Insect Biochem. Vol. 19, No. 5, pp. 445-455, 1989 0020-1790/89 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1989 Pergamon Press pie

PROTEIN EXPRESSION DURING PARASITE REDIRECTION OF HOST (TRICHOPLUSIA NI)

BIOCHEMISTRY

DAVY JONES

Department of Entomology, University of Kentucky, Lexington, KY 40546, U.S.A.

(Received 18 August 1988; revised and accepted 27 February 1989)

Abstract--Expression of proteins during normal egg and larval development of Trichoplusia ni was compared with that occurring in hosts stung as eggs by the parasitic wasp Chelonus sp. near curvimaculatus. Those stung hosts which produced a parasite (truly parasitized), precociously expressed proteins associated with larval-pupal metamorphosis, as did those stung hosts which did not contain a developing endoparasite (pseudoparasitized). No highly abundant, low-intermediate molecular weight hemolymph proteins were observed in truly or pseudoparasitized larvae which did not also occur at some point in the development of normal larvae. A low abundance, high molecular mass (160,000 Da) protein was observed in the hemolymph of truly parasitized larvae, but not of normal or pseudoparasitized larvae. The protein is glycosylated and very acidic (pI near 4.5). The data show that any parasitization proteins injected or induced by the ovipositing female parasite are in low abundance, in contrast to situations reported for parasitic wasps which sting hosts as larvae.

Key Word Index: Chelonus, metamorphosis, immune response

INTRODUCTION

Insect parasites produce a variety of developmental effects on their insect hosts (Beckage, 1985; Thomp- son, 1984; Vinson and Iwantsch, 1980). It has been noted that insect host-parasite systems in general offer a richer array of parasite manipulations of host development than has been reported in vertebrate systems (Jones, 1985). It has become apparent that regulatory agents from parasites can be powerful experimental probes in addressing questions in devel- opmental biology that would otherwise be intractable (Jones et al., 1986).

In several insect parasite systems involving parasitic wasps unique "parasitization proteins" or mRNAs appear in the host within 2-72 h of ovi- position by the adult female (Beckage et aL, 1987; Blissard et al., 1986a, b; Cook et al., 1984; Ferkovich et al., 1983; Fleming et al., 1983). These proteins of primarily low-intermediate molecular weight become highly abundant in the hemolymph and maintain their high concentrations during much of the period of parasitization. They are considered to be possible agents of parasite regulation of host biochemistry. Each of the above systems in which abundant, low- intermediate molecular weight parasitization proteins and mRNAs have been observed involve wasps which sting larval hosts.

The Trichoplusia ni-Chelonus spp (Braconidae) insect host-parasite system is an interaction in which a number of manipulations of host development are evident. In contrast to the above described systems,

Abbreviations used: SDS-PAGEIsodium dodecyl sulfate polyacrylamide gel electrophoresis; IEF--isoelectric focusing.

the female oviposits into the differentiating embryo of the host. The host then precociously initiates meta- morphosis several instars later. However, further metamorphic development is suppressed during the precocious prepupal stage. The parasite then emerges from these "truly parasitized" hosts. These redirec- tions in host development also occur in "pseudopara- sitized" hosts which, although stung, possess no live internal parasite at the time the redirected develop- ment becomes manifest. It has recently been shown that the source of the regulatory material is the adult female wasp (Jones, 1987).

The mechanism by which female-derived factors redirect insect host biochemistry is unknown. How- ever, it can be postulated that the mechanisms by which egg-larval parasites regulate their hosts are different than the strategies used by strictly larval parasites. In order to test this hypothesis, we have examined both truly parasitized and pseudopara- sitized larvae for unique low-intermediate molecular weight parasitization proteins, since they appear to be necessarily associated with host manipulation in host-parasite systems involving larval parasites.

MATERIALS AND METHODS

Insects Host Trichoplusia ni and the parasite Chelonus sp. near

curvimaculatus were reared and staged as described pre- viously (Jones, 1986). in some experiments the endoparasite larva was surgically removed from the host during the host's 2nd or 3rd larval stadium. The host was then placed back on food and allowed to continue development. Some of these surgically prepared larvae were bled again on the last day of feeding prior to metamorphosis. In other experiments pseudoparasitized larvae were generated by several other methods (Jones, 1987). First, host eggs were stung on day 1

m IO/S--A 445

446 DAVY JONES

of embryonic development, a procedure in which pseudo- parasitism is low (<10% become pseudoparasitized). Second, host eggs were stung on late day 3, when embryonic development is nearly complete, and when the host has reached the pharate first instar larval stage, a procedure which produces > 50% pseudoparasitism. Third, host eggs were stung on day 1 and then held at 4°C for 5 days, before being placed at 28°C for resumed development. This pro- cedure yields very high levels of pseudoparasitism.

Chemicals Silver nitrate protein staining reagent was purchased from

Eastman. Chemical reagents were obtained from Sigma. Ampholine for isoelectricfocusing (IEF) was purchased from Pharmacia-LKB. Concanavalin A/horseradish peroxi- dase and chromatofocusing supplies were from Sigma.

Electrophoresis and chromatofocusing Sodium dodecyl sulfate (SDS)-polyacrylamide gel elec-

trophoresis (PAGE) was performed as described by Laemmli and Favre (1973). Wide range (pH 3.5-9.5) and narrow range (pH 4-6.5) IEF were performed as described previously (Jones et al., 1986; Winter et al., 1977). After preparative electrofocusing was complete, the gel was sliced into 0.5 mm sections and the proteins eluted overnight in 150mM phosphate buffer (pH7.4) at 4°C. Preparative chromatofocusing of approximately 0.5 ml of hemolymph from day 2, penultimate instar truly parasitized larvae was performed using a pH gradient of 445 as described by Pharmacia instructions. Fractions from preparative IEF or chromatofocusing were concentrated with a Centricon device (YM membrane, 10,000 mol. wt cut-off), and sub- jected to analytical SDS-PAGE. Silver staining was used to visualize proteins after SDS-PAGE, and Coomassie blue was used for staining following isoelectric focusing.

Protein samples Host eggs which were less than 24 h old were each stung

a single time. Stung eggs, as well as unstung control eggs, were homogenized after 24h (homogenization buffer (pH 6.4): 60 mM Tris/l mM phenylmethylsulfonyl fluoride/0.1% NP40). The homogenate was centrifuged at 10,000g for 10min, and the proteins in the supernatant subjected to electrophoretic analysis. Normal, truly para- sitized or pseudoparasitized larvae of selected ages were bled and the hemolymph centrifuged at 10,000g for l rain before the hemolymph proteins were subjected to electrophoretic analysis.

Concanavalin A/horseradish peroxidase staining The glycosylation state of certain proteins was tested

following SDS-PAGE and transfer to nitrocellulose by staining with concanavalin A conjugated to horseradish peroxidase (Lin, 1986).

RESULTS

Hemolymph protein expression during egg and larval development

Examination of embryonic or hemolymph proteins of Trichoplusia ni by SDS-PAGE (5-20% acryl- amide) gradient showed very similar profiles for control and stung eggs, and for normal and truly parasitized 1st and 2nd instar larvae (Fig. 1). No highly abundant proteins unique to parasitized eggs or larvae were observed. As a positive control that the electrophoretic and staining conditions were of suffi- cient resolution to detect such "parasitization proteins", hemolymph proteins were compared for normal and parasitized larvae in another braconid/ noctuid host-parasite systems. Larvae of Heliothis

virescens parasitized by Microplitis croceipes in inde- pendent experiments consistently showed a high abundance protein with a tool. wt of 120,000, a situation not observed in normal larvae, as well as two highly enriched proteins (Fig. 2b). These data suggest if there exist abundant parasitization proteins in the C. near curvimaculatus system, they would be detectable under our conditions.

When the hemolymph of 4th instar normal and truly parasitized larvae, and of4th instar pseudopara- sitized larvae, were subjected to SDS--PAGE (5-20% acrylamide gradient), the profiles were very similar for low-intermediate molecular weight proteins (Fig. 3A). During the 4th larval stadium truly and pseudoparasitized larvae precociously expressed proteins which are normally expressed only during final larval stadium, such as the T. ni arylphorin and juvenile hormone suppressible proteins (sub-units 73-76,000Da, Jones et al., 1987). However, no intermediate or low molecular weight proteins were observed from truly or pseudoparasitized larvae which were not also expressed in normal larvae.

Examination of high molecular weight proteins showed the presence of a distinct protein of ca. 160,000 mol. wt in the hemolymph of day 1 and day 2 4th stadium, truly parasitized larvae (Fig. 3A). This size region of proteins was examined in more detail using SDS-5% PAGE. The protein was clearly visible in the sample from truly parasitized larvae (Fig. 3B, lanes 4, 5), running just slower than a much lower abundance protein found in all the hemolymph samples. The 160,000 mol. wt protein was not found in hemolymph from pseudoparasitized or normal larvae (Fig. 3B, lanes 3, 6, 7). Under conditions of heavier protein loading truly parasitized 3rd instar larvae were also seen to possess a parasitization protein not found in normal larvae (Fig. 3A, lane 6; Fig. 3B, lanes 1, 2). However, it migrated slightly faster than the protein from 4th instar truly para- sitized larvae. These patterns were confirmed on numerous replicate gels using independent hemolymph samples.

Isoelectric focusing of hemolymph proteins from normal, truly and pseudoparasitized larvae did not show any detectable "parasitization" proteins. Fol- lowing preparative isoelectric focusing, gel regions were sliced, eluted and the recovered proteins sub- jected to SDS-PAGE. The parasitization protein was recovered in a fraction with pH 4.5, indicating the protein is very acidic (Fig. 4). Preparative narrow range (pH 4-6) chromatofocusing was therefore em- ployed. The procedure provided very high enrich- ment of the parasitization protein (Fig. 5, lanes 1, 3).

As a further characterization of the protein, a sample of preparative eluate from chromatofocusing was subjected t o SDS--PAGE, the proteins trans- ferred to nitrocellulose, and the blots were probed with concanavalin A/horseradish peroxidase conju- gate. Although most protein bands in the higher molecular region did not detectably bind con- canavalin A, a few did, including the parasitization protein (Fig. 5, lanes 1, 2).

The absence of the parasitization protein in pseudoparasitized larvae arising from stung day 1 host eggs suggested a relationship between a live parasite larva and the protein. As a test of this

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Fig. 1. SDS-PAGE (5-20% polyacrylamide gradient) of proteins from eggs and larvae of stung and normal T. hi. Lane l, proteins from eggs stung 24 h earlier; lane 2, proteins from control eggs (lane 2); lanes 3 and 5, hemolymph proteins from normal 1st and 2nd instar larvae; lanes 4 and 6, hemolymph proteins from truly parasitized 1st and 2nd instar larvae. No abundant proteins unique to stung eggs or

parasitized larvae were observed.

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Fig. 2. SDS-PAGE (5-20% polyacrylamide gradient) of hemolymph proteins from last instar larvae of H. virescens parasitized by Microplitis croceipes during the 4th stadium. Parasitized last instar H. virescens (lane 1) possessed an abundant hemolymph protein (ca 120,000 mol. wt, indicated by arrow), a situation not observed in the hemolymph of early (lane 2) or late (lane 3) feeding stage last instar control larvae. The parasitized larvae also possessed two abnormally abundant proteins of 50-55,000 mol. wt. These data show that the experimental conditions used will detect abundant "parasitization proteins" in the

hemolymph of hosts of larval parasites.

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Fig. 3. SDS-PAGE of hemolymph proteins from normal and stung T. ni. (A) 5-20% polyacrylamide gradient analysis of hemolymph proteins from (1) day 2, 5th (last) stadium normal larvae, (2) pseudoparasitized, feeding larvae, on day prior to precocious wandering behavior, (3) truly parasitized, feeding larvae one day prior to precocious wandering behavior, (4) truly parasitized, feeding larvae two days prior to precocious wandering behavior, (5) normal, feeding larvae, one day prior to ecdysis to the ultimate instar, (6) truly parasitized, feeding 3rd instar larvae and (7) normal feeding 3rd instar larvae. For each lane, a minimum of five larvae of each developmental stage were bled and the hemolymph pooled. All lanes received 1/3 #1 of hemolymph. Although premature expression of 73-76,000 mol. wt last instar storage proteins during the late feeding stage is seen in truly parasitized or pseudoparasitized larvae (lines on left, lanes I-3), no abundant low-intermediate molecular weight proteins unique to these larvae, and not normal larvae, are seen. However, a low abundance, high molecular weight protein was observed in the hemolymph of truly parasitized larvae (arrows). (B) Region of high molecular weight hemolymph proteins (region in bracket from A) following 5% polyacrylamide gel electrophoresis. (1) Normal feeding 3rd instar larvae, (2) truly parasitized, feeding 3rd instar larvae, (3) normal feeding penultimate instar larvae, (4) truly parasitized, penultimate instar larvae two days before precocious wandering behavior, (5) truly parasitized, penultimate instar larvae one day before precocious wandering behavior, (6) pseudoparasitized penultimate instar larvae and (7) normal 5th (last) instar. All larvae were selected on the last day of feeding in the given stage, except for lane 4. A unique protein was observed in the hemolymph of truly parasitized 3rd instar larvae (arrow on left) and a unique protein was observed in the hemolymph of 4th instar truly parasitized larvae (arrow on right), just above a very low abundance protein observed in the samples from all the experiments groups of larvae. Lanes 1 and 2 received 1 #1

of hemolymph, while the rest received 1/3 #1.

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Fig. 4. SDS-5% PAGE of the preparative IEF (wide range) fraction (pH 4.5) containing the high molecular weight parasitization protein. (A) Photograph of the entire gel containing, from left to right, proteins in ~he preparative IEF fraction; hemolymph proteins from normal, 4th instar larvae (NL4D2); hemolymph proteins from 4th instar truly parasitized larvae (TL4D2); hemolymph proteins from normal 5th instar larvae (NL5D2). The arrow on the left indicates the position of the high molecular weight protein in the preparative fraction, and it is highly enriched relative to its concentration in the hemolymph of truly parasitized larvae. (B) The high molecular weight region containing the parasitization protein. Arrow on left indicates the position of the parasitization protein in the preparative fraction (IEF) and

in the hemolymph of truly parasitized larvae (TL4D2).

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CONCANAVALIN SILVER A STAIN STAIN

Fig. 5. Concanavalin A/horseradish peroxidase staining of the parasitization protein following SDS-5% PAGE (figure shows the high molecular weight region of the gel). Lanes 1 and 3, fraction from preparative chromatofocusing (pH 4-6) containing the parasitization protein. Lanes 2 and 4, hemolymph from truly parasitized larvae. Lanes 3 and 4 were silver stained for total protein. Lanes I and 2 were stained for glycoproteins. The highly enriched parasitization protein in lane 1 (arrow) gave a very strong signal, while several other proteins did not. Also, inclusion of alpha-methyl mannose in the incubation with concanavalin A/horseradish peroxidase completely blocked staining of all glyeoproteins (not shown). On the right are shown the positions of molecular size markers. The protein has an approximate molecular

mass of 160,000 daltons.

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Fig. 6. SDS-PAGE (5-20% polyacrylamide gradient) of hemolymph protein from truly parasitized larvae and from pseudoparasitized larvae generated by three different methods. From left to right: truly parasitized larvae; pseudoparasitized larvae arising from hosts stung on the third day of embryonic development (D3); pseudoparasitized larvae arising from hosts stung early in embryonic development, and then held at 4°C for 5 days (4°C); pseudoparasitized larvae arising from initially truly parasitized hosts from which the parasite larva was surgically removed during the prior host stadium (surgery). The parasitization protein (arrow) was not observed in the hemolymph of any of the pseudoparasitized treatments. The lines of the left indicate three proteins in that region of the gel which were common to

truly and pseudoparasitized larvae.

452

Parasite redirection of host development 453

relationship, pseudoparasitized larvae were generated by several alternative methods. It was found that when the parasite was surgically removed during the host's 2nd or 3rd larval stadium, the protein was absent when the host prematurely initiated metamor- phosis one or two instars later (Fig. 6). Alternatively, when the stung host egg was subjected to cold, which resulted in the death of the parasite during the host's 1st larval stadium (Jones, 1987), the parasitization protein was missing during premature host metamor- phosis (Fig. 6). Finally, when host eggs were stung late during embryonic development, which also leads to early death of the parasite (Jones, 1987), the parasitization protein was missing during precocious host metamorphosis (Fig. 6). It should also be noted that all pseudoparasitized larvae generated by the alternative methods precociously initiated metamor- phosis, and then stopped development as precocious prepupae.

DISCUSSION

The data presented here have implications for parasite manipulation of host developmental pro- grams, and for possible differences in mechanisms by which egg-larval insect parasites regulate host devel- opment, as compared with parasites which oviposit in host larvae.

Manipulation of host developmental program

In a previous study all tested metamorphosis- associated proteins appeared prematurely in the hemolymph of larvae pseudoparasitized by Chelonus spp approximately 10 days after stinging (Jones et al., 1985). Especially conspicuous were the highly abun- dant hemolymph storage proteins which normally occur late in the feeding stage of T. ni (Jones et al., 1987). The data from the present study confirm that result, as well as document the precocious appearance of the metamorphosis-associated proteins in truly parasitized larvae. Thus, the premature appearance of the proteins is a characteristic of the T. ni-Chelonus spp interaction, rather than being an atypical result unique to the pseudoparasitized con- dition. This result validates the use of pseudopara- sitized larvae as a model for the T. ni-Chelonus spp interaction (Jones et al., 1986). Pseudoparasitized hosts exhibit the developmental symptoms of regula- tion by the parasite in the absence of a live internal parasite. Thus, they are attractive experimental in- sects for the biochemistry of metamorphosis because the internal parasite is not present as a confounding variable during precocious metamorphic commit- ment and suppressed prepupal development. The demonstration that the altered developmental path- ways in pseudoparasitized hosts are similar to those altered in truly parasitized hosts increases the attrac- tiveness of experimental use of pseudoparasitized hosts.

Regulation of host development by egg-larval vs larval parasites

A number of insect host-parasite systems have been described in which abundant, persistent, para- sitization proteins appear within 2-72 h of stinging. Oviposition by the braconid Cotesia marginiventris

induces the appearance of a new glycoprotein of slightly less than 66,000 Da (Ferkovich et al., 1983). Oviposition by the ichneumonid Campoletis sonoren- sis induces a 55,000 Da glycoprotein within several hours. The induction of the protein does not require the presence of the parasite, as injection of the wasp polydnavirus can duplicate the induction process (Cook et al., 1984). A protein of 33,000 mol. wt appears in host Manduca sexta within several hours of oviposition by Cotesia congregata, and proteins of 60, 90 and 120 kDa appear within several days. At least the 33,000 mol. wt one is a glycoprotein. Again, the induction process does not require the presence of the parasite, and can be duplicated by injection of the wasp polydnavirus (Beckage et al., 1987). At present, the charges of the unique, induced proteins are unreported. In a number of these host-parasite sys- tems, the parasite manipulates the host developmen- tal program, such as for postponement of expression of metamorphic commitment (Beckage and Temple- ton; 1986) or for suppression of ecdysteroid driven prepupal development (Dover et al., 1988). It is also though that the wasp polydnavirus suppresses the immune response of the host larva (Edson et al., 1981; Guzo and Stoltz, 1985; Stoltz et al., 1986). All of these systems involve parasites which sting host larvae.

In contrast, the parasite of the present study is a representative of a group of parasites (Cheloninae) which sting host eggs and emerge later from host larvae. Such a pattern of development confronts the internal parasite with a more complex environment (that of both the host egg and host larva) than is faced by strictly larval parasites. The occurrence of the parasite in the developing host embryo, in which tissue differentiation and programming is occurring, also presents the egg-larval parasite with opportun- ities for host regulation not accessible to strictly larval parasites. Thus, it could be anticipated that parasites such as Chelonus spp would use different strategies in host regulation than larval parasites. The present study supports this hypothesis, in that the parasite effectively redirects host biochemistry by a mechanism not associated with rapid production of abundant, low-intermediate molecular weight para- sitization proteins. Another species closely related to Chelonus, Ascogaster quadridentatus, also redirects host development (Jones, 1985b), yet also does not induce a detectable rapid change in host protein profiles (J. Brown, personal communication). It was shown that the adult female is the source of the regulatory material (Jones, 1987), and that she injects a number of proteins, and other components, into the host which are detectable immunologically, but not by protein staining (Leluk and Jones, 1989). In as much as these regulatory materials are injected into embryonic hosts which are incompletely differ- entiated, latent alteration of host developmental pro- grams is possible without concurrent production of abundant parasitization proteins. This interpretation is consistent with the premature appearance of certain proteins 10 days and several larval stadia later. These abundant metamorphosis-associated proteins appear after the developmental pathway for larval-pupal metamorphosis has been precociously initiated (Jones et al., 1986). Thus, the premature

454 DAVY JONES

expression of genes for those proteins is a conse- quence of redirection of development, rather than a cause.

High molecular weight, low abundance parasitization protein

Although the host-parasite interaction of the Chelonus egg-larval parasite does not involve proteins in the 30-60,000 tool. wt range, a protein of approximately 160,000 was observed. This protein was observed during the feeding stage of the stadium during which metamorphosis was initiated. A protein of similar, but not identical, size was observed during the 3rd larval stadium of the host. However, the protein was not in detectable levels in the parasitized host egg, or 1st or 2nd instar larva. The parasite egg does not hatch within 24h of oviposition. Thus, it appears that high molecular weight parasitization protein(s) do not appear rapidly, but do appear by the time of the host 3rd instar (which is prior to the molt the parasite final larval instar).

The parasitization protein from the 4th host larval stadium is a glycosylated, very acidic protein (pI near 4.5). Acidic chromatofocusing provided a prep- aration very highly enriched for the protein. The contaminating proteins in the 100,000-200,000 mol. wt range present were not detected to be glycosylated. Therefore, combination of chromatofocusing and concanavalin A column chromatography should be a fruitful approach to two-step purification of the protein. Further biochemical and immunological studies will be required to assess the relationship of this protein to the parasitization protein occurring during the host's prior larval stadium.

The data presented here suggest that there is a very strong relationship between the presence of a live parasite larva and the presence of the protein. How- ever, it is still not known whether the protein is encoded by the host, the parasite, or the polydnavirus injected by the ovipositing female (Jones et al., 1986). It is possible that the protein is encoded by the host or the polydnavirus, but that the presence of the parasite larva is required for trans-regulation of its expression. The data do permit the conclusion that if the protein is trans-regulated, the regulatory signal from the parasite larva does not persist from one host larval stadium to the next.

Wasp virus as a tool for host regulation

Research on wasp parasites which sting host larvae has shown that injection of purified wasp polyd- navirus results in the appearance of the same para- sitization proteins as appear in normally stung hosts. These segmented-genome viruses apparently do not replicate in host Lepidoptera (Stoltz et al., 1984). However, virus mRNA has been isolated from larval hosts of parasitic wasps (Blissard et al., 1986a, b; Fleming et al., 1983) and these mRNAs have been translated in vitro (Blissard et al., 1986b). However, no proteins known to be transcribed from viral DNA have as yet been identified in vivo. The present study has identified a protein as a possible end-point of measurement in future experiments on injection of Chelonus poydnavirus, venom, or other parasite materials into host eggs.

CONCLUSIONS

The host-parasite systems which have been studied in biochemical detail until now have all involved parasites which sting host larvae. In these host-parasite interactions (1) primarily high abun- dance, low-intermediate molecular weight glyco- proteins appear, (2) these proteins appear within 2-72 h and are persistent for several days or longer, and (3) the appearance of these proteins does not require the presence of the parasite, in that their induction can be duplicated by injection of the cognate wasp polydnavirus. In contrast, the parasite used in the present study stings host eggs, and later emerges from host larvae. No high abundance, low- intermediate molecular weight proteins were detected either soon after oviposition or much later during host and parasite larval development. A low abun- dance protein of 160,000 mol. wt was detected during host and parasite larval development. The appear- ance of this highly acidic glycoprotein requires the presence of the parasite larvae. These comparative data suggest that the nature of host regulation for unique protein induction used by this group of egg-larval parasites is different than the mechanism of host regulation used by strictly larval parasites studied thus far.

Acknowledgements--This research was supported, in part, by NIH grant GM33995. Appreciation is extended to Dr Douglas Dahlman for his provision of Heliothis virescens and Microplitis croceipes. This paper is published in connec- tion with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director.

Note added in proofnRecently Beckage et al. reported a number of basic and acidic proteins unique to parasitized Manduca sexta larvae (Arch. Insect Biochern. Physiol. I0, 29-46).

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