Pseudoparasitism of host Trichoplusia ni by Chelonus spp. as a new model system for parasite regulation of host physiology

J. Insecz Physiol. Vol. 32, No. 4, pp. 315-328, 1986

0022-1910/X6 $3.00+ 0.00 Copyright Q 1986Pergamon Press Ltd

Printed in Great Britain. All rights reserved

PSEUDOPARASITISM OF HOST TRICHOPLUSIA NI CHELONUS SPP. AS A NEW MODEL SYSTEM FOR PARASITE REGULATION OF HOST PHYSIOLOGY

BY

DAVY JONES, GRACE JONES, MARIA RUDNICKA, ANITA CLICK, VIKTORIA RECK-MALLECZEWEN and MAKOTO IWAYA* Department of Entomology, University of Kentucky, Lexington, KY 40546-0091 and *School of Biological Sciences, University of Kentucky, Lexington, KY 40506, U.S.A. host insect species parasitized as eggs by wasps of the subfamily Cheloninae initiate precocious metamorphosis many days later as preultimate-instar larvae. However, development is then suppressed early in the precocious prepupal stage. These events occur even in “pseudoparasitized” hosts containing no live or functional parasite at the time the endocrine lesions become manifest. The endocrine bases for these effects have been determined using a Trichoplusia ni-Chelonus spp. model system. Using hosts pseudoparasitized by C. near curvi~~aculutus or C. insularis it was determined that the entire feeding stage developmental pattern of the final instar is precociously expressed in penultimate-instar hosts. The precocious expression of the final-instar developmental pattern may be due to alteration or reprogramming of tissues involved in establishment of critical growth thresholds. The second endocrine lesion is due to a lower than normal titre of juvenile hormone in precocious prepupae, leading to a lower than normal ecdysteroid titre. The validity of the concept of parasite regulation of host physiology, and its necessary distinction from indirect stress effects, are discussed. In few systems suggestive of parasite regulation of the host has the hypothesis been tested at a biochemical level. It is proposed that the T. ni-Chelonus spp. interaction is an excellent model system for such an analysis. The current results using this system already have implications for basic knowledge on host-parasite interactions, development of chemicals to disrupt insect growth, biological control strategies and new experimental probes for investigations of normal insect physiology. Abstract-Most

Key Word Index: Trichoplusiu ni, Chelonus, pseudoparasitism, egg-larval parasitism, metamorphosis, juvenile hormone esterase, ecdysteroids, juvenile hormone

INTRODUCTION Insect ho&-parasite physiological interaction has been a somewhat neglected area of research in comparison with the effort expended on the study of normal insect physiology. Yet, there are advances to be made and which can only be made through studies of host-parasite interaction. Recent progress in this field, as exemplified by this symposium, demonstrates that the field is now ripe with fruits of insight that will have considerable impact not only in this field but in others as well. This paper centres on a model system we have been developing using Trichoplusia ni (Hiibner) as a host and several wasps from the genus Chelonus as parasites. Hosts stung by these egg-larval parasites precociously initiate metamorphosis during their penultimate stadium, and spin a cocoon as if for pupation. The parasite emerges from and kills the developmentally stationary host prepupa, and then spins its own cocoon within the cocoon or pupation cell of its host. About 10% of stung hosts prematurely initiate metamorphosis, and then cease development as prepupae, but yet contain no live or functional parasite during the coccon spinning stadium (“pseudoparasitism”). Thus, this host-parasite model system offers excellent opportunities concerning the hypothesis that some parasites regulate their host’s physiology.

precocious

There are currently some controversies in parasitology concerning the concept of parasite regulation of its host. One school of thought places a definition on “regulation” that is broad, perhaps too broad. Another school questions the validity of the regulation concept as “out-moded”. These positions question the very premise of the T. ni-Chelonus interaction as a model system of parasite regulation of host physiology. Thus, some space will be devoted to answering these two schools of thought in such a way as to firmly validate the study of the T. ni-Chelonus and other host-parasite interactions from a logical “host regulation” perspective. This paper is intended primarily as a review of our research on this host-parasite system, and its implications for concepts in parasitology and other fields. Some new data will also be presented, so as to facilitate a more complete delineation of this host-parasite system. It is hoped that the present paper will stir additional interest in an exciting field which has moved from the era of natural history observations to one of high-resolution experimental scrutiny. MATERIALS AND

METHODS

Insects

Host insects of T. ni were reared under conditions of 14 h: 10 h light:dark, at 28”C, as described by 315

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316

D. Jones (1986) and Shorey and Hale (1965). The parasite Chelonus near curvimaculatus and Chelonus insularis Cresson were reared as described by D. Jones (1986). The former species is from Ethiopia (D. Jones et al., 1981a), and in the interests of space will be tentatively referred to in this review as C. curvimaculatus. The latter species is indigenous to North America. Pseudoparasitized larvae were used for experimentation and were selected on the basis of size, head capsule width and other criteria (Fig. 1). Larvae of Manduca sexta were reared essentially as described by Bell and Joachim (1976). Chemicals Radiolabelled juvenile hormone was purchased from NEN. Cold juvenile hormone was obtained from Sigma or Cal-Biochem. Hot and cold hormone was verified by HPLC analysis by the supplier to be at least a 97% pure racemic preparation of the given juvenile hormone. The juvenile hormone analogues fenoxycarb and epofenonane were provided by Hoffmann-LaRoche. The juvenile hormone esterase inhibitor TFT (l,l, 1 trifluoro-tetra-deca-2-one) was synthesized in our laboratory by the method of Hammock et al. (1982) and shown by GC analysis to be > 99% pure. Radiolabelled ecdysone was obtained from NEN, while cold ecdysteroids were obtained from Simes (Milan, Italy). Juvenile hormone was C- 10, 3H-labe11ed.

al.

Chemical

treatments

Topical application of juvenile hormone, analogues, and esterase inhibitors was done in 1~1 ethanol. Control larvae were treated identically with ethanol alone. Induction of esterase in topically treated wandering larvae was measured 12 h after the previous application at 15 h after lights on. Protein electrophoretic

techniques

Narrow range (pH 4-6.5) isoelectric focusing was performed on an LKB multiphor apparatus with Ampholine”, as described by Winter et al. (1977). Twenty ~1 of haemolymph diluted 1: 1 with distilled H,O were applied to the gel on paper wicks. After 3 h the run was terminated and the gel sliced into 0.25 cm sections along the pH gradient. The gel sections were eluted overnight at 4°C in distilled H,O (for pH or phosphate buffer (pH 7.4, measurement) I = 0.2 M). The eluted esterase activity was then analyzed kinetically by Lineweaver-Burk plots. Agarose gel electrophoresis of viral DNA. The reproductive tract of female Chelonus curvimaculatus was dissected into 40mM Tris-20 mM sodium acetate-2mM EDTA buffer, pH 7.8. The ovaries were homogenized and centrifuged. The supernatant was treated with proteinase K (15 min, 37°C) and 2% SDS (15 min, 60°C) and then layered onto agarose gels. The gels were run at 25 V for 8-16 h, and the DNA stained with ethidium bromide.

Juvenile hormone esterase assay Total haemolymph juvenile hormone esterase activity was measured in vitro as described by Hammock and Sparks (1977), using a final substrate concentration of juvenile hormone III of 5 x low6 M. Kinetic analysis of the esterase activity in crude diluted blood or IEF-purified preparations was performed using up to 23 different substrate concentrations, ranging from 5 x 10e9 M to 9 x 1O-6 M. Juvenile hormone concentration

RESULTS

Increasedjuvenile hormone esterase activity in pseudoparasitized larvae Haemolymph esterase activity increases dramatically during the end of the feeding stage of larvae pseudoparasitized by either C. curvimaculatus (Fig. 2) or C. insularis (unpubl. data). The levels of activity

determination 50

The haemolymph juvenile hormone concentration was measured by the M. sexta black mutant bioassay (Fain and Riddiford, 1976). The standard curve was developed using juvenile hormone II, since this is the natural homologue in T. ni (G. Jones et al., unpubl. data). Extraction efficiency was monitored using radiolabelled juvenile hormone III as an internal marker. This hormone extracts with the same efficiency as juvenile hormone II, and was added at a concentration approaching the limit of detectability by bioassay. The low response of the bioassay larvae to the tracer was still subtracted from the overall juvenile hormone II equivalents detected. Ecdysteroid

40

30 20 10 0 PSEUDOPARASITZED

titre

The haemolymph ecdysteroid titre was determined by RIA, using well-characterized antiserum according to the procedure of Bollenbacher et al. (1975). The antibodies react with both ecdysone and 20-hydroxyecdysone, although with a 2-fold greater affinity for ecdysone (Bollenbacher, pers. commun.). The radiolabelled ligand in the assay was ecdysone and the standard curve was generated using various concentrations of unlabelled 20-hydroxyecdysone. Inferred haemolymph ecdysteroid concentrations are expressed in 20-hydroxyecdysone equivalents.

w-2

w-1

WANDER

W+l

Fig. 2. Haemolymph juvenile hormone esterase activity during the final feeding stage in larvae pseudoparasitized by C. curvimaculatus, 1 day prior to wandering. Variance is

represented as standard error. Three haemolymph pools of 3 larvae each are represented in each data point.

Fig. 1. Comparison of size differences of normal last-instar (the largest), truly parasitized penultim tateinst, ar (the smallest) and pseudoparasitized penultimate-instar (intermediate in size) Trichoplusia ni (4 Feel ding larvae 1 day prior to wandering and cocoon spinning. (b) Larvae on the day of wandering and cocoon spinning.

317

Fig. 5. Electrophoretic banding patterns of DNA extracted from virus extracted from the reproduc :tive tract of female. C. curvimaculutus. From left to right, each lane contains 2, 4, 8 and 16 female equivalents, respectively. Preliminary experiments showed that host chromosomal DNA does not stain sufficiently to be visible on these gels.

318

Fig,. 6. Precocious pupae obtained from larvae pseudoparasitized by C. insularis. The upper figure sho WS the commonly shortened wings of such pupae. The lower figure shows the commonly observed failure to shed the larval cuticle from around the pupal head parts.

319

321

Pseudoparasitism are very similar to that found during the normal last stadium. In contrast, the esterase activity during a normal penultimate stadium does not show this dramatic rise. On narrow range Ampholine@ isoelectric focusing gels, sliced into’ 0.5 cm fractions, the esterase activity focuses near pH 5.4 (5.4 + 0.1 in normal larvae, 5.4 + 0.1 in larvae pseudoparasitized by C. curvimaculatus). On very narrow range Immobiline” gels, the activity focuses into 2 peaks [PI = 5.6 and pI = 5.3 for normal larvae and p1 = 5.6 and p1 = 5.3 for pseudoparasitized larvae, respectively] (Rudnicka and Jones, 1985). When the esterase activity in diluted haemolymph from normal and pseudoparasitized larvae is analyzed kinetically, similar apparent K,‘s are found (D. Jones, 1983). Diluted haemolymph of both normal and pseudoparasitized larvae possesses juvenile hormone esterase activity with two K,%, one in the IO-*M range and one in the lo-’ M range, whereas both lose the lower K,,, following isoelectric focusing. Juvenile hormone concentration in normal and pseudo parasitized larvae Current study in our laboratory on the titre of juvenile hormone has found the activity in normal, penultimate instar larvae is at least lo-’ M (juvenile hormone II equivalents). Preliminary data on normal last instar and penultimate instar, pseudoparasitized larvae indicate that the hormone titre in these two groups, 1 day prior to ecdysteroid-induced wandering behaviour, is similar (approx 10esM) which is much less than that occurring in normal, penultimate-instar larvae. This bioassay titre is Mimilar to that observed by bioassay in another noctuid

~ L5D2

L5D4 --NORMAI m-C. ins. pi l --c. cur”. PI l

80

DOSE

Ro- 103108

lnml

Fig. 4. Dose-dependent increase in haemolymph juvenile hormone esterase activity in larvae pseudoparasitized by C. curuimaculatus, following topical application with the juvenile hormone analogue epofenonane (Ro 10-3108). Variance is represented as standard deviation. Five to eight larvae are represented in each point.

species, Spodoptera litura (Yagi, higher than some physico-chemical

1976), although measurements.

Prepupal ecdysteroid titre In liormal prepupae a large increase of haemolymph ecdysteroids begins to occur by the early photophase on the day of wandering. This increase peaks the following scotophase, at the marker of dense cocoon formation (G. Jones et al., 1985b). Prepupae pseudoparasitized by C. insularis show a similarly timed peak in ecdysteroids that is only about half the normal level (Fig. 3). The prepupal ecdysteroid peak in larvae pseudoparasitized by C. curvimaculatus is but one tenth that of normal prepupae (Fig. 3), although sometimes similar to that is C. insularis. Induction of prepupal juvenile hormone esterase by juvenile hormone application

I

When prepupae pseudoparasitized by C. curvimaculatus are treated with a epofenonane, haemolymph juvenile hormone esterase activity was increased in a dose-dependent manner (Fig. 4). Preliminary experiments with different juvenile hormone analogues or juvenile hormone II have yielded similar results (unpubl. data). Viral DNA

L4D2

L4D3

L4D4

Fig. 3. Haemolymph ecdysteroid titre in prepupae of normal larvae (a) and those pseudoparasitized by C. insularis (b) or C. curvimaculatus (c). Variance is represented as standard error. The averages plotted in each curve were determined from (a) 3 haemolymph pools, 3 insects/pool; (b) 6 insects per data point, and (c) 4-5 haemolymph pools, 3 insects/pool.

When DNA from the reproductive tract of female C. curvimaculatus was subjected to agarose electrophoresis, a number of bands were separated (Fig. 5). The DNA banding pattern of virus from C. insularis is different from that shown in Fig. 5 (D. Jones et al., unpubl. data). Experiments are currently underway to analyze the bands from within species and between species for the degree of sequence homology. DISCUSSION

The results obtained using the T. ni-Chelonus spp. model system are of relevance to a number of basic

322

DAVYJONESet al.

and applied considerations. In the following discussion we will review the T. ni-Chelonus system and consider the implications of the results for basic research on the nature of host-parasite interactions, the use of parasites as experimental probes of normal physiology, increased flexibility in biological control efforts and finally parasites as sources of novel insect control agents. Overview of the T. ni-Chelonus Precocious

initiation

system

of metamorphosis

Corpus allatum activity in pseudoparasitized larvae. Bioassay of corpus allatum activity has demonstrated that the activity of the glands in pseudoparasitized larvae, 1 day prior to precocious wandering, is significantly lower than that in normal penultimate instar larvae, 1 day prior to head-capsule slippage (D. Jones, 1985b). The activity of the former corpora allata was more similar to that occurring in normal last-instar larvae, 1 day prior to their wandering. Thus, decline in gland activity is at least a component of the precocious metamorphosis symptomology. Increased juvenile hormone esterase activity in pseu doparasitized larvae. The increase in haemolymph esterase activity in larvae pseudoparasitized by either species indicates several things. First, the similar timing of its appearance before wandering and of its kinetic properties to juvenile hormone esterase of normal, last-instar larvae demonstrates that it is the esterase of the host, T. ni, which is being expressed. It also suggests that increased juvenile hormone metabolism may be playing a role in the premature initiation of metamorphosis, but this must be tested. Effect of juvenile hormone esterase activity. The esterase activity in pseudoparasitized larvae functions in a manner similar to that in normal larvae. The rate of metabolism of endogenous juvenile hormone during the peak of esterase activity, as indicated by metabolism of small amounts of radiolabelled, injected juvenile hormone III, is similar in normal and pseudoparasitized larvae (6.2 Ifr 0.4% min and 6.0 &-0.3% min, resp.). The rate of juvenile hormone metabolism in normal penultimate-instar larvae is much lower at 4.9 + 0.2% min (D. Jones, 1985b). The peak of esterase activity in normal last-instar larvae is necessary to drive juvenile hormone sufficiently low to ensure metamorphic commitment at the next ecdysteroid peak. Inhibition of esterase in vivo with EPPAT in normal larvae will cause an extra larval moult (D. Jones and Sreekrishna, 1984; G. Jones, 1985). These supernumerary-instar larvae show aberrations in crochet, antennae and mouthpart development similar to supernumerary larvae generated from juvenile hormone treatment (G. Jones, 1985). The accumulation of endogenous juvenile hormone in larvae treated with EPPAT has been demonstrated by biosassay and physicochemical tecniques (G. Jones et al., unpubl. data). Treatment of pseudoparasitized larvae with juvenile hormone or EPPAT blocks precocious rnetamorphosis, and the larvae instead moult to another larval instar (D. Jones and Sreekrishna, 1984). These pseudoparasitized larvae show the same morphological aberrations as those in supernumerary juvenile hormone or EPPAT-treated, normal larvae. Treatment

of pseudoparasitized larvae with juvenile hormone or EPPAT also results in an abnormally large increase in size due to the extended feeding period (Jones and Jones, 1985; D. Jones et al., unpubl. data). Prothoracic gland sensitivity to juvenile hormone. During the feeding stage of the last stadium of Lepidoptera, juvenile hormone can inhibit ecdysone production by the prothoracic glands (Cymborowski and Stolarz, 1979; Safranek et al., 1980). There is also evidence that this hormone can act to inhibit prothoracicotropic hormone release by the brain (Nijhout and Williams, 1974; Roundtree and Bollenbather, 1984). Both actions of juvenile hormone would serve to postpone wandering behaviour as long as the hormone titre remains sufficiently high. The following discussion will refer to sensitivity of the prothoracic glands, although it is realized that the brain may be involved. During the final stadium of T. ni, wandering behaviour can be postponed by topical treatment with juvenile hormone, or by accumulation of endogenous juvenile hormone through treatment with a juvenile hormone esterase inhibitor (G. Jones, 1985; Sparks and Hammock, 1980). Treated larvae continue to grow beyond the normal fold increase in size, due to the delay in ecdysone production (G. Jones, 1985). Juvenile hormone content in the haemolymph of normal andpseudoparasitized larvae. Use of the black mutant bioassay showed the juvenile hormone titre in penultimate-instar, pseudoparasitized larvae is below that in normal, penultimate-instar larvae. Thus, the suppression of corpus allatum activity and increase in juvenile hormone metabolism act to prematurely lower the hormone content of pseudoparasitized larvae. The last instar increase in sensitivty of the glands to juvenile hormone is not caused by the last-instar decline in this hormone, since this increase in sensitivity occurs both in normal, last-instar larvae with a low titre of juvenile hormone and those continuously treated with this hormone beginning at the moult to the final instar. Pseudoparasitized larvae precociously show a similar increase in juvenile hormone inhibition of the prothoracic glands. Haemolymph proteins. There are a number of feeding stage, haemolymph proteins which appear or increase dramatically only during the final-larval stadium. Other proteins occurring during the penultimate stadium disappear or decrease dramatically during the final stadium. These changes in haemolymph proteins are seen on gels resulting from several electrophoretic techniques, such as electrophoresis, isoelectric focusing (wide range and narrow range Ampholine’& and very narrow Immobiline” gels) and SDS-PAGE electrophoresis (D. Jones and Sreekrishna, 1984; D. Jones, 1985b; D. Jones et al., unpubl. data). When haemolymph from feeding, prewandering larvae pseudoparasitized by either C. curvimaculatus or C. insularis is analyzed on such gels, it is seen that these penultimate-instar larvae are precociously showing the last-instar haemolymph protein profile. These results are obtained by either Coomassie Blue or silver staining or by staining for cr-napthyl acetate esterase activity. Growth threshold associated with jinal instar. The data presented above demonstrate the precocious

Pseudoparasitism expression of the last-instar feeding stage developmental pattern in penultimate-instar, pseudoparasitized larvae. This alteration of host physiology is not just a result of chemical allatectomy by the parasite, since several of the precocious events (high juvenile hormone esterase activity; high prothoracic gland sensitivity to juvenile hormone) are not driven by a decline in the juvenile hormone titre. The question remains: What causes the precocious expression of the last-instar developmental pattern? Normal larvae continue the pattern of larval-larval moults until reaching a threshold head capsule width associated with attainment of the final instar of 1.66mm (D. Jones et al., 1981b). In larvae pseudoparasitized by C. curvimaculatus this critical threshold has been lowered to N 1.02 mm (D. Jones, 1985b). Thus, pseudoparasitized larvae attain this lowered threshold at a preultimate instar, and are then triggered to precociously switch to the last-instar developmental pattern. This hypothesis is enhanced by the analysis of the morphology of normal and pseudoparasitized larvae. We have found morphological characters suitable for distinguishing each instar of T. ni from the other (D. Jones and Neary, unpubl. data). Using these characters it has been determined that no instar is “skipped” during development of pseudoparasitized larvae. Rather, it is the actual penultimate instar which precociously metamorphoses. The use of pseudoparasitized larvae offers new opportunities in probing the regulation of normal development in feeding larvae. For example, surgical or chemical allatectomy of penultimate-instar larvae, to cause precocious juvenile hormone decline, has been a popular approach to the study of certain last-instar biochemical events. However, a number of biochemical events during the final stadium are not regulated by juvenile hormone decline. Larvae pseudoparasitized by chelonine wasps offer, for the first time, a tool with which to utilize the precocious induction of last-instar events not regulated by the decline of juvenile hormone. Also, the mechanism by which Lepidopteran larvae gauge their size, and perceive attainment of the final instar, is a problem with which insect physiologists have grappled for a decade. The apparent alteration of the last-instar growth threshold in pseudoparasitized larvae may present us with a new approach to answering this difficult question. Suppressed development in pseudoparasitizedprepupae Juvenile hormone esterase activity in prepupae. During the feeding stage of the final instar of T. ni juvenile hormone esterase is under the regulation of an inducing factor released by the brain and suboesophageal ganglion (G. Jones et al., 1981). Once larvae have reached a sufficient fold increase in size, a head-centred inhibitory factor acts during the late photophase before ecdysone release and shortly thereafter to drive the esterase activity down to a very low level (G. Jones et al., 1981; G. Jones and Click, unpubl. data). When the ecdysteroid titre rises to cause cellular reprogramming for metamorphosis, it also reprogrammes the source of haemolymph juvenile hormone esterase (fat body, Wing et al., 1981; Click et al., 1985) to produce esterase when challenged with juvenile hormone (G. Jones and Click,

323

unpubl. data). Juvenile hormone from active corpora allata will cause haemolymph esterase activity to appear in T. ni prepupae (G. Jones and Hammock, 1983; Sparks, 1984), as will exogenous juvenile hormone (G. Jones and Hammock, 1983; Sparks and Hammock, 1979). If prepupal production of juvenile hormone is stopped by surgical (G. Jones and Hammock, 1983) or chemical allatectomy (Sparks, 1984), then the normal prepupal peak of esterase will not occur. It can be restored by juvenile hormone application (G. Jones and Hammock, 1983; Sparks, 1984). Thus, the level of juvenile hormone esterase activity is an index of the amount of prepupal juvenile hormone present. In prepupae pseudoparasitized by either C. curvimaculatus or C. insularis the titre of juvenile hormone esterase is very low. Application of exogenous juvenile hormone will induce esterase activity in a dosedependent manner in larvae pseudoparasitized by either the former (Fig. 4) or latter (D. Jones, 1985a) parasite. In view of the data summarized above on the esterase in normal prepupae, this situation implies an abnormally low titre of juvenile hormone in pseudoparasitized prepupae. It should be noted that in those pseudoparasitized by C. insularis there is a slight, but much below normal, increase in haemolymph juvenile hormone esterase at the same physiological time as occurs in normal prepupae (D. Jones et al., 1981b; D. Jones, 1985a). Role of juvenile hormone in prepupal development.

When normal larvae are allatectomized, they cease or greatly slow development at the developmental marker of ocellar pigment withdrawal (G. Jones and Hammock, 1984, 1985). Pseudoparasitized larvae which are left untreated also show suppression of development at this same physiological marker (D. Jones et al., 1981b; D. Jones, 1985a). When either allatectomized normal prepupae or prepupae pseudoparasitized by either parasite are treated with juvenile hormone or an analogue, development toward the pupal moult is enhanced (G. Jones and Hammock, 1984, 1985; D. Jones, 1986). Treated prepupae pseudoparasitized by C. insularis develop much more towards the pupal moult than those pseudoparasitized by C. curvimaculatus (D. Jones, 1985a,b). When normal prepupae of T. ni or some other Lepidoptera are allatectomized, those few which pupate often fail to completely shed the larval cuticle from the head. Those which do ecdyse usually possess shortened wings (G. Jones and Hammock, 1985; Williams, 1961; Kiguchi and Riddiford, 1978). Similar effects occur in precocious pupae generated by allatectomy of penultimate-instar T. ni (n = 8). Occasionally untreated prepupae pseudoparasitized by C. insularis reach the pupal moult, and they show symptoms similar to those in allatectomized, normal insects (Fig. 6). These and the above data strongly suggest that the juvenile hormone titre in pseudoparasitized prepupae is below that in normal prepupae. Juvenile hormone-driven ecdysteroid peak. In normal T. ni the prepupal peak of juvenile hormone acts to prompt an increase in the haemolymph ecdysteroid titre. Exogenous application of juvenile hormone or endogenous hormone accumulation due to EPPAT

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treatment causes an increase in the haemolymph ecdysteroid titre (G. Jones et al., 1986). Similarly, application of juvenile hormone to pseudoparasitized prepupae causes an increase in ecdysteroid concentrations (D. Jones et al., unpub. data). These data corroborate the hypothesis that the suppression of prepupal development involves an interaction between juvenile hormone and ecdysteroids. Ecdysteroids in normal and pseudoparasitized prepupae. The prepupal peak in ecdysteroids which drives normal prepupal development was measured in this study to reach nearly 800 ng/ml of haemolymph, while that in larvae pseudoparasitized by C. insularis was approximately half that. These results are similar to the values of approx 700 ng/ml and 350 ng/ml obtained for such larvae in another study (D. Jones, 1985a). The data presented here for larvae pseudoparasitized by C. curvimaculatus show the haemolymph ecdysteroid titre in these prepupae is far below normal. If suppression of prepupal ecdysteroids causes suppression of prepupal development, then ecdysteroid injection would be predicted to cause resumed development. Indeed, injection of either ecdysone or 20-hydroxyecdysone promoted additional development in either kind of pseudoparasitized larva (D. Jones, 1986). We are occasionally able to induce complete pupal moults in injected larvae, more often in those pseudoparasitized by C. insularis. These completely ecdysed pupae possess shortened wings. This morphology is seen in allatectomized unparasitized larvae and symptomatic of a low prepupal juvenile hormone titre. These pupae do not eclose to adults, although dissections reveal well developed, pharate moths. The development of these insects to the pharate adult prior to their death provides clues as to the endocrine lesions in pseudoparasitized pupae. We are attempting to obtain pseudoparasitized adults, and are eager to test the possibility that such adults will give rise to pseudoparasitized offspring. It is also of interest that injection of very high levels of ecdysteroids initially accelerates prepupal development through early prepupal markers, but then blocks development through subsequent markers. This situation probably has a basis similar to that found in experiments on normal M. sexta, in which high ecdysteroid levels block release of eclosion hormone (Truman, 1981). Endocrine basis of suppressedprepupal development. The above data indicate that the primary lesion in developmentally stationary prepupae is an abnormally low ecdysteroid titre. This effect is in turn probably due to a suppression in the titre of juvenile hormone. This hypothesis is corroborated by the intermediate endocrine and developmental events in larvae pseudoparasitized by C. insularis. These pseudoparasitized prepupae, although showing suppressed development, naturally approach closer to the pupal moult than those pseudoparasitized by C. curvimaculatus. Prepupae pseudoparasitized by C. insularis show a titre of juvenile hormone esterase activity intermediate between normal larvae and those pseudoparasitized by the other parasite. These prepupae also show an ecdysteroid titre intermediate between normal prepupae and those pseudoparasitized by the other parasite. The correlation

between the degree of prepupal development and the level of biochemical and endocrine components regulating prepupal development is strong evidence in favour of the above hypothesis for the basis of developmental effects in pseudoparasitized prepupae. The use of pseudoparasitized prepupae offers an independent means of testing hypotheses on regulation of normal prepupal development. For example, earlier work using classical endocrine techniques indicated that juvenile hormone was necessary for completion of ecdysteroid-driven prepupal development (G. Jones and Hammock, 1983, 1985). The new results obtained from the pseudoparasitized system are completely consistent with this hypothesis. The induction of prepupal esterase by the juvenile hormone from active corpora allata has led to propositions of its use as a bioassay of corpus allatum activity (D. Jones and Sreekrishna, 1984), and it has been used to measure endogenous gland activity (G. Jones and Hammock, 1983; Sparks, 1984). Pseudoparasitized prepupae are a system in which the activity of implanted corpora allata or topically applied juvenile hormone agonists can be assayed with little interference from endogenous juvenile hormone. These pseudoparasitized prepupae may be similarly used to probe the interaction between prepupal juvenile hormone and ecdysteroids, or ecdysteroids and eclosion hormone. Since surgical removal of prothoracic glands is logistically difficult, researchers must resort to thoracic ligations to obtain abdomens with a low ecdysteroid titre. Such ligations unfortunately remove the brain and other head structures from contact with the abdomen. Pseudoparasitized larvae offer an attractive system in which the in situ interaction between the head and abdomen can be investigated under conditions of low ecdysteroids. In fact, test injections of various ecdysteroid concentrations can be replaced by use of a species of Chelonus which causes the desired reduction of endogenous ecdysteroids. Pseudoparasitism in host-parasite interactions The phenomenon of pseudoparasitism adds an intriguing aspect to the study of parasite regulation of host physiology. We have defined pseudoparasitism as the expression of the physiological lesions of parasitism at a time when the parasite is not present (D. Jones et al., 1984a; D. Jones, 1985a,b,c). More specifically, the lesions are manifest when there is at least no live or functional parasite present. Pseudoparasitism may have a number of possible bases including: (1) early release of regulatory factors by the parasite egg or immature parasite and subsequent parasite death (2) release of regulatory factors by teratocytes independently of the presence or absence at that time of a live or functional parasite (3) the action of venom or other soluble components in the oviposition fluid (4) the action of viruses in the oviposition fluid which replicate in the reproductive tract of the adult female parasite (5) mechanical injury to the host as a result of insertion of the female ovipositor. The occurrence of host regulation in the absence of a live or functional parasite may also be due to an interaction of some combination of the above factors. In our system pseudoparasitism occurs

Pseudoparasitism

naturally. In some other host-parasite interactions it apparently must be artificially induced (Shaw, 1981). We are currently investigating the above as possible bases of pseudoparasitism in larvae stung by Chefonus species. Mechanical injury to the eggs of T. ni by pin-pricking, to simulate oviposition, has failed to induce pseudoparasitism. Likewise stresses of temperature or malnutrition have failed to induce pseudoparasitism in T. ni. Teratocytes appear to be a possibility, although we and another laboratory have been unable to document their occurrence in Chelonus (Strand, pers. commun.). Also, Hawlitsky has not been able to demonstrate their presence in the closely related genus Phanerotoma (Hawlitsky, pers. commun.). Both species of Chelonus used in this study possess DNA-viruses in the female reproductive tract (e.g. Fig. 5). We have found that these viruses have entered hosts destined to be pseudoparasitized, and injection of these viruses into eggs by females is now being studied. Our studies have found that the percentage of stung hosts in which the parasite dies is subject to experimental manipulation, but the role of the virus is uncertain. Implications of results from T. ni-Chelonus system Controversies in interpretation of host-parasite interactions. The concept of parasite regulation of host physiology to the benefit of the parasite is at present a very controversial hypothesis. Validation of this hypothesis entails both (1) identification of specific mechanisms of intervention in host physiology by the parasite, including target biochemical or endocrine pathways, and (2) demonstration that such induced changes in the host confer an advantage to the parasite, either ecologically or physiologically. We have used the T. ni-Chelonus system to test the hypothesis of host regulation from both physiological and ecological perspectives. However, two schools of thought within parasitology maintain positions that question the validity of such an approach. Before discussing the relevance of our findings to the hypothesis, we must first address these two schools of thought. Parasite regulation of host physiology: A valid con cept. One school of thought, most promoted within entomological circles by Thompson (1984, 1985), places extreme emphasis on the “integrative” nature of host-parasite interaction. This school maintains (1) the notion of parasite regulation of the host to the parasite’s benefit embraces the “outmoded” concept of benefit-harm (2) the concept of benefit-harm is anthropomorphic, subjective and difficult to demonstrate (3) some parasites enhance, rather than retard, host growth which is to the host’s benefit (4) the integrative nature of host-parasite associations is the “essence” of parasitism, and (5) the concept of parasite regulation of the host fails to consider whether or not the host-parasite complex forms a distinct, integrative entity significant to the success of the association. These objections must be addressed, since they undercut the validity of the very hypothesis we are testing, that Chelonus wasps regulate their host’s physiolagy. First, many parasitologists COnStrUCtiVdy use harm-benefit criteria in their studies (Pappas, 1980; Wood, 1983; Goff and Coleman, 1984; Moul-

325

der, 1979), including one cited by Thompson as against the benefit-harm concept (Starr, 1975). Second, there is no anthropomorphism or subjectivity in describing the venom used by a cobra to subdue its prey as benefitting the cobra and harming the prey. Similarly, there should be no objection to applying benefit-harm criteria to the interaction where an endoparasite secretes a chemical mediator which regulates the physiology of its host to its own benefit. Use of quotes around words like “direct” in the phrase “direct” regulation of the host will signify, if it is not already clear from the context, that anthropomorphic intent is not present. Also, the difficulty in testing a hypothesis should not be a criterion for whether or not it was valid to advance the hypothesis in the first place. Third, “enhanced growth” of hosts should not be synonymized with benefit to the host and not the parasite since, for example, a normalsized gregariously parasitized host may not satisfy the food requirements of its parasites (Beckage and Riddiford, 1983). Fourth, the integrative nature of some host-parasite associations may be an important, or even dominant, aspect of the interaction (e.g. mitochondria as symbiotes of eukaryotic cells). However, if a regulatory signal from the parasite is required to move the physiology of the unparasitized host toward the new (coupled) physiology of the host-parasite association, then the importance of parasite regulation of the host should not be minimized. Finally, the concept of parasite regulation of the host compliments the concept on “integrated physiologies” by enabling us to understand why the physiology of the host is changed so as to permit the new, coupled, integrative aspect to even occur. Regulation versus stress effects: qualitatively dQj%rentphenomena. Another school of thought, promoted most vigorously within entomological circles by Vinson (Vinson, 1975; Vinson and Iwantsch, 1980) maintains that (1) “indirect stress-effect” is a catch-all for uninvestigated or unexplained phenomena (2) whether the action is direct manipulation or an indirect stress effect, the parasite is “subverting” some aspect(s) of the host’s developmental programme (3) both direct regulation and indirect stress effects should be collectively referred to as host regulation (4) parasite regulation of the host (as in 3) is necessary for successful parasitism, and (5) parasite regulation of the host is a feature of insect parasites (parasitoids) which separates them from other (nonparasitoid) parasite-host associations. These objections must also be dealt with, since they severely detract from the usefulness of the concept of regulation of the host. First, slower than normal growth in parasitized hosts has been demonstrated to be due to nutrient stress, in a number of systems (e.g. vitamin B12 deficiency during tapeworm infection, Nyberg, 1963). Second, the use of adjectives such as “subverting” in the above context implies “direct” manipulation by the parasite without first testing for such an effect. Third, collectively lumping together efIects of stress and regulation will (a) hinder elucidation of possible evolutionary pathways a host-parasite relationship may take (b) hinder our ability to reconstruct evolutionary phylogenies between parasite groups (c) prevent predictive ability on the anticipated results of novel host-parasite combi-

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nations (d) hinder identification of which parasites can serve as novel sources of bioactive substances (e) eliminate the use of parasites which cause specific biochemical lesions as probes of these biochemical pathways in normal development, and (f) prevent heuristic comparisons between host-parasite systems (e.g. why an insect parasite can induce precocious host metamorphosis but no metazoan parasite of vertebrates causes precocious host metamorphosis or puberty). Fourth, just because parasite regulation of the host occurs in one system it does not follow that it must occur in all host-parasite systems. Finally, the release of a mimic of the vertebrate growth hormone by the tapeworm, Spirometra masonoides, (Mueller, 1980) shows that regulation of the host can occur in associations other than insect host-parasitoid associations. The above discussion shows the validity of the concept of parasite regulation of its host, provided the term “regulation” is not defined to include indirect stress effects. T. ni-Chelonus spp. as a model system for parasite regulation of host The T. ni-Chelonus spp. interaction is a particularly suitable model system for testing the hypothesis that parasites regulate the host to their own advantage. The interaction is rich in regulatory aspects in that two different acts of regulation of the host are involved: Precocious initiation of the last-instar developmental programme and then suppression of development of the precocious prepupae. The occurrence of these events in pseudoparasitized hosts may indicate that parasite defence plays an important role. The kinds of regulations of the host which occur are interesting and have important implications for a number of other fields (see below). Since much of the physiological work involved in this system involves use or the occurrence of pseudoparasitized hosts, and since pseudoparasitism may occur in other host-parasite systems, the nomenclature to be used should be defined. If it is shown that viruses cause the endocrine effects seen in pseudoparasitized hosts, then the phenomenon is “viral-pseudoparasitism”. If the cause is found to originate with wasp venom, it is “venomouspseudoparasitism”, and so on. Implications

of current

knowledge

of T. ni-Chelonus

system The insect host-parasite systems involving regulation of the host offer new opportunities for bringing together this discipline and that of insect control. Parasites which use a paralyzing venom to immobilize their hosts have been considered as sources of insecticidal factors (Beard, 1971), but use of parasite factors regulating insect endocrinology seems not to have been vigorously addressed. Location of an anti-juvenile hormone factor suitable for commercial use has remained one of the most elusive applied entomological goals in the last 2 decades (Williams, 1967). The lepidopteran host range of the Cheloninae includes at least 15 families (D. Jones, 1985c) and the occurrence of precocious pupation in pseudo-

parasitized larvae enhances the prospect for use of chelonine factors as model insecticides or agents engineered into crop plants. Some parasitologists have felt “ . . . it seems nigh impossible to predict how a parasite may behave in a new host. . . ” (Read, 1970). In fact, many biological control efforts have probably failed because of physiological incompatibility of the host and parasite (Debach and Bartlett, 1964). However, an understanding of the regulatory offences of the parasite and the host biochemical pathways which must be present as templates to be regulated for parasite survival will enable predictions on the physiological incompatibility on a proposed new host-parasite combination. Such knowledge will also be useful in situations where, of the different effects of host regulation (e.g. precocious pupation, extra larval instars, etc.), one effect would be most desirable (D. Jones, 1985d). wish to thank Drs Tom Ashley, Davies for supplying some of the Chelonus insularis used in this study. Dr Fred Legner provided the original females for the colony of Che/onus near curvimaculatus. Dr Carroll Williams has provided stimulating discussion and helpful assistance on several aspects of this study. Drs Larry Gilbert and Walter Bollenbacher kindly provided the ecdysteroid antiserum. Dr Philip Magee, Shell Chemical Co., generously provided the juvenile hormone esterase inhibitor EPPAT. Dr Albert Ellingboe provided a stimulating presentation on the concepts of host and parasite offence and defence at a Cold Spring Harbor seminar attended by the senior author. Our present colony of the black mutant of M. sexta began with eggs provided by Dr Lynn Riddiford, although Zoecon Corp. provided some larvae used in preliminary experiments. Numerous other researchers offered suggestions on examples of parasite regulation of host physiology, and another provided important lessons in science relative to this parasite project. This paper is in connection with a project of the Kentucky Agricultural Experiment Station (No. 85-7-135) and is published with approval of the Director, and was supported, in part, by NIH Grant No. l-ROl-GM3399501 and USDA Cooperative Agreement No. 58-7B30-3-539. Acknowledgements-We

‘Joe Lewis and Frank

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