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Chemicals affecting the encapsulation of foreign material in an insect
JOURNAL
OF
INVERTEBRdTE
Chemicals
PATHOLOGY
18,
Affecting Material
287-289
(1071)
the
Encapsulation in
The immune mechanism in invertebrates is an intriguing problem. A hemocytic reaction resulting in the formation of a capsule has been considered the primary defense reaction of insects against metazoan parasites while melanin deposition has been considered a secondary development (G. Salt, Parasitology 53, 527-528, 1963). G. Salt (Proc. Roy. Sot. Ser. B, 162, 303-318, 1965) reported that melanin deposition on the encapsulated egg of the ichneumonid Nemeritis canescens could be prevented by phenylthiourea, but he did not investigate the effect of phenylthiourea on the initiation of egg encapsulation. Attempts to block encapsulation by the iniection of parasite homogenates, suscept.ible host blood, or repeated parasitization were unsuccessful (CT. Salt, Biol. Rev. Cambridge Phil. Sot. 43, 200-232, 1960). Inhibition of the defensive reaction in Drosophila melalzogaster by a substance injected by the female parasite Pseudocoila bochei has been reported (F. A. Streams and L. Greenberg, J. Invert&r. Pathol. 13, 371-377, 1969). It has recently been proposed that a major act’ive role for the polyphenol-phenoloxidase system in invertebrates might be one of immunity (R. L. Taylor, J. Invertebr. Pathol. 14, 427428, 1969). W. J. Lewis and S. B. Vinson (J. Insect Physiol. 14, 613-626, 1968) reported the complete encapsulation of eggs of the parasite Cardiochiles nigriceps when oviposit,ed within Heliothis zea larvae. This present investigation was carried out to determine whether certain chemicals that inhibit melanin formation might block the encapsulation reaction against foreign materials. To determine the effect of selected chemicals on the encapsulation of parasite eggs, early fourth-instar H. zea larvae were nar-
of Foreign
an Insect’ cotized with CO2 and injected intrahemocoelically between the head and cervical shield wit,h 2 ~1 of one of the following chemicals: distilled water, reduced glutathione (0.1 g/ml distilled water), a saturated aqueous solution of 1-phenyl-Zthiourea (PTU), 2 % aqueous solution of disodium ethylenediaminetetraacetate (EDTA), 2,4dinitrophenol (0.001 g/ml distilled water), and corn oil. One group of larvae were injected with 4 ~1 of reduced glutathione solution. Chemical injected and control larvae were subjected to parasitism by C. nigriceps 1 hr following inject)ions. A second series of experiments employed small 1 mm nylon threads which were surface sterilized \\.ibh alcohol, dried, and mechanically inserted into the hemocoel through a small puncture between the head and cervical shield of na,rcotized larvae. One hour after the introduction of the nylon thread the larvae were narcotized and injected with one of the chemicals. Both groups of larvae were held at 28°C and dissected 22-24 hr after the final manipulation. Parasitized control larvae were dissected at various intervals to determine the encapsulation rate. In parasit#ized noninjected H. zea larvae, 50% of C. xigriceps eggs were encapsulated in 12 hr while 95% egg encapsulation was observed in 24 hr (Fig. 1). I-I. zea larvae subjected to induced physical injury (via sterile forcep punctures) did not show a detectable difference in the encapsulation rate when compared to controls. H. xea larvae that had been injected with reduced glutathione and PTU displayed the greatest number of nonencapsulated eggs and nylon threads 22-24 hr after injection (Table 1). It is of interest to note that these two chemicals are known inhibitors of mela-
287
288
NOTES
nin formation. These results st,rongly support the suggestion that the polyphenolphenoloxidase system is involved in invertebrate immunity (R. L. Taylor, J. Invertebr.
0
1
4
6
*
10 Hovrr
FIG. 1. The chiles nigriceps instar Heliothis
12
14
16
,I
20
22
24
after oriposi,~ion
rate of encapsulation eggs by hemocytes zea.
of Cardioin early fourth
Pathol., 14, 427-488, 1969) and suggest that melanin formation may either precede or accompany (or both) the encapsulation process. The high percentage (70%) of nonencapsulated parasite eggs observed from larvae injected with 2,4-dinitrophenol may be due to this chemical’s action as a respiratory inhibitor. The reaggregation of dissociated pupal fat body cells by plasmatocytes of saturniid moths has been reported blocked when 2,4dinitrophenol was added to the culture (D. R. Walters and C. M. Williams, Science 154, 516-517, 1966). Injections of EDTA, a known chelator of divalent cations, also reduced encapsulation when compared to larvae receiving distilled water. These results suggest that divalent cations may play a role in the encapsulation process, e.g., rearrangement. or redistribution of ionic charges upon cellular membranes or alien surfaces which could reduce plasmato-
TABLE ENCAPSULATION INSTAR
Controls
OF Cardiochiles nigriceps EGGS Heliothis zea LARVAE FOLLOWING
Chemical treatment
Foreign object
(noninjected)
J&g
1 AND NYLON THREADS WITHII\’ EARLY FOURTH THE INJECTION OF CERTAIN CHEMICALS
Number of larvae used
% Encapsulated
“; Nonencapsulated
No. of hours in host
Thread
20 10
95 100
5 0
24 24
2 pl distilled
water
Egg Thread
29 11
79 73
21 27
24 24
2 pl reduced
glutathione
Egg Thread
22 13
10 15
90 85
24 24
4 ~1 reduced
glutathione
Egg
27
0
100
23
Egg Thread
29 14
0 21
100 79
24 24
Egg Thread
19 10
68.5 50
2 /Al PTU” 2 ~1 2% disodium
EDTAb
2 ~1 2,4-dinitrophenol 2 ~1 corn
oil
Shams0 a 1-Phenyl-2 thiourea b Ethylenediaminetet,raacetic c Physically induced
22 24
Egg
18
30.0
70.0
23
Egg Thread
12 5
GG.6 67
33.4 33
24 24
Thread
5
0
24
(saturated
solution). acid.
injury.
31.5 50
100
289
NOTES
cyte attachment. Distilled water, although having the least effect’, did reduce encapsulation. Distilled water, as EDTA, may have upset the ionic balance in the host. Corn oil also reduced encapsulation. In larvae cont.aining nonencapsulated eggs, it was observed that the corn oil droplet had not dispersed and was surrounded by a melanized capsule. The production of melanin around the corn oil droplet may have reduced the level of tyrosine cont’aining proteins or polyphenols in the hemolymph. Such proteins or polyphenols may be necessary for opsonizing the alien surface, thus preparing the surface for attachment of the hemocytes. H. R. Bullock (Diss. Abstr. 24, 5499, 1963) showed that melanization removed hemolymph proteins and was detrimental to insect resistance to bacterial infection. Eggs of C. nigriceps when oviposited in H. zea become “sticky” before any evidence of hemocytes on the surface can be
seen. However, eggs oviposited in H. virestens, where encapsulation does not occur are not sticky (S. B. Vinson, unpublished). Eggs from H. zea injected with PTU or reduced glutathione were not sticky, indicating the possible absence of an opsonin. This study was supported in part by ARS Cooperative Agreement No. 12-14-100-10, 359 and conducted in cooperation with the Entomology Research Division, U.S.D.A. It was approved for publication as TA 8997 by the Director, Texas Agricultural Experiment Station. FRANKLIN
D. BREWER
Department of Biochemistry Piississippi State University State College,Mississippi S. BUDLEIGH Departmevd of Entomology Texas A & M University CollegeStation, Texas 778&Y ReceivedFebruary 27, 197.1
VINSON
OF
INVERTEBRdTE
Chemicals
PATHOLOGY
18,
Affecting Material
287-289
(1071)
the
Encapsulation in
The immune mechanism in invertebrates is an intriguing problem. A hemocytic reaction resulting in the formation of a capsule has been considered the primary defense reaction of insects against metazoan parasites while melanin deposition has been considered a secondary development (G. Salt, Parasitology 53, 527-528, 1963). G. Salt (Proc. Roy. Sot. Ser. B, 162, 303-318, 1965) reported that melanin deposition on the encapsulated egg of the ichneumonid Nemeritis canescens could be prevented by phenylthiourea, but he did not investigate the effect of phenylthiourea on the initiation of egg encapsulation. Attempts to block encapsulation by the iniection of parasite homogenates, suscept.ible host blood, or repeated parasitization were unsuccessful (CT. Salt, Biol. Rev. Cambridge Phil. Sot. 43, 200-232, 1960). Inhibition of the defensive reaction in Drosophila melalzogaster by a substance injected by the female parasite Pseudocoila bochei has been reported (F. A. Streams and L. Greenberg, J. Invert&r. Pathol. 13, 371-377, 1969). It has recently been proposed that a major act’ive role for the polyphenol-phenoloxidase system in invertebrates might be one of immunity (R. L. Taylor, J. Invertebr. Pathol. 14, 427428, 1969). W. J. Lewis and S. B. Vinson (J. Insect Physiol. 14, 613-626, 1968) reported the complete encapsulation of eggs of the parasite Cardiochiles nigriceps when oviposit,ed within Heliothis zea larvae. This present investigation was carried out to determine whether certain chemicals that inhibit melanin formation might block the encapsulation reaction against foreign materials. To determine the effect of selected chemicals on the encapsulation of parasite eggs, early fourth-instar H. zea larvae were nar-
of Foreign
an Insect’ cotized with CO2 and injected intrahemocoelically between the head and cervical shield wit,h 2 ~1 of one of the following chemicals: distilled water, reduced glutathione (0.1 g/ml distilled water), a saturated aqueous solution of 1-phenyl-Zthiourea (PTU), 2 % aqueous solution of disodium ethylenediaminetetraacetate (EDTA), 2,4dinitrophenol (0.001 g/ml distilled water), and corn oil. One group of larvae were injected with 4 ~1 of reduced glutathione solution. Chemical injected and control larvae were subjected to parasitism by C. nigriceps 1 hr following inject)ions. A second series of experiments employed small 1 mm nylon threads which were surface sterilized \\.ibh alcohol, dried, and mechanically inserted into the hemocoel through a small puncture between the head and cervical shield of na,rcotized larvae. One hour after the introduction of the nylon thread the larvae were narcotized and injected with one of the chemicals. Both groups of larvae were held at 28°C and dissected 22-24 hr after the final manipulation. Parasitized control larvae were dissected at various intervals to determine the encapsulation rate. In parasit#ized noninjected H. zea larvae, 50% of C. xigriceps eggs were encapsulated in 12 hr while 95% egg encapsulation was observed in 24 hr (Fig. 1). I-I. zea larvae subjected to induced physical injury (via sterile forcep punctures) did not show a detectable difference in the encapsulation rate when compared to controls. H. xea larvae that had been injected with reduced glutathione and PTU displayed the greatest number of nonencapsulated eggs and nylon threads 22-24 hr after injection (Table 1). It is of interest to note that these two chemicals are known inhibitors of mela-
287
288
NOTES
nin formation. These results st,rongly support the suggestion that the polyphenolphenoloxidase system is involved in invertebrate immunity (R. L. Taylor, J. Invertebr.
0
1
4
6
*
10 Hovrr
FIG. 1. The chiles nigriceps instar Heliothis
12
14
16
,I
20
22
24
after oriposi,~ion
rate of encapsulation eggs by hemocytes zea.
of Cardioin early fourth
Pathol., 14, 427-488, 1969) and suggest that melanin formation may either precede or accompany (or both) the encapsulation process. The high percentage (70%) of nonencapsulated parasite eggs observed from larvae injected with 2,4-dinitrophenol may be due to this chemical’s action as a respiratory inhibitor. The reaggregation of dissociated pupal fat body cells by plasmatocytes of saturniid moths has been reported blocked when 2,4dinitrophenol was added to the culture (D. R. Walters and C. M. Williams, Science 154, 516-517, 1966). Injections of EDTA, a known chelator of divalent cations, also reduced encapsulation when compared to larvae receiving distilled water. These results suggest that divalent cations may play a role in the encapsulation process, e.g., rearrangement. or redistribution of ionic charges upon cellular membranes or alien surfaces which could reduce plasmato-
TABLE ENCAPSULATION INSTAR
Controls
OF Cardiochiles nigriceps EGGS Heliothis zea LARVAE FOLLOWING
Chemical treatment
Foreign object
(noninjected)
J&g
1 AND NYLON THREADS WITHII\’ EARLY FOURTH THE INJECTION OF CERTAIN CHEMICALS
Number of larvae used
% Encapsulated
“; Nonencapsulated
No. of hours in host
Thread
20 10
95 100
5 0
24 24
2 pl distilled
water
Egg Thread
29 11
79 73
21 27
24 24
2 pl reduced
glutathione
Egg Thread
22 13
10 15
90 85
24 24
4 ~1 reduced
glutathione
Egg
27
0
100
23
Egg Thread
29 14
0 21
100 79
24 24
Egg Thread
19 10
68.5 50
2 /Al PTU” 2 ~1 2% disodium
EDTAb
2 ~1 2,4-dinitrophenol 2 ~1 corn
oil
Shams0 a 1-Phenyl-2 thiourea b Ethylenediaminetet,raacetic c Physically induced
22 24
Egg
18
30.0
70.0
23
Egg Thread
12 5
GG.6 67
33.4 33
24 24
Thread
5
0
24
(saturated
solution). acid.
injury.
31.5 50
100
289
NOTES
cyte attachment. Distilled water, although having the least effect’, did reduce encapsulation. Distilled water, as EDTA, may have upset the ionic balance in the host. Corn oil also reduced encapsulation. In larvae cont.aining nonencapsulated eggs, it was observed that the corn oil droplet had not dispersed and was surrounded by a melanized capsule. The production of melanin around the corn oil droplet may have reduced the level of tyrosine cont’aining proteins or polyphenols in the hemolymph. Such proteins or polyphenols may be necessary for opsonizing the alien surface, thus preparing the surface for attachment of the hemocytes. H. R. Bullock (Diss. Abstr. 24, 5499, 1963) showed that melanization removed hemolymph proteins and was detrimental to insect resistance to bacterial infection. Eggs of C. nigriceps when oviposited in H. zea become “sticky” before any evidence of hemocytes on the surface can be
seen. However, eggs oviposited in H. virestens, where encapsulation does not occur are not sticky (S. B. Vinson, unpublished). Eggs from H. zea injected with PTU or reduced glutathione were not sticky, indicating the possible absence of an opsonin. This study was supported in part by ARS Cooperative Agreement No. 12-14-100-10, 359 and conducted in cooperation with the Entomology Research Division, U.S.D.A. It was approved for publication as TA 8997 by the Director, Texas Agricultural Experiment Station. FRANKLIN
D. BREWER
Department of Biochemistry Piississippi State University State College,Mississippi S. BUDLEIGH Departmevd of Entomology Texas A & M University CollegeStation, Texas 778&Y ReceivedFebruary 27, 197.1
VINSON