Archives of Insect Biochemistry and Physiology 21 :155-165 (1 992)

Hemolymph Concentrations of Host Ecdysteroids Are Strongly Suppressed in Precocious Prepupae of Trichoplusia ni Parasitized and Pseudoparasitized by Chelonus Near Curvimaculatus Davy Jones, Dale Gelman, and Marcia Loeb Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky (D.J.); Insect Neurobiology and Hormone Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, Maryland (D.G., M.L.)

Regulation of ecdysteroid production in lepidopteran prepupae was studied using a parasitic wasp (C. near curvimaculatus) which specifically suppresses host prepupal ecdysteroid production after the induction of precocious host mctamor- phosis. At the developmental stage at which the hernolymph of the unparasitized metamorphosing host has its maximum titer of prepupal ecdysteroids, the hemolymph of 4th instar ”truly parasitized” hosts (hosts with a surviving endo- parasite) had a strongly reduced ecdysteroid titer. However, during the pho- tophase about 12 h later, just prior to emergence of the parasite larva, an ecdysteroid peak was observed in the host hemolymph. Fourth instar pseu- doparasitized prepupal hosts (in which the endoparasite was not present or died early in development) exhibited a sustained suppression in the hemolymph ecdysteroid titer. Small 5th instar pseudoparasitized hosts, which normally would molt to a 6th instar prior to metamorphosis, but which precociously attained the prepupal stage, also had a strongly reduced ecdysteroid titer. The late increase observed in truly parasitized hosts could be completely prevented by surgical removal of the parasite 24 h earlier, resulting in a titer similar to that in pseudoparasitized hosts. HPLC analysis of ecdystcroids in normal, truly parasit- ized, and 4th or 5th instar pseudoparasitized prepupae showed that both ecdysone and 20-OH ecdysone* were suppressed in truly and pseudoparasitired prepupae, with ecdysteroid levels being lowest in pseudoparasitized hosts. These data, and

Acknowledgments: We express our appreciation to Dr. Grace Jones for her suggestions during this project. This work was supported in part by NIH Grant GM 33995.

Received February 14, 1992; accepted July 16, 1992.

Address reprint requests to Davy Jones, Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40506.

*Abbreviations used: 20-OH ecdysone = 20-hydroxyecdysone; 20, 26-OH ecdysone = 20, 26 di hydroxyecdysone; PTTH = prothoracicotropic hormone.

0 1992 Wiley-Liss, Inc.

156 Jones et al.

those of Brown and Reed-Larsen (Biol Contr I , 136 [19921), showing endoparasite secretion of ecdysteroids just prior to its emergence from the host, strongly indicate that: (1) the prepupal peak in truly parasitized hosts originates from the endopar- asite, and (2 ) the low level of ecdysteroids in pseudoparasitized hosts results from the host’s intrinsic inability to express a normal level of prepupal ecdysteroid titer. While precocious 4th or 5th instar prepupae of similar size had similarly suppressed ecdysteroid titers, smaller 4th instar prepupae had a lower ecdysteroid titer than larger, precocious 5th instar prepupae. Rare 5th instar pseudoparasitized prepupae that were of nearly normal size showed a prepupal ecdysteroid titer distinctly greater than those of the usual smaller, precocious 5th instar prepupae. The data suggest that the competence of the host to express a normal hemolymph titer of prepupal ecdysteroids is more closely correlated with the size of the prepupae than with the instar attained. D 1992 WiIey-Liss, inc.

Key words: commitment, developmental program, ecdysteroids, metamorphosis, parasite

INTRODUCTION

A major area of interest in the developmental biology of insects and other invertebrates is the biochemical nature of the mechanism by which the differentiating organism assesses its developmental status. Of particular interest are the mechanisms underlying the decision to change the develop- mental trajectory away from the program of successive larval molts, and instead express a genetic program uniquely associated with metamorphic development. The expression of prepupal ecdysteroids by metamorphosing final instar larvae occurs in a qualitatively different physiological milieu than the expression of ecdysteroids which occurs during each prior larval stadium. In addition, it is the first time after larval eclosion that ecdysteroid production is not expressed in a circumstance of cellular commitment for a larval program, but rather is expressed at a time of commitment of the organism for expression of a pupal cellular program.

Parasites of the subfamily Cheloninae redirect prepupal ecdysteroid ex- pression in a two-stage manner [l]. First, host embryos that are injected with regulatory material by the adult wasp are later induced to precociously initiate metamorphic development to the prepupal stage during a preulti- mate instar [2]. Then, prepupal ecdysteroid expression is suppressed, al- though prior ecdysteroid expression in each prior instar is not affected [3]. Although there are chemical agents which inhibit ecdysteroid biosynthesis or action [MI, such compounds do not lend themselves to probing the basis for acquisition of the ability of the biosynthetic tissues to produce ecdy- steroids. Thus, this host-parasite system provides an excellent and unique tool with which to address questions on developmental acquisition of com- petence of tissues to contribute to the hemolymph ecdysteroid peak. In this paper we report investigations concerning the differential effects of the injected regulatory agents versus the endoparasite larva on the hemolymph ecdysteroid levels in prepupae.

Competence to Express Prepupal Ecdysteroids 157

MATERIALS AND METHODS

Insects

C. insularis were reared as described previously [3,7,8].

Staging and Hemolymph Collection Following the final larval molt, the feeding stage usually lasts 2-3 days,

followed by one day of wandering. Wandering and prepupal larvae were staged for hemolymph collection using developmental markers described previously [8]. In some experiments, hosts were stung as old host embryos (several h before hatching), a condition which promotes the occurrence of “pseudoparasitized” hosts (endoparasite is not present or dies just after hatch of the host egg). Such hosts will precociously initiate metamorphosis as either a 4th instar or a stunted 5th instar (that would normally molt to a 6th instar). In some experiments, truly parasitized larvae (those containing a surviving parasite) were selected on the day of wandering and the parasite was surgi- cally removed. Other wandering larvae received sham surgery in which the parasite was not removed. Surgically manipulated larvae were bled 24 h later.

Ecdysteroid HPLC and RIA Methods used to determine ecdysteroid titers were similar to those of Borst

and OConner 191 and Bollenbacher et al. [lo], as adapted by Gelman and Woods [ll]. The relative affinities (pg ecdysone required to displace 50% of the radiolabeled ecdysone ligand)/(pg ecdysteroid required to displace 50% of the radiolabeled ligand) of ecdysone, 20-hydroxyecdysone and 20,26-dihy- droxyecdysone for the antibody are 1,0.77 and 0.81, respectively [ll]. There- fore, to convert 20-dihydroxyecdysone equivalents to ecdysone equivalents or to 20, 26-dihydroxyecdysone equivalents, values reported should be di- vided by factors of 1.3 and 1.16, respectively.

Host Trichoplusiu ni and parasitic wasps Chelonus near curvirnaculatus and

RESULTS

Ecdysteroid Titers in Normal and Pseudoparasitized Prepupae The ecdysteroid titer in normal prepupae reaches a peak of ca. 1,800 pglpl

early in the scotophase of the day of wandering behavior (Fig. la). This peak then falls to ca. 300 pg/pl over the next 18 h, as these prepupae molt to the pupa. In contrast, the ecdysteroid titer in pseudoparasitized 4th instar preco- cious prepupae is strongly suppressed, with little if any detectable peak occurring at the time of the normal peak (Fig. lb). During the next several days there is no peak in the titer, merely a slow rise to a level which is still less than 10% that of the highest level shown by normal prepupae. The hemolymph ecdysteroid titer in pseudoparasitized 5th instar prepupae showed a pattern similar to that of 4th instar pseudoparasitized prepupae; that is, there was no peak at either the normal time or at any subsequent time (Fig. lb). However, the titers in these 5th instar prepupae were consistently higher than the even lower levels seen in 4th instar prepupae.

158 Jones et al.

100’ , b

0

50 --

0 5 t h instor prepupa 4th instar prepupa

2 5 - 9

OJ

Fig. 1. Ecdysteroid titers in (a) normal (triangles) and truly parasitized larvae (open circles) and (b) pseudoparasitized 4th instar (closed circles) and 5th instar (open circles) larvae, on the day of wandering behavior and the subsequent prepupal stage. Darkened sections of x-axis indicate scotophase of each day. Each point represents the average of at least 3 samples, each consisting of at least 6 larvae. DW = day of wandering, DW + 1 = one day after the day of wandering, and so on.

Truly parasitized precocious prepupae, which contain the additional vari- able of a living endoparasite, also showed a much suppressed ecdysteroid titer at the time of the normal prepupal peak although the titer in the hemolymph (250 pg/pl) was distinctly higher than that in pseudoparasitized hosts (ca. 50 pg/pl hemolymph) (Fig. 1). These hosts live only for an additional 12-18 h, since the parasite emerges from and kills the host on the day following what should be the normal prepupal peak. Shortly before the parasites emerge from the lethargic host, and at a time when the parasite fills much of the internal space of the host, the concentration of ecdysteroids in what remains of the hemolymph rose to 1,300 pg/pl (Fig. 1).

To further assess the physiological basis of this peak, parasites were removed from the host 24 h prior to the peak, with the result that this peak did not appear, and the ecdysteroid titer was the same as in pseudoparasitized hosts (Fig. 2, TD4PR). The role of the endoparasite was tested in yet another way, by making use of the occasional occurrence of late death of the parasite in 4th instar hosts.

Competence to Express Prepupal Ecdysteroids 159

= 1 3 30001

I N5D3 P4D3 PSI33 TD3 'ID4 TD4 TD4

D SHAM PR PREPUPAE SAMPLED

Fig. 2 . Hemolymph ecdysteroid titer in normal (N5D3), pseudoparasitized 4th instar (P4D3) or 5th instar (P5D3), and parasitized 4th instar larvae in which the large endoparasite recently died (TD3). These hernolymph samples were taken at the time of the normal day 3 ecdysteroid peak. Also shown I S the hernolymph ecdysteroid titer in various categories of truly parasitized 4th instar (TD4) prepupae, at the time of the peak of hernolymph ecdysteroids in truly parasitized prepupae (Fig. 1). PR = truly parasitized larvae from which the parasite had been removed 24 h previously; SHAM = truly parasitized larvae surgically shammed 24 h previously; D = the large endoparasite had recently died in these larvae. The titer in truly parasitized larvae from which the parasite has been removed is essentially the same low level found in pseudoparasitized prepupae. Variance is expressed as standard error.

Such hosts are in the smallest size class of pseudoparasitized prepupae (20-30 mg), and are further distinguished from other pseudoparasitized forms by the presence of a conspicuous, but dead, 3rd instar parasite in the host prepupae. The ecdysteroid titer in these hosts (TD3), at the time of the normal peak, was reduced to the level found in other pseudoparasitized larvae (50 pg/pl, P4D3). It was also observed that after bleeding, the hemolymph of these hosts rnelanized much more quickly than did hemolymph from truly parasitized hosts which contained a live endoparasite.

A qualitative profile of ecdysteroids found in the hemolymph of normal larvae during the prepupal ecdysteroid peak is shown in Figure 3A. The predominant ecdysteroids observed were ecdysone, 20-OH ecdysone, 20,26- OH ecdysone and a peak of unknown polar ecdysteroids. These same major ecdysteroids were observed in hemolymph from pseudoparasitized prepupae (Fig. 3B). However, although ratios among these ecdysteroids were similar for each type of larva, the concentration of each of these ecdysteroids was much lower in the pseudoparasitized larvae. Thus, it is evident that the concentration of the physiologically active ecdysteroid (20-OH ecdysone), and its precursor, as well as of other ecdysteroid metabolites, are markedly suppressed in the hemolymph of pseudoparasitized prepupae.

A qualitative analysis was made of ecdysteroids present in hemolymph (Fig. 3A) from parasitized hosts at the time of the normal prepupal peak (when the level in truly parasitized hosts is low, "parasitized A") and during the peak occurring in host hernolymph just prior to parasite emergence (parasitized B).

160 Jones et al.

1000 A m n - 800 o *- - * Pseudoparasitized $ 0-a Parasitized A 2 600 *-• Parasitized B z 0

400 0 W

E 200 I 0 FJ

0 10 20 30 40 50

FRACTION NUMBER

A m n v

W z 0

E 0 w I 0

I 0 FI

Pseudo parasitized Parasitized A 60 0

(v

10 20 30 40 50 FRACTION NUMBER

Fig. 3. Qualitative profile of ecdysteroids in the hemolymph of normal, pseudoparasitized and truly parasitized prepupae. Normal, pseudoparasitized and Parasitized A samples were taken 1-3 h after initiation of the scotophase, late on the day of wandering. Parasitized B are samples in which truly parasitized larvae were bled at the time of the ecdysteroid peak just before parasite emergence. Samples were extracted in 75% aqueous methanol. Profiles were determined by RIA of eluate fractions from HPLC (Waters’ microBondapak C18 column; fraction size 0.6 ml). Ecdysteroid titer expressed as pg 20-hydroxyecdysone equivalents per fraction. 20-OH = ecdysteroid with retention time of 20- hydroxyecdysone; 20,26-diOH = ecdysteroid with retention time of 20,26-hydroxyecdysone; E = ecdysteroid with retention time of ecdysone. Lower figure b utilizes an expanded scale to show the results for the two groups with a very low ecdysteroid titer. The qualitative nature of ecdysteroids in pseudoparasitired and truly parasitized prepupae is the same as that in normal prepupae.

The results show that the same etdysteroids were present at both times. However, titers for each major ecdysteroid were much higher in the hemo- lymph taken from the host just prior to parasite emergence.

The basis for the slightly higher average ecdysteroid titer in 5th instar as compared to 4th instar pseudoparasitized prepupae (Fig. 1) was investigated further. Prepupae were separated into discrete size classes and their ecdy- steroid titer determined. Fourth instar prepupae usually range in size from 25 mg to 70 mg, while 5th instar prepupae range from 60 mg to a nearly normal

Competence to Express Prepupal Ecdysteroids 161

700:

120/

i O o l rn

0

O' 25 35 45 55 65 75 85 95 110 130 IsOl70lsii" WEIGHTOF PREPUPA imgi

Fig. 4. Ecdysteroid titer as a function of the size of the pseudoparasitized 4th instar (circles) or 5th instar (open squares) or normal (solid square) prepupa. Circles within squares represent samples of similar size, mixed 4th and 5th instar prepupae. The indicated weights are the midpoint of the size class into which prepupae were pooled (e.g., 25 mg corresponds to the 20-30 rng size class). The weight of normal 5th instar prepupae (240-280 mg) is designated by 'N.' Hernolymph samples were taken 1-3 h after initiation of the scotophase. These data establish a clear relationship between the size of the precocious prepupae and its hemolymph ecdysteroid titer.

size of 200 mg. As shown in Figure 4, small 4th instar prepupae (2540 mg) had a distinctly lower titer (35-50 ng/ml) than larger (90-100 mg) 5th instar pseudoparasitized prepupae (80-100 ng/ml).

However, this result cannot be explained on the basis of the number of molts experienced by the larva, since the larger 4th instar prepupae (50-70 mg) had an ecdysteroid titer similar to that of 5th instar pseudoparasitized prepupae that were in the same size range (Fig. 4). Further, 5th instar prepupae that were smaller (50-70 mg) had a lower ecdysteroid titer than those which were larger ( 100 mg) (Fig. 4). These results suggest that the ecdysteroid level in prepupal hemolymph is correlated with the mass attained by the insect, and is not determined by the number of instars per se.

This hypothesis was further tested by generating a larger number of host larvae, and selecting for analysis some rarely occurring, larger, 5th instar pseudoparasitized prepupae (150-200 mg) whose size was much closer to that of normal-size larvae (24&280 rng; [8]). As shown in Figure 4, these prepupae had an ecdysteroid titer higher than their more typically sized pseudoparasitized counterparts. Additionally, ecdysteroids were analyzed in the hemolymph of 5th instar pseudoparasitized hosts of C. insularis, a related parasite whose 5th instar pseudoparasitized host prepupae are larger than that typical of 5th instar host prepupae of C. curvimaculafus [3]. Although reduced below normal, the concen- tration of both ecdysone and 20-OH ecdysone in the larger hosts of C. insularis (as determined by HPLC-RIA) was about twice that occurring in typically sized, 5th instar pseudoparasitized hosts of C. curvimaculatus (not shown).

162 Jones et al.

DISCUSSION

For both truly and pseudoparasitized T . ni we report a strong suppression of total ecdysteroids at the time of the normal prepupal ecdysteroid peak characteristic of unparasitized larvae. HPLC analysis showed that 20-OH ecdysone, the physiologically active ecdysteroid (molting hormone), as well as the other predominant hemolymph ecdysteroids were suppressed at this time (Fig. 3). In truly parasitized prepupae, there was a sharp increase in ecdysteroid titers just prior to parasite emergence, as observed by Grossni- klaus-Burgin et al. [12]. The ecdysteroid peak in truly parasitized animals occurred in the early photophase, while the ecdysteroid peak in normal insects was seen in early scotophase, suggesting that this displaced peak is under the control of the parasite. This hypothesis is further supported by the fact that pseudoparasitized T. ni hemolymph ecdysteroids do not rise at the time that this peak occurs in truly parasitized hosts, and that removal or death of the endoparasite 1-2 days earlier eliminates the peak seen in hemolymph of truly parasitized prepupae. Brown and Reed-Larsen have shown [13] that the parasite larva releases ecdysteroids into the host shortly prior to parasite emergence. We interpret these results as suggesting that the level of sup- pressed host ecdysteroids in pseudoparasitized prepupae reflects the basic effect of regulatory materials injected by the ovipositing female, while any departure from that observed in truly parasitized hosts is due to the added participation of the endoparasite larvae in regulating the final ecdysteroid titer in the host’s hemolymph.

Normal and Parasite-Altered Ecdysteroid Expression Several endocrine components have been identified which are necessary

for expression of the prepupal peak of ecdysteroids. Secretion of PTTH, and the modulation of its actions by juvenile hormone are examples [14]. A hernolymph factor has also been identified which is capable of stimulating the production of ecdysteroids [15], and there is also the participation of an oxoecdysteroid ketoreductase which converts the 3-dehydroecdysone pro- duced by the prothoracic glands to ecdysone [16]. The timely production and/or release of all of these factors results in the large increase in ecdysone and 20-OH ecdysone observed in lepidopteran prepupae [17].

Several different kinds of alteration in normal ecdysteroids have been observed in lepidopteran hosts of insect parasites. In host Manduca sexta, the parasite Cotesia congregata causes postponement of the prepupal stage, so that expression of ecdysteroids in a prepupal milieu does not occur, even after emergence of the parasites [MI. A component in the venom of Euplectrus spp. inhibits apolysis by a mechanism which blocks receptivity of tissues to ecdysteroids [ 191. Suppressed prepupal development by larval parasites such as Microplitis croceipes has been reported [20,21] and changes in ecdysteroid production have been implicated.

Correlation Between Size and Ecdysteroid Titer In this study, we also have explored the relationship between size of the

pseudoparasitized prepupa and the ability of the prepupa to produce ecdy-

Competence to Express Prepupal Ecdysteroids 163

steroids, Under conditions of normal growth, larvae of T. ni initiatemetamor- phosis and attain the prepupal state during the 5th larval stadium. Precocious 4th instar pseudoparasitized prepupae do not exhibit a prepupal ecdysteroid peak. However, this result cannot be attributed to an insufficient number of molts, since small penultimate 5th instar larvae that would normally molt to a sixth final instar 181, when induced to precociously attain the prepupal state, also exhibit suppressed titers of prepupal ecdysteroids.

There is a clear correlation, however, between the size of the prepupa and the ecdysteroid production observed. Thus the ecdysteroid titer in small 4th instar precocious prepupae (2540 mg) is distinctly less than that of larger (90-100 mg) 5th instar prepupae. Further, 4th and 5th instar pseudopar- asitized prepupae of similar size (50-70 mg) have similar ecdysteroid titers. In addition, we have shown that rare, very large pseudoparasitized hosts whose size (150-200 mg) closely approaches the size of normal 5th instar prepupae [8], have a distinctly higher ecdysteroid titer than all of the other sizelinstar pseudoparasitized prepupae. In a previous study, we analyzed total prepupal ecdysteroids in T. ni which were parasitized by a related parasite(C. insularis). Fifth instar pseudoparasitized prepupal hosts of that parasite are characteris- tically larger as prepupae (yet still under normal size), and are close in size to the rare, largest pseudoparasitized hosts stung by the parasite used in the present study [3]. Consistent with the present data, the titer of ecdysone and 20-OH ecdysone in pseudoparasitized hosts of C. insularis is about twice that typically observed in the smaller prepupal hosts of C. curvimaculatus. These data establish a strong correlation between the mass attained by the insect, and the prepupa’s ability to produce ecdysteroids.

If the observed ecdysteroid production by pseudoparasitized larvae is indeed an attempt to produce the normal premolt ecdysteroid peak, then our data suggest that the ability (competence) of ecdysteroid-producing tissues to synthesize the prepupal ecdysteroid peak is related to size rather than number of molts. With respect to Lepidoptera, Nijhout [22] firmly established that the decision by Manduca sexta larvae that they had attained the metamorphic instar was closely correlated with the size of the insect at ecdysis to that instar. This result has been confirmed for T. ni [8]. However, injection of space-filling materials into M. sexfa larvae did not induce precocious metamorphosis, nor did manipulations directed toward the increased tracheal diffusion distances in larger larvae (C. M. Williams, personal communication). The inability to genetically or otherwise manipulate the experimental system has prevented investigators from determining the identity of the initial “switch” triggering subsequent events such as JH decline. However, the remarkable ability of parasites in the subfamily Cheloninae to induce precocious pupation in preultimate instar hosts [23] may present us with a unique opportunity to address the question of acquisition of competence to express a prepupal program, such as prepupal ecdysteroid production.

Conclusions

In conclusion, our results show that ecdysteroid production is suppressed in precocious prepupae parasitized by C. curvimaculatus. At a time in the scotophase corresponding to the prepupal ecdysteroid peak in unparasitized

164 Jones et al.

larvae, concentrations of all ecdysteroids are low in hernolymph of truly parasitized and pseudoparasitized precocious prepupae. In truly parasitized larvae a peak in ecdysteroid titers occurs in the early photophase just prior to the emergence of the parasite. Data from this study and other reports suggest that this peak is probably due to production of ecdysteroids by the parasite itself. Our results also suggest that the competence of the host to produce prepupal ecdysteroids is related to the size or mass of the host rather than to the number of instars completed by that host.

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15. Watson RD, Bollenbacher WE: Juvenile hormone regulates the steroidogenic competence of Manduca sexfa prothoracic glands. Mol Cell Endocrinol57, 251 (1988).

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17. Riddiford LM: Interaction of ecdysteroid and juvenile hormone in regulation of larval growth and metamorphosis of the tobacco hornworm. In: Progress in Ecdysone Research. J.A. Hoffman, ed. ElseviedNorth Holland Biomedical Press, Amsterdam, pp 409 (1980).

18. Beckage NE, Riddiford LM: Effects of parasitism by Apunteles congregatus on the endocrine physiology of the tobacco hornworm, Munduca sextu. Gen Comp Endocrinol47, 308 (1982).

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