between serum sects would be more susceptible to bacterial proteins and foreign materials is not known infection after melanization.
EFFECT
OF
ZYMOSAN
ON
PHAGOCYTOSIS
REFERENCES BERNHEIMER, A. W., CASPERI, E., AND KAISER, A. D. 1952. Studies on antibody formation in caterpillars. .I. Exptl. Zool., 119, 23-25. BRIGGS, J. T. 1958. Humoral immunity in lepidopterous larvae. J. Exptl. ZooI., 138, 135-188. BRIMS, J. T. 1964. Immunological responses. In “The Physiology of Insecta,” (Morris Rockstein, ed.) Vol. III, pp. 259-283. Academic Press, New York. BULLOCK, H. R. 1963. A study of hemolymph proteins of insects in relation to melanization and natural defense against microorganisms. Ph. D. thesis, 160 pp. DUTKY, S. R., AND FEST, W. C. 1942. Microinjector. U. S. patent 2,270,804.
IN
AN
INSECT
179
DUTKY, S. R., THOMPSON, J. V., AND CANTWELL, G. E. 1962. A technique for mass rearing the greater wax moth ( Lepidoptera: Galleridae) . Proc. Entomol. Sot. Wash., 64, 56-58. GINGRICH, R. E. 1964. Acquired humoral immune response of the large milkweed bug, Oncopeltus fusciutus (Dallas), to injected materials. J. Insect Physiol., lo, 179-194. STEPHENS, J. M. 1959. Immune responses of some bacterial antigens. Can. J. Microbial., 5, 20% 228. STEPHENS, J. M. 1962. Bactericidal activity of the blood of actively immunized wax moth larvae. Can. J. Microobiol., 8, 491499. STEPHENS, J. M., AND MARSHALL, J. H. 1962. Some properties of an immune factor isolated from the blood of actively immunized wax moth larvae. Can. I. Microbial., 8, 719-725.