Immunological relationships between the parasite Cardiochiles nigriceps Vierick and certain Heliothis species

J. Insect Physiol., 1968, Vol. 14, pp. 613 to 626. Pergamon Press. Printed in Great Britain

IMMUNOLOGICAL RELATIONSHIPS BETWEEN THE PARASITE CARLUOCHILES NIGRICEPS VIERICK AND CERTAIN HELIOTHIS SPECIES* W. J. LEWISt Department of Entomology,

and S. BBADLEIGH

VINSON

Mississippi Agriculture Experiment Station, State College (Receiwed 11 December 1967)

Abstract-Heliothis oirescens (F.) made no apparent defensive reaction against the braconid parasite Cardiochilesnigriceps Vierick. The parasite was found to be encapsulated by haemocytes in H. zea (Boddie). The interrelationships between C. nigriceps and each of the Heliothis species were found to be such that the parasite possesses some passive characteristic by which it did not initiate a humoral response in H. virescens but did initiate this humoral response in H. zea. The presence of this humoral response resulted in encapsulation by haemocytes in H. zea and the absence of this humoral response was demonstrated as the reason for the parasite not being encapsulated in H. vimscens. The principle of heterophile antigens is suggested as an explanation for the differing responses of these two HeZiothis species to this parasite. INTRODUCTION

THREE Heliothis species H. zeu (Boddie), H. wirescens (F.), and H. subpexa (Guen.), occur in the southeastern United States. LEWIS and BRAZZEL(1966) demonstrated that a braconid parasitoid, Cardiochiles nz&iceps Vier., would. oviposit in both H. zea and H. virescens in the laboratory and in nature. The egg successfully developed, however, only in H. virescens. LEWIS et al. (1967) showed that H. subflexa was also a host for this parasitoid. These authors pointed out that the relationship between C. nigficeps and these Heliothis species was of special interest in that HARDWICK(1965) has placed H. xeu in a separate genus from H. wirescens and H. subjlexa. Testing the ability of this parasite to develop in other Heliothis species may be of help in evaluating Hardwick’s splitting of the genus He&this. The study reported herein was undertaken to determine the interrelationships between C. nipiceps and H. virescens and H. zea which result in H. virescens being susceptible and H. zeu being immune to the development of this parasite. Insect immunity is still poorly understood. Humoral defence reactions by insects to micro-organisms have been demonstrated by several authors (BRIGGS, 1958; STEPHENS, 1959, 1962; GINGRICH, 1964), but the mechanisms involved in such reactions have gone virtually unexplained. * Publication No. 1505, Mississippi Agricultural Experiment Station, in co-operation with the Agricultural Research Service, United States Department of Agriculture. Supported in part by Cooperative Agreement No. 12-14-100-8411(33). t Present address, Southern Grain Insects Research Laboratory, ARS, USDA, Tifton, Georgia 31794. 613

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W. J. LEWISANDS. BRADLEIGH VINSON

SALT (1963) has made an extensive review of defensive reactions of insects to metazoan parasites. He shows that the reaction made by haemocytes is the only primary defence reaction made by insects against internal metazoan parasites. He distinguished four such reactions by haemocytes: (1) phagocytosis, in which small particles are engulfed by amoebocytes; (2) segregation, in which small particles are collected and stored within fixed cells, such as the pericardial cells; (3) nodule formation, in which haemocytes, sometimes already containing particles in their cytoplasm, aggregate about clumps of bacilli or other small particles and so prevent their dispersion; (4) encapsulation, in which haemocytes collect and form a capsule about a foreign body too large to be engulfed by a single cell. The mechanisms of these reactions and whether or not a humoral response is involved have not been shown. MATERIALS AND METHODS General Heliothis larvae were laboratory reared on the conventional Heliothis media described by BRAZZELet al. (1961) and modified by BEHGER(1963). Adult female parasites used in the study were for the most part collected flying around tobacco plants, although some were laboratory reared. Field collected, the adult parasites were placed in small cages 2 x 15 x 1.5 ft until ready for use. Food was supplied in the form of a honey-and-water mixture absorbed in cotton plugs. All studies were conducted at approximately 80°F. Oviposition by the parasitoid was induced by placing the female in a vial with a Heliothis larva and tapping the female into contact with the larva until oviposition was observed. Dissections verified that actual oviposition occurred under these conditions. Parasite eggs for the experiment were obtained by superparasitizing third instar donor larvae and dissecting the eggs from the donor larvae immediately following ovipositions. The dissecting procedure described by LEWIS and BRAZZEL(1966) was used. Artificial injections of the parasite eggs and brush bristle into the Heliothis larvae were made with a small capillary pipette just large enough for the inoculation material to pass. A small amount of saline was used as a carrier solution. Injections into the haemocoele were always made through one of the posterior abdominal prolegs. The brush bristle chips were cut from a test-tube brush and were about 0.15 mm long and 0.05 mm wide. Ink injections were made by a tuberculin syringe fitted with a 27-gauge needle and mounted on a microapplicator. These injections were made through the integument into the body cavity. About 1.2 ~1 of ink were delivered to each individual. I?lectrophoresik Cellulose acetate electrophoresis was used for studying the proteins of the haemolymph. Haemolymph for electrophoresis was collected by making a small incision in the dorsal integument of the larvae and collecting it as it exuded in a

IMhWNOLoGICAL RELATIONSHIPS BETwBEN PARAsITB ANDHOSTS

615

watch-glass containing a few granules of phenylthiourea. The haemolymph from four to six individuals was combined and applied to 25 x 15.9 cm cellulose acetate strips (Sepraphore III, Gelman Instrument Company), using a standard applicator. Electrophoreais was conducted for a period of 45 min at 250 V with a veronal buffer (pH 8.6, 0.07 M). Following electrophoresis, the strips were immediately stained in Nigrosin for about 2 hr. Agar gel d$ksion

The double diffusion (Ouchterlony) technique was used to test for the possibility of a specific antibody response by H. zea to the egg of C. nigrkeps. Eggs (antigen) used in the immunodiffusion were prepared by dissecting the ovaries from several females and placing them in a small amount of distilled water and grinding them thoroughly in a fine tissue grinder. This material was then placed in the agar wells by pipette. The antiserum was prepared by parasitizing fourth instar H. zea five to ten times and holding the larvae for 24 hr. The haemolymph (antiserum) was then collected by making a small incision in the dorsal integument of the larvae and allowing the haemolymph to exude into the designated agar well. The haemolymph from several of these larvae was combined in each well. A few granules of phenylthiourea were placed in the wells with the haemolymph to prevent melanization. The prepared plates were incubated in a dark place for about a week before examination for precipitin. Histochemkal

Parasite eggs were prepared for staining by placing them in distilled water for a few minutes to cause inflation of the chorion. This procedure allows a clear view of the chorion after staining. Whole mounts of eggs were then fixed by placing them in Carnoy’s fixative for about 15 min. The eggs were then stained for lipids with Sudan Black B and for proteins with ninhydrin as described by HUMA~ON 1 ‘s reagent was used according to the procedure described by (1962). S c h’ff CASSELMAN(1959) to test for carbohydrates. Antiserum to eggs C. nigriceps eggs were injected into a chicken for the purpose of obtaining antibodies to the eggs. A 7 per cent suspension, v/v, of the parasite eggs in saline was used for the injection. An intravenous injection of 1 ml of suspension was made into the radial vein of the wing. One week subsequent to the injection 10 ml of blood were collected from the bird by a cardiac puncture. The serum was then collected from the blood sample by allowing the blood to coagulate for 2 or 3 hr, spinning the coagulant down in a cold centrifuge, and collecting the supemate.

RESULTS Description of deface reactions of Heliothis species to the parasite and foreign particles

A large number of parasitized H. virescem and H. zea larvae were dissected at various intervals after oviposition, and the parasites were examined to determine

616

W. J. LEWIS ANDS. BRADLEIGHVINSON

responses of the host to the parasite and to describe these responses. An occasional cluster of haemocytes attached to a few first instar parasite larvae was the only apparent defence reaction observed in H. wkescens. Haemocytes were found to always encapsulate the parasite in H. zea larvae. There was a considerable amount of variation in the time required for encapsulation to begin. In second instar and older H. zea larvae, at least 4-6 hr from oviposition was always required. Encapsulation of the egg had taken place in approximately 80 per cent of the cases after 24 hr (Fig. 1). In a few cases the parasite was

1

100

so

2

4

6

8 Number

IO

12

hours after

14

16

18

20

22

24

oviposition

FIG. 1. Per cent C. nigriceps eggs encapsulated in H. sea intervals.

larvae at different time

observed to reach the first instar before being encapsulated. No parasite was ever observed to develop beyond the early first instar in H. mu. When early first instar H. zea were parasitized, encapsulation did not begin until late in this instar or early in the second instar, often resulting in the parasite reaching the early larval stage before being encapsulated. A number of first instar H. zea larvae were parasitized, a single egg to each individual, and dissected 24 hr later. A parasite egg was recovered from nine of these larvae. There was slight encapsulation on only three of the eggs, these three eggs being from late first instar H. xeu larvae. A number of H. zeu larvae were parasitized during the first instar and dissected three days later. Heavily encapsulated first instar parasites were found in several of the larvae, demonstrating that these parasites had reached the first instar before being encapsulated. These data indicate that the immune mechanism does not begin functioning efficiently during the early first instar.

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617

Beginning with the second instar, encapsulation follows the pattern shown in Fig. 2. The manner in which encapsulation took place varied. Encapsulation began uniformly over the egg in some cases whereas in others it began at one or more spots and spread over the entire egg. The capsule rapidly increased in thickness once encapsulation began and took on the same general form as that described for other parasites and as summarized by SALT (1963).

IO0

so P 80 ‘ii

20 70 I?

SW 550 0)

f 40 s 6

30 20 IO

2

4

6

14 16 16 6 IO 12 Number hours ofter oviposition

20

22

24

FIG. 2. Compariaona of the time required for encapsulation of C. ni~iceps egga by H. zeu at different periods in the moulting cycle.

One possible reason for the wide range of variation in the time required for encapsulation may be connected to the moulting cycle of H. zeu larvae. The time required for encapsulation then would depend on the stage in the moulting cycle of the parasitized larvae. .To test for this possibility, a number of H. zeu early in the fourth instar and a number late in the third instar were parasitized and the eggs dissected from them at various intervals and examined. The percentage of eggs encapsulated by each stage at various time intervals was determined. Fig. 2 shows that there was no apparent difference in the time required for these two stages to react to the egg. The time required for each stage varied considerably. One possible reason that H. zea is able to encapsulate the egg whereas H. virescenslacks this ability is that H. zea has a better overall ability to react to and encapsulate foreign particles in the body. To test for this possibility, chips of brush bristles were injected into the larvae of both species. These chips were dissected out at various intervals and examined. Both species always responded to 40

618

W. J. LEWIS AND

S. BRADLEIGHVINSON

these

bristles very quickly and encapsulation was always clearly under way within O-5 hr. These data eliminate the possibility that the H. zea encapsulates eggs of C. @r+r% because of a better overall encapsulation ability. The parasite’s mechanism of avoiciing encapsulation

Since foreign particles in the body of HeZiothis larvae are generally encapsulated, it is evident that a successful endoparasite must have some means of avoiding this process. SALT (1965) h as shown that the ichneumonid parasite, Nemeritis canescens (Grav.), avoided encapsulation by some character of the outer surface of the egg, which was obtained as the egg passed through the calix. In order to determine the type of mechanism possessed by C. n&riceps that adapts it for avoidance of encapsulation in H. virescens, eggs were altered by various means and injected into H. oirescens larvae. These eggs were later dissected from the host to determine their fate. Physical alterations. A number of C. ntg/iceps eggs were thoroughly abraded with sand, injected singly into H. virescens larvae, and dissected out 6-10 hr later. Six of the eggs were recovered. Five of them showed no signs of encapsulation. One was encapsulated, but it was one that had been allowed to set in saline for 2 or 3 hr before being injected into H. virescens and was probably unhealthy. A number of eggs were thoroughly abraded with fine iron filings. The iron filings stuck to the egg and could not be cleaned off, so eggs coated with these filings were injected singly into H. wirescens larvae. Three of the eggs were recovered 6 hr after injection into H. oirescens. They were all clean. Not only was there no capsule but all of the filings had been removed from the eggs and encapsulated by haemocytes. Several eggs of C. nQriceps were cut in half or one end chipped off. These portions were injected into H. oirescens larvae-two or three per larva. Later they were dissected out and examined. Three were recovered 6 hr after injection and two were recovered 24 hr after injection. There was no evidence of encapsulation on any of them. Physiological alterations. A number of eggs were heat-treated by placing the eggs in saline in a test-tube and holding the test-tube in a water-bath raised to 60°C for 2 min. These heat-treated, non-viable eggs were injected singly into H. virescens larvae. The eggs were dissected out 6-10 hr after injection. Three of the four eggs recovered were heavily encapsulated. Eggs were treated with chemicals which would render them non-viable without causing major chemical alterations. Several eggs were treated with sodium cyanide or chlorohydrate. A few granules of the appropriate chemical were placed into saline near the eggs and allowed to set for 2 min. The eggs then were transferred to fresh saline and rinsed. These eggs were injected one egg per individual into H. virescens larvae and dissected out 5 hr later. Three eggs treated with sodium cyanide and two of the eggs treated with chlorohydrate were recovered. All five were encapsulated.

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Active or passive character. An experiment was conducted to test whether or not the egg of C. nigriceps actively repelled the haemocytes of H. virescen.sor whether their immunity to encapsulation was passive. Ovarioles containing eggs were dissected from the adult female and injected into H. virescens larvae. The host larvae were dissected several hours later and the parasiteeggs were examined. The walls of the ovariole were encapsulated. Eggs that were free of the ovariole or were protruding from the ovariole were clean. Also, as mentioned earlier, haemocytes in H. virescens were found to move in and encapsulateiron filings adhering to the surface of the egg, but would not encapsulatethe egg. These data indicate that the egg does not actively repel the haemocytes. Opsonization with chicken antiserum. Eggs were treated with antiserum from a chicken previously injected with a suspension of parasite eggs to test whether H. virescens can encapsulate C. nigriceps eggs when they have been opsonized by antibodies to the eggs. A number of eggs were exposed to the antiserum in a watch-glass for 2 hr, rinsed in saline, injected into H. virescens, and dissected out 6 hr later. Twelve of the eggs thus treated were recovered, of which three were encapsulated. Other eggs were exposed to the antiserum for 25 hr and left in H. virescens larvae for a period of 7 hr. Of the eggs treated in this manner, five were recovered of which three were encapsulated. As a control, eggs were treated with serum from chickens which had not been exposed to C. nigriceps eggs. Several eggs were treated with the control serum for 2 hr and exposed to H. virescens for 6 hr. Nine of the eggs treated in this manner were recovered. None of them were encapsulated. Other eggs were exposed to the control serum for 2.5 hr and even allowed to remain in H. virescens for 8 hr. Seven of these eggs were recovered and all of them were clean. These data indicate that the antiserumcontained specific antibodies to the eggs, which altered or opsonized the eggs, thereby filling the missing link to enable H. virescem to encapsulate the eggs. Encapsulation of the parasite in H. zea

Experiments were conducted in an attempt to determine the factors in H. zea that result in encapsulation. Taking into consideration the delayed encapsulation of the egg in H. zea as compared to the quick encapsulation of the brush bristle chips, there are four possible processes that may be takingplace : (1) the egg undergoes no change and the haemocytes slowly respond to it as a foreign materialin its original state; (2) the egg is actively altered in H. sea by humor-almechanism and is then recognized by the haemocytes as being foreign; (3) the egg goes through certain changes during development which causes it to become susceptible to encapsulation in H. zea; (4) there is no active alterationof the egg by H. zea, but the egg soon becomes unhealthy in this species because of the lack of certain critical requirements and causes haemocytes to act on the egg. Eggs which were allowed to develop in H. virescens to the stage where they were usually encapsulatedin H. zea were found still to require severalhours before encapsulation began, thus eliminating the thiid possibility.

620

W.J.

LBWIS ANDS. BRADLRIGH

VINSON

Several H. zea larvae were injected with India ink 24 hr prior to parasitization. In these larvae encapsulation was delayed much more than normally. A parasite egg was recovered from each of eight larvae dissected 24 hr after parasitization. All of these eggs were clean except for a small patch of haemocytes on one egg. Seven of the ink-treated H. zea were dissected 2-3 days following parasitixation. The parasite was recovered from five of these larvae. All of these parasites reached the first instar. One was clean, one was heavily encapsulated, and the others were partially encapsulated. The clean one and those partially encapsulated were active and healthy looking. These data indicate that the parasite does not become unhealthy prior to encapsulation and thus eliminates the fourth possibility. The question then becomes whether H. zea haemocytes can eventually respond to the egg as a foreign material in its original state or whether H. xea possesses some mechanism for actively altering the egg to make it susceptible to encapsulation by haemocytes. To determine which of these is the case, a number of H. zea larvae were parasitized and the eggs dissected from them at various intervals and transferred to H. wikescens. An effort was made to include some eggs which had been in H. zea long enough for any changes to take place but just before encapsulation had begun. Since there is so much variation in the time required for encapsulation to begin in H. zea, eggs at this stage could not be picked readily. From Table 1 it can be seen that several eggs which were left in H. zea for a period of time and were clean when dissected from H. zea were encapsulated in H. oirescens. The table clearly shows that those left in H. zea for a longer period

TABLE 1--RBSULTSOFBNCAPSULATIONOFC.nigricepsE~WHICHWeRBLEFTLNH. INDICATED PERIOD AND THRNTRANSFRRRRD TO H. Ui~.?s~c?tU Period egg was in H. zea before transfer @r) 2 4 10 20 24

ZeUFOR

No. of eggs recovered and condition after S-10 hr in H. virescens 3 6 3 1 1

clean clean; clean; clean; clean;

1 1 3 1

encapsulated encapsulated encapsulated encapsulated

All eggs were clean when transferred.

of time ,were more often encapsulated when transferred to H. oirescens. Eggs which were being encapsulated when dissected from H. zea and were cleaned and then transferred to H. virescem were always encapsulated. When encapsulation had begun at certain places on the egg in H. zea, the encapsulation tended to take place at these same places in H. virescens. The data demonstrate that H. xea possesses some mechanism, probably a humoral reaction, lacking in H. oirescens for altering the egg to make it susceptible

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to encapsulation by haemocytes. Once this alteration has occurred, H. vi~escens can also encapsulate the egg. Acquired immunity. To further demonstrate the humoral response prior to encapsulation, and to determine if the humoral response was a specific reaction, as an antibody reaction produced in response to the presence of the parasite egg, an experiment was conducted to determine if H. SW larvae which had been previously exposed to the C. rn&‘ceps egg could encapsulate the eggs faster. A series of third instar H. aeu larvae were parasitized with three or four parasite eggs and held for a 24 hr period. At the end of this period each larva was again parasitized with a single egg. These larvae were then dissected at various intervals and the younger egg examined for encapsulation. In these larvae there was a 10-20 per cent increase at each time interval in the percentage of the younger eggs on which encapsulation had begun as compared to eggs in larvae without a prior exposure to the parasite egg (Table 2). TABLE 2--INCREASE

IN THE INCIDENCR

OF ENCAPSULATION

OF c.

nigriC@S

EGGS IN

H. Zea

LARVAJi WHICH I-IAVJ3HAD A PREVIOUS EXPOSURE TO THE EGGS

Per cent encapsulated Hours after oviposition

Initial exposure

Larvae with prior exposure

4 6 10 20

8 26 43 68

27 47 57 87

Passike transfm of immunity. Attempts were made to transfer this immunity from parasitized H. zea to H. viresceru via transfer of haemolymph. H. zea larvae were parasitized with three or four eggs and held for 24 hr. At this time haemolymph was transferred from these individuals to H. virescens by inserting a micropipette into H. zeu, collecting the pipette full of haemolymph, and inserting this haemolymph into H. virescens larvae. The H. virescens were then parasitized with eggs. The eggs were dissected from H. virescens 24 hr later and examined. There was no encapsulation of eggs found at the end of this period. Although many of the H. virescexs were still alive at the end of the 24 hr, they were all unhealthy. Electrophoresis. In an effort to determine what this humoral response consisted of, comparative electrophoresis was conducted on the haemolymph of normal and parasitized H. zea larvae. A series of early fourth instar H. zeu larvae were parasitized with four or five eggs and held for 24 hr. At the end of this period electrophoresis was conducted on the haemolymph of the parasitized and nonparasitized larvae of similar size. No apparent difference could be detected in the electrophoretic pattern of the parasitized and non-parasitized larvae.

622

W. J. LEWISANDS. BRADLEIGH VINSON

Agar gel u%@sion. Ouchterlony diffusion was run with haemolymph from parasitized H. zea versus a homogenate of C. nigriceps eggs obtained from the ovaries of adult female parasites. The plates were prepared from 4, 6, 8, and 10 per cent saline solutions in an attempt to ensure that the electrolyte concentration was correct. The haemolymph was from H. zea larvae that had been parasitized for 24 hr. All of the Ouchterlony plates yielded negative results. Hi&chemical staining. Comparative stainings of normal and encapsulated eggs, or eggs just before encapsulation, were made in an effort to determine what type of alteration H. zea was making on the egg to condition it for encapsulation. Stainings of whole mounts of the eggs were made in a search for changes on the surface of the chorion. The ninhydrin test for protein and S&ii’s stain for carbohydrates were both negative on the chorion of both normal and encapsulated eggs. There was no detectable difference in the chorion of normal and encapsulated eggs when stained for lipids by Sudan Black B. DISCUSSION The data presented demonstrate that C. najyiceps is adapted to H. virescens to the extent that it develops with practically no apparent inhibiting defensive reactions on the part of the host. Though H. zea fits all of the other host requirements, the last link of the host selection chain is broken in that H. zea larvae are unsuitable for development of the parasite (LEWIS and BRAZZEL,1966). The observations reported herein show that the parasite is encapsulated by haemocytes in H. zeu. Apparently due to some quantitative factor, the time required for this encapsulation to take place varied considerably. In the second instar and older H. zeu larvae, the time varied from 3 or 4 hr after oviposition to much longer periods of time in which the parasite occasionally reached the early first instar. The immune mechanism did not appear to begin functioning efficiently until late in the first instar of H. zeu or early in the second instar, as the parasite often reached the early first larval stage before encapsulation in H. zeu parasitized during the early first instar. H. zea and H. virescem larvae immediately encapsulated chips of brush bristles. These reactions demonstrate that both species possess a strong ability to react to and encapsulate foreign particles. The fact that C. nigriceps is able to survive in H. virescens but not in H. zeu is apparently not due to a difference in the overall Instead the interrelationships between ability to react to foreign particles. C. nz&%ps and each of the Heliothis species is such that the parasite can successfully avoid this defence mechanism in H. virescens but not in H. zeu. The encapsulation of the parasite in H. zeu began much later than did the encapsulation of the brush bristles, which indicates that there is at first a factor or condition present which gives the egg some degree of immunity in H. zeu. This immunity is eventually lost in H. zeu but persists in H. virescem. Abrading or cutting of the egg did not result in the egg being susceptible to encapsulation in H. virescens. These data demonstrate that the means by which the

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parasite avoids encapsulation in this species is not by virtue of the physical nature of the surface of the chorion as suggested by SALT (1965) as being the means by which Nemeritis canescens avoided encapsulation in Ephestzk kuehnida. Furthermore, the factor is distributed throughout the egg rather than being confined to the surface of the egg as was the case demonstrated by Salt. Rendering eggs of Cl ++z+ non-viable with heat or treating the ege with antiserum from egginoculated chickens does result in the eggs being susceptible to encapsulation. The fact that the haemocytes will move in and encapsulate the walls of the ovariole with eggs just inside, and the fact that the haemocytes will pick off and encapsulate iron filings adhering to the surface of the egg, indicates that the egg’s means of avoiding encapsulation in H. virescens is a passive characteristic of the egg rather than an active characteristic, such as actively repelling the haemocytes. One would expect that the means by which the parasite delays encapsulation in H. zecr is similar to the character by which they avoid encapsulation in H. virescens. The ability of H. zeu to overcome this character would seem to be attributable to either of two causes. Either H. zeu is capable of changing the egg by some means so that it no longer possesses the character or, else, the egg remains unchanged and the delayed encapsulation is because of slowness in haemocytes to react to the egg as a foreign material. Eggs which were allowed to remain in H. zea for a number of hours and transferred to H. virescens before encapsulation began were often encapsulated in H. ti~escens. These data indicate that a humoral reaction occurs prior to encapsulation, which alters the egg in such a way that it becomes susceptible to encapsulation, even in H. virescetrs. The absence of this humoral reaction and not the lack of encapsulation ability allows the survival of the parasite in II. tiescens. The fact that treating the eggs with chicken antiserum fills the missing link necessary for encapsulation in H. virescens tends to confirm this conclusion. There was a 10-20 per cent increase in the incidence of encapsulation at each given time interval in larvae which had had a previous exposure to the parasite eggs. This previous contact with the eggs must have caused them to acquire an increased immunity to the egg. These data further demonstrate a humoral response prior to the encapsulation of the egg and indicate that the humoral response is a specific reaction in response to the presence of the parasite egg. Apparently the initial exposure to the eggs resulted in the production of the humoral substance, which is then already present upon the introduction of the next egg and, consequently, results in quicker encapsulation of this egg. Attempts to passively transfer this substance from parasitized H. zeu to H. virescens larvae failed. There are a number of possible reasons for this failure. There may have been incompatibility between the haemolymph of these species, resulting in secondary effects. The concentration of the humoral material may have been diluted too much when transferred into H. virescerzs. Also the health of H. virescens may have been affected too much as a result of the injection injury and thereby caused the in v&o environment to be unsuitable for the humoral material to carry out its function.

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Comparative

electrophoresis

of the haemolymph of normal and parasitized this humoral substance as a protein. However, cellulose acetate electrophoresis only demonstrates the more concentrated proteins. The humoral material produced by H. zea against C. nz&L+s may be in too small an amount to be detected by cellulose acetate. Further studies with the more sensitive disk electrophoresis would be worth while. Comparative staining of encapsulated egg and eggs not encapsulated failed to demonstrate an opsonin on the surface of the encapsulated eggs or on eggs just before encapsulation. These stainings were not conclusive, however, and further such stainings with a wide variety of stains and staining of sectioned material may be more fruitful. Testing of haemolymph from parasitized H. zeu vs. egg homogenates by the double-diffusion (Ouchterlony) method yielded negative results. These data indicate that the humoral material will not precipitate with the antigen under the experimental conditions used, as do mammalian antibodies. These results were not surprising, however, as several other investigations have had similar results (BRIGGS, 1958; STEPHENS, 1959). Also it is very possible that the eggs were not homogenated fine enough to diffuse well in the agar plates. Much more data must be collected before a complete explanation can be given for the mechanism by which C. nigriceps avoids the defence reactions in H. virescens and the means by which H. xeu is immune to the parasite. However, a conjecture may be offered at this point. What seems to be a very plausible explanation can be offered on the basis of ‘heterophile antigens’, similar to the explanation suggested by ROWLEYand JENKIN (1962) for the means by which certain bacteria were adapted for survival in mice. Heterophile antigens* can be defined as antigens, ‘. which are sunilar enough in some respect, that antibodies to one can cross-react with others. In order for a host to make a defensive reaction against a parasite, they must have some means of recognizing the parasite as a foreigner and distinct from ‘self’. Recent studies in vertebrate immunology have indicated that a humoral response of antibody or some other type of opsin is required for recognition by phagocytic cells of all that is foreign, including effete and damaged selfcomponents. If this argument is accepted, then a host would be unable to encapsulate a parasite if there was an insufficient humoral response to the antigens of the parasite. On the other hand, an animal with the ability to initiate a humoral response of suflicient magnitude to antigens of the parasite would be capable of encapsulating the parasite. From what has been said about heterophile antigens it is obvious that such antigens closely related to host antigens may not elicit a humoral response when presented in an animal because antibodies to them will also act against antigens of the host. Parasites which possess such antigens in the proper quantities and groupings on the surface which come into intimate contact with the body fluids and

H. zea failed to demonstrate

* Readers are referred to JENKIN (1963) for an excellent discussion of heterophile antigens.

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AND HOSTS

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tissue of the host would not stimulate humoral reactions. Consequently, phagocytosis or encapsulation of the parasite could not take place. Under such conditions, host responses would be quantitative rather than ‘all or none’. The quantitative relationship(s) of heterophile antigen(s) to other surface antigens would determine the rate and magnitude to which the parasite is reacted. The present data indicate that heterophile antigens may well explain the differing immunological relationships of C. nz&iceps with H. uiresce12s and C. n@ceps with H. zeu. If such is the case, C. nigricepseggs and larvae possess antigens which are similar in certain determinant groups to antigens possessed by H. virescens. These antigens are present in the parasite in sufficient quantity and location so that H. virescens larvae are unable to make sufficient humoral reactions and, consequently, are unable to encapsulate the parasite. These antigens must be distributed throughout the chorion and on the surface of the embryonic cells, as abrading or cutting of the egg does not elicit encapsulation. On the other hand, the parasite does not possess such antigens thus related to antigens of H. zea in sufficient quantity, or in the proper location. Consequently, H. zea larvae respond to the parasite with humoral reactions sufficient in magnitude to condition it for encapsulation. The parasite does possess some such antigens, however, in that the encapsulation is delayed for several hours, Such an explanation would also be plausible for the relationship between Nemeritis canescens and Ephestia kuehniella reported by SALT (1965). In this case, however, the heterophile antigens of the parasite would appear to be confined to the outer surface of the chorion as abrading of the chorion elicited encapsulation. AcKnowledgernext-The authors are grateful to Dr. BRUCE GLICK of the Department of Poultry Husbandry, Mississippi Agriculture Experiment Station, for important suggestions and advice, and use of certain facilities during this study. REFERENCES BURGERR. S. (1963) Laboratory techniques for rearing Heliothis species on artificial medium. U.S.D.A., ARS-33-84, October. BRAZZELJ. R., CHAMBBR~ H., and HAMMONDP. J. (1961) A laboratory rearing method and dosage mortality data on the bollworm, Heliothis zeu. J. econ. Ent. 64, 949-952. BRIGCSJ. D. (1958) Humoral immunity in lepidopterous larvae. J. exp. Zool. 138, 155-188. CA~~ELMAN W. G. B. (1959) Histochemicul Technique. John Wiley, New York. GINGRICHR. E. (1964) Acquired humoral response of the large milkweed bug, Oncopeltus &c&us (Dallas), to inject materials. J. Insect Physiol. 10, 179-194. HARDWICKD. F. (1965) The corn earworm complex. Mem. ent. Sot. Can. 40, 247 pp. HUMASONG. L. (1962) Animal Tissue Technique. W. H. Freeman, San Francisco. JENKIN C. R. (1963) Heterophile antigens and their significance in the host-parasite relationship. In Advances in Immunology (Ed. by DIXON F. J., JR. and HUMPHREYJ. H.) 3, 351-376. Academic Press, New York. LEWIS W. J. and BRAZZELJ. R. (1966) Biological relationships between Curdiochiles nigriceps and the Heliothis complex. J. econ. Ent. 59, 820-823. LEWIS W. J., BRAZZELJ. R., and VINSONS. B. (1967) Heiiothis subflexa, a host for Curdiochiles nigriceps. J. econ. Ent. 69, 615-616.

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ROWLEY D. and JENKIN C. R. (1962) Antigenic cross-reaction between host and parasite as a possible cause of pathogen&y. Nowe, L.ond. 193,151-154. SALT G. (1963) The defense reactioti of insects to metazoan parasites. Para.ddogy 53, 527#2. SALT G. (1965) Experimental studies in insecf parasitism-XIII. The haemocytic reaction of a caterpillar to eggs in its habitual parasite. PYOC.R. Sot. (B) lsS, 303-319. STBPHBNSJ. M. (1959) Immune responses of some insects to some bacterial antigens. Can. g. Mkrobiol. 5,203-228. STEPHENSJ. M. (1962) Bactericidal activity of the blood of actively immunized wax moth larvae. Can. g. iWcrobio1. 8,491-499.