Growth-blocking peptide titer during larval development of parasitized and cold-stressed armyworm

Growth-blocking peptide (GBP) has been isolated for the first time from the haemolymph of the host armyworm Pseudaletia separata whose development was halted in the last larval instar stage by parasitization with the parasitoid wasp Cotesia kariyai. Recent studies demonstrated that GBP not only exists in the plasma (haemolymph without cells) of parasitized last instar larvae, but also in the plasma of nonparasitized penultimate (5th) instar larvae. Monoclonal antibodies were prepared to measure the titers of GBP in nonparasitized and parasitized larval plasma. One of three monoclonal antibodies raised against GBP, which is the most specific for GBP, was used to quantify the concentration of plasma GBP. As this antibody recognized two plasma peptides other than GBP in crude plasma fractions, each plasma peptide fraction was separated by a reversed phase HPLC, and then plasma GBP level was measured by ELISA. The highest level of plasma GBP detected on Day 0 of the penultimate instar larvae was gradually decreased throughout the larval growth except for the temporary increase on Day 0 of last larval instar. After parasitization on Day 0 of last larval instar, two peaks of plasma GBP titer were detected during the last larval instar, one day and six days after parasitization. This characteristic increase and decrease in plasma GBP level was also observed by transferring last instar larvae of the armyworm from 25 to 10°C, as a result of which larvae delayed pupation by more than 15 days. From these results, it is reasonable to propose that plasma GBP in lepidopteran larvae might control certain upstream steps in a cascade of events leading to pupation; thus, an elevated level of plasma GBP interferes with normal metamorphosis from larvae to pupae.     

Influence of the braconid Glyptapanteles liparidis on the juvenile hormone titer of its larval host,the gypsy moth,Lymantria dispar

The increase in the juvenile hormone (JH) III titer in the hemolymph of Lymantria dispar larvae that were parasitized by the endoparasitoid braconid, Glyptapanteles liparidis, during the host's premolt to third instar, coincided with the molt of the parasitoid larvae to the second instar between day 5 and 7 of the fourth host instar. It reached a maximum mean value of 89 pmol/ml on day 7 of the fifth instar while it remained below 1 pmol/ml in unparasitized larvae. Only newly molted fifth instar hosts showed a low JH III titer similar to that of the unparasitized larvae. JH II, which is the predominant JH homologue in unparasitized gypsy moth larvae, also increased relative to controls in the last two samples (days 7 and 9) from parasitized fourth and fifth instars. Compared to unparasitized larvae, a generally reduced activity of JH esterase (JHE) was found in parasitized larvae throughout both larval stages. The reduction in enzyme activity at the beginning and at the end of each instar, when the JHE activity in unparasitized larvae was high, may be in part responsible for the increased JH II and JH III titers in parasitized larvae. Ester hydrolysis was the only pathway of JH metabolism in the hemolymph of unparasitized and parasitized gypsy moth larvae as detected by chromatographic assays. © 1996 Wiley-Liss, Inc.     

Endocrine alteration and precocious premetamorphic behaviors in the greater wax moth larvae,Galleria mellonella,parasitized by an endoparasitoid,Apanteles galleriae

Parasitization of Galleria mellonella (Lepidoptera: Pyralididae) larvae by a larval endoparasitoid Apanteles galleriae (Hymenoptera: Braconidae) leads to the precocious expression of premetamorphic behavior in the sixth (normally penultimate) instar host larvae prior to the parasitoid's emergence. We investigated the role of parasitization with A. galleriae on the alteration of development and/or behavior of its host. The ecdysteroid titer in the hemolymph of parasitized sixth instar larvae (the last instar of parasitized larvae) was higher than that of unparasitized ones, and the high ecdysteroid concentrations induced premetamorphic behaviors such as wandering and cocoon spinning. However, the epidermis of the parasitized larvae was not pupally committed through this stage. The activity of JH esterase in the parasitized larvae remained low, and application of a JH analogue to these larvae caused the production of a larval-type cocoon. These facts suggest that the parasitization by A. galleriae induces precocious premetamorphic behaviors of G. mellonella larvae by changing host endocrine conditions without causing the typical larval-pupal metamorphosis. Arch. Insect Biochem. Physiol. 34:257–273, 1997. © 1997 Wiley-Liss, Inc.     

Endokrinologische Untersuchungen an dem Wirt-Parasit-System: Pieris brassicae-Apanteles glomeratus

Die von Apanteles glomeratus L. parasitierten Raupen von Pieris brassicae zeigen in Abhängigkeit des Parasitierungstermins eine deutliche Veränderung des Juvenilhormon (JH)-Titer-Verlaufs während des letzten Larvenstadiums. Dabei tritt ein steiler Anstieg des JH-Gehaltes der Wirtshämolymphe im Zusammenhang mit der Häutung der Parasitenlarven vom 1. zum 2. Larvenstadium auf. Aufgrund von Ligations-experimenten konnte nachgewiesen werden, daß die Parasitenlarven selbst für den erhöhten JH-Titer ihrer Wirtsraupen verantwortlich sind, indem sie während ihrer Häutungsphase anscheinend JH in die Wirtshämolymphe abgeben.Eine durch die Parasitierung gesteigerte Syntheseaktivität läßt sich aus den Befunden histologischer Schnitte der Corpora allata frühparasitierter Raupen nicht feststellen. Dagegen weisen die Prothoraxdrüsen parasitierter Raupen zur Mitte des letzten Stadiums eine deutlich kleinere Querschnittsfläche auf als unparasitierte Tiere. Eine dadurch im Zusammenhang mit dem erhöhten JH-Titer bestehende Beziehung zur Häutungsunfähigkeit parasitierter Pieris-Raupen am Ende des letzten Larvenstadiums wird diskutiert.

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Summary The effects of parasitism by Apanteles glomeratus on the hemolymph juvenile hormone (JH) titers of Pieris brassicae during the last larval instar were determined using the Galleria bioassay.Depending on the time of parasitization, a significant increase of the JH titer could be observed when moulting of the parasites from the first to the second larval instar occurred.As neck-ligatured, parasitized Pieris larvae showed a similar increase of the JH titer at this time, it is concluded that the parasite larvae themselves are responsible for the elevation of the titer by delivering JH during their ecdysis into the host's hemolymph.This is supported by histological results from the corpora allata of parasitized and unparasitized caterpillars at the first and third day of the last larval instar, indicating no differences in its secretory activity. The prothoracid glands of parasitized host larvae, however, appear significantly smaller than those of comparable unparasitized ones in the middle of the last instar. A reduced secretory activity at this time, which is assumed from their decreased size, combined with an elevated JH titer may explain why parasitized larvae fail to moult at the end of their larval development.

     

Juvenile hormone synthesis, metabolism, and resulting haemolymph titre in Heliothis virescens larvae parasitized by Toxoneuron nigriceps

Last instar larvae of the tobacco budworm, Heliothis virescens F., fail to pupate and have little 20-hydroxyecdysone when parasitized by Toxoneuron nigriceps (Viereck). In this paper, we extend these observations to juvenile hormone (JH) to determine if parasitism by this wasp affects other endocrine systems. To this end, we compared the production of JH by corpora cardiaca-corpora allata complexes (CC-CA), the metabolism of JH by haemolymph enzymes, and the haemolymph titre of JH in parasitized and non-parasitized control larvae of H. virescens during the last larval instar. CC-CA from parasitized and control larvae had similar peaks of JH synthesis on day 1 of the fifth instar, with JH II accounting for more than 90% of total JH in both groups. On subsequent days, JH synthesis dropped to undetectable levels more quickly in non-parasitized controls than in parasitized larvae. JH metabolism by haemolymph of parasitized and control animals increased from low levels on day 1 of the fifth instar to high levels on days 2 and 3 of the instar. JH metabolism was significantly higher in control larvae than in parasitized larvae. After day 3, JH metabolism decreased in both groups, but was significantly higher in parasitized larvae. The major metabolite of JH in both groups was JH acid, though traces of JH diol and JH acid diol were also detected. The haemolymph titre of JH in both groups peaked on day 1 of the fifth instar and, similar to the synthesis of JH by CC-CA, decreased more rapidly in control larvae. As a result, non-parasitized animals had significantly lower JH titres on day 2. The higher JH titres observed in parasitized larvae during the early fifth instar may contribute to their developmental arrest. The possible role of these JH alterations in the host developmental and metabolic redirection is discussed and a more comprehensive physiological model accounting for host-parasitoid interactions is proposed.     

Tissue-specific regulation of juvenile hormone esterase gene expression by 20-hydroxyecdysone and juvenile hormone in Bombyx mori

Juvenile hormone esterase (JHE) is the primary juvenile hormone (JH) metabolic enzyme in insects and plays important roles in the regulation of molt and metamorphosis. We investigated its mRNA expression profiles and hormonal control in Bombyx mori larvae. JHE mRNA was expressed at the end of the 4th and 5th (last) larval instars in the midgut and in all the three (anterior, middle, posterior) parts of the silk gland. In the fat body, JHE expression peaked twice in the 5th instar, at wandering and before pupation, while it gradually decreased through the 4th instar. When 20-hydroxyecdysone (20E) was injected into mid-5th instar larvae, JHE mRNA expression was induced in the anterior silk gland but suppressed in the fat body. Topical application of a juvenile hormone analog fenoxycarb to early-5th instar larvae induced JHE expression in both tissues. In the anterior silk gland, JHE expression was accelerated and strengthened by 20E plus fenoxycarb treatments compared with 20E or fenoxycarb single treatment, indicating positive interaction of 20E and JH. JHE mRNA is thus expressed in tissue-specific manners under the control of ecdysteroids and JH.     

Inhibition of juvenile hormone esterase activity in Lymantria dispar (Lepidoptera, Lymantriidae) larvae parasitized by Glyptapanteles liparidis (Hymenoptera, Braconidae)

Glyptapanteles liparidis is a gregarious, polydnavirus (PDV)-carrying braconid wasp that parasitizes larval stages of Lymantria dispar. In previous studies we showed that parasitized hosts dramatically increase juvenile hormone (JH) titers, whereas JH degradation is significantly inhibited in the hemolymph. Here we (i) quantified the effects of parasitism on JH esterase (JHE) activity in hemolymph and fat body of penultimate and final instars of L. dispar hosts and (ii) assessed the relative contribution of individual and combined wasp factors (PDV/venom, teratocytes, and wasp larvae) to the inhibition of host JHE activity. The effects of PDV/venom was investigated through the use of gamma-irradiated wasps, which lay non-viable eggs (leading to pseudoparasitization), while the effects of teratocytes and wasp larvae were examined by injection or insertion of these two components in either control or pseudoparasitized L. dispar larvae. Parasitism strongly suppressed host JHE activity in both hemolymph and fat body irrespective of whether the host was parasitized early (premolt-third instar) or late (mid-fourth instar). Down-regulation of JHE activity is primarily due to the injection of PDV/venom at the time of oviposition, with only very small additive effects of teratocytes and wasp larvae under certain experimental conditions. We compare the results with those reported earlier for L. dispar larvae parasitized by G. liparidis and discuss the possible role of JH alterations in host development disruption.     

Relationship between an increase of juvenile hormone titer in early instars and the induction of diapause in fully grown larvae of Sesamia nonagrioides

The larvae of Sesamia nonagrioides (Lepidoptera: Noctuidae) grown at 25 degrees C and long photoperiod (16:8h light:dark) pupate in the 5th or 6th (mostly) larval instar, while the larvae reared under a short photoperiod (12:12h) enter diapause during which they consume some food and undergo up to 12 (usually 3-4) stationary larval molts. Diapause programming includes an increase of juvenile hormone (JH) titer in the hemolymph from about 20 to 50 nM in the 4th and 5th instar larvae (titer in earlier instars was not measured). JH I, II, and III are present in approximate ratio 1-2:10:1. The JH titer drops to zero before pupation but remains around 20 nM during diapause. Perfect extra larval molts associated with a body weight increase can be induced in the non-diapausing larvae with a JH analogue (JHA). The weight rise is due to accumulation of reserves and not to a general body growth. The timing of extra molts is similar to the molting pattern of the diapausing larvae only when JHA is present since early larval instars. In the diapausing larvae, JHA application affects neither molting periodicity nor the body weight. It is concluded that (1) Increased JH titer in early larval instars is a part of diapause programming; (2) The extension of larval stage in the diapausing larvae, but not the timing pattern of extra molts, is due to continuously high JH titer; (3) The diapause program includes low food intake, maintenance of a certain body weight, and periodic larval molts.     

Structure of a growth-blocking peptide present in parasitized insect hemolymph

Last instar larvae of the insect armyworm, Pseudaletia separata, parasitized with the parasitoid wasp, Apanteles kariyai, do not initiate metamorphosis and, ultimately, the wasp larvae emerge from the host larvae about 10 days after parasitization (Tanaka, T., Agui, N., and Hiruma, K. (1987) Gen. Comp. Endocrinol. 67, 364-374). It is necessary for the parasitoid wasp to perturb the armyworm's endocrinological processes that control normal metamorphosis from larvae to pupae. This endocrinological perturbation allows the parasitoid to complete its larval growth before emerging from the host larvae. It is obligatory for the parasitoid larvae to emerge while the host is still in a larval stage because the sclerotized pupal cuticle is impenetrable for the parasitoid larvae. A growth-blocking peptide with repressive activity against juvenile hormone esterase has been proven to exist in the parasitized host larval plasma (Hayakawa, Y. (1990) J. Biol. Chem. 265, 10813-10816). Here, I describe the detailed structure of this peptide and also the corresponding synthetic peptide to confirm this structure.     

Chilled Galleria mellonella larvae: mechanism of supernumerary moulting

ABSTRACT. Supernumerary larval instars were produced when Galleria mellonella L. (Lepidoptera) larvae were chilled at 0°C. Although sensitivity to cooling stress of the last instar and younger larvae were generally the same, only penultimate and the last instar larvae showed a significant correlation between their age and the number of additional larval moults. Chilling stress induced a rapid and persistent increase in the JH titre of the last instar larvae. Severing the ventral nerve cord resulted in a predictable loss of the ability to produce supernumerary moults in chilled last instar larvae. The data suggest that sensory input stimulates allatotropic hormone secretion by the brain of chilled larvae. The possible mechanism controlling supernumerary moulting is discussed.     

Endocrine changes associated with metamorphosis and diapause induction in the yellow-spotted longicorn beetle, Psacothea hilaris

At 25 degrees C and under a long-day photoperiod, all 5th instar Psacothea hilaris larvae pupate at the next molt. Under a short-day photoperiod, in contrast, they undergo one or two additional larval molts and enter diapause; the 7th instar larvae enter diapause without further molt. The changes in hemolymph juvenile hormone (JH III) titers, JH esterase activity, and ecdysteroid titers in pupation-destined, pre-diapause, and diapause-destined larvae were examined. JH titers of the 5th instar pupation-destined larvae decreased continuously from 1.3 ng/ml and became virtually undetectable on day 13, when JH esterase activity peaked. Ecdysteroids exhibited a small peak on day 8, 1 day before gut purge, and a large peak on day 11, 2 days before the larvae became pre-pupae. The two ecdysteroid peaks are suggested to be associated with pupal commitment and pupation, respectively. JH titers of the 5th instar pre-diapause larvae were maintained at approximately 1.5 ng/ml for 5 days and then increased to form a peak (3.3 ng/ml) on day 11. JH esterase activity remained at a low level throughout. Ecdysteroid levels exhibited a large peak of 40 ng/ml on day 18, coincident with the larval molt to the 6th instar. JH titers of the 7th instar diapause-destined larvae peaked at 1.9 ng/ml on day 3, and a level of approximately 1.1 ng/ml was maintained even 30-60 days into the instar, when they were in diapause. Ecdysteroid titers remained approximately 0.02 ng/ml. Diapause induction in this species was suggested to be a consequence of high JH and low ecdysteroid titers.     

Stimulated synthesis of the female-specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog

Application of methoprene to fourth (penultimate) instar larvae of the silkworm Bombyx mori induced the appearance of the feeding dauer larvae at the fifth (last) instar and prevented pupal metamorphosis. Methoprene also increased the protein concentrations of hemolymph last instar larvae by preventing sequestration of storage proteins by the fat body. Usually, the female-specific storage protein 1 (SP1)* disappears from the male hemolymph at the time of the last larval instar. However, exposure of male larvae to methoprene at the penultimate instar enhanced the accumulation of SP1 in the hemolymph. The SP1 accumulated in males did not differ in molecular weight and immunoreactivity from the SP1 produced in female larvae. Both sexes of fourth instar larvae allatectomized on day 1 instantly accumulated SP1 in the hemolymph, and methoprene application after allatectomy suppressed the hemolymph accumulation of the SP1. In contrast, if allatectomy was carried out at a later stage of the fourth larval instar, SP1 concentration in hemolymph of fifth instar larvae did not increase, suggesting the different juvenile hormone action for regulation of SP1 synthesis in the penultimate instar larvae of silkworms.     

Parasitoid-host endocrine relations: self-reliance or co-optation?

High titers of juvenile hormone (JH) maintain developmental arrest in Manduca sexta larvae parasitized by Cotesia congregata. Parasitized hosts exhibit up to 9.5 times greater amounts of total hemolymph JH (from 0.6±0.09 to 2.51±0.43 ng/ml) compared to non-parasitized controls. Elevated titers are observed throughout the fifth instar, even beyond egression of the parasitoids on day 5. GC–MS analysis revealed that in hemolymph of unparasitized control larvae, JH I is the major homolog and levels of JH III are negligible; in parasitized individuals the amounts of JH I, II, and III rise, and JH III predominates. Neck ligation ensured separation of M. sexta’s corpora allata from the posterior section, which contained most of the parasitoids in the infected insects. When the posterior region was sampled, JHs were not detected in the non-parasitzed larvae, but in those parasitized, JH III was found (1.98±0.29 ng/ml, 24 h post-ligation). JH III was the only homolog produced and secreted by the parasitoid in in vitro culture. This is the first report stating that a parasitoid secretes JH III and may contribute, at least in part, to the circulating titer in the host hemocoel, concurrently promoting host production of JH I and II.     

Juvenile hormone esterase activity repressive factor in the plasma of parasitized insect larvae

A proteinaceous factor that represses plasma juvenile hormone esterase activity in parasitized insect larvae has been isolated and partially characterized from last instar larvae of the armyworm Pseudaletia separata parasitized with the wasp Apantales kariyai. Purification procedures consisted of extraction with 25% ethanol, gel filtration and reversed phase high performance liquid chromatography. Plasma juvenile hormone esterase activity in Day 3 last instar larvae was repressed by 50% when larvae were injected on Days 1 and 2 with 6.5 pmol of the purified peptide, which has a molecular weight of about 4,500 Da. The application of the factor also causes more than a 2-day delay in the onset of pupation. The sequence of 23 amino acid residues at the amino terminus of the factor was determined as follows: H-Glu-Asn-Phe-Ser-Gly-Gly-Xaa-Val-Ala-Gly-Tyr-Met- Arg-Thr-Pro-Asp-Gly-Arg-Xaa-Lys-Pro-Thr-Phe-Tyr-Gln-.     

Juvenile hormones and their titer regulation in Galleria mellonella

Juvenile hormones I, II and III occur in Galleria but JH II is dominant. Its concentration reaches a peak of 3 pmol/g body wt in the penultimate instar, drops to zero in the last larval instar and, except for a small peak in prepupae (0.2 pmol/g), remains undetectable until pharate adults. After emergence the titer reaches over 5 pmol/g in both sexes. Presence of JH II is associated with JH II acid; JH III acid occurs even more often, including stages lacking JH III. Brain implantation into freshly ecdysed last instar larvae effects a similar JH peak as in the penultimate instar and causes an extra larval molt. The opposite treatment, i.e. decerebration of fresh last instar larvae, elicits a continuous rise of JH II to 10 pmol/g and an increase of otherwise rare JH I to 3 pmol/g. Sham operations of these larvae or decerebration of old larvae elevate practically only JH II titer to 1–1.5 pmol/g. Implanted brain-corpora cardiaca-corpora allata complexes maintain in various hosts 0.14–1.6 pmol/g of JH II. The significance and regulation of these fluctuations in JH titer are discussed.     

Correlations between juvenile hormone esterase activity,ecdysone titre and cellular reprogramming in Galleria mellonella

Juvenile hormone esterase (JHE) activity, ecdysone titre, and developmental competence of the epidermis were determined in last instar larvae and pupae of Galleria mellonella. Haemolymph JHE activity reaches a peak before increases are observed in ecdysone titre both during larval-pupal and pupal-adult metamorphosis. JHE activity is low during the penultimate larval instar although general esterase activity is relatively high. In last instar larvae two ecdysone peaks are noted after the increase in JHE activity. Furthermore, epidermal cell reprogramming occurs just after the increase in haemolymph JHE activity and possibly before the first increase in ecdysone titre. This was tested by injection of high doses of β-ecdysone into last instar larvae of different ages resulting in rapid cuticle deposition. Reprogramming occurred if the resulting cuticle was of the pupal type. These correlative observations may increase our understanding of the relative importance of an ecdysone surge in the absence of JH in reprogramming of the insect epidermis.     

The juvenile hormone titer of Trichoplusia ni and its potential role in embryogenesis of the polyembryonic wasp CopidosomaFloridanum

The hemolymph juvenile hormone (JH) titer of third through fifth stadia Trichoplusia ni parasitized by the polyembryonic parasitoid, Copidosoma floridanum, was measured by radioimmunoassay and compared to the titers of unparasitized larvae. The JH titer of parasitized larvae fluctuated from 28 pg/μl to undetectable levels. Maximum levels of hormone were present at ecdysis to the fourth and fifth stadium, and at the prepupal stage. Qualitatively, similar fluctuations were observed in unparasitized larvae. However, the titers in unparasitized larvae were much lower than those of parasitized larvae in the third and early fourth stadia, and the titer fell to undetectable levels in the fifth stadium 24 h earlier (48 h) than in parasitized larvae (72 h). Preventing the JH titer from falling during the fourth and fifth stadia by topical application of (RS)-methoprene or JH II had a juvenilizing effect on parasitized T. ni, and inhibited C. floridanum embryo morphogenesis. The effect of exogenous methoprene and JH on C. floridanum development depended on timing of application and dosage. Application of 100 pmol per day of methoprene beginning at 2 h of the host fourth stadium, prior to the large drop in the endogenous JH titer, inhibited morphogenesis in the majority of C. floridanum embryos. Application of methoprene at later times of host development did not inhibit morphogenesis although other developmental alterations were observed. The potential significance of host JH and ecdysteroid titers on polyembryonic development are discussed.     

Endocrine interrelationship between the parasitoid Chelonus sp. and its host Trichoplusia ni

The egg-larval parasitoid Chelonus sp. induces the precocious onset of metamorphosis in the 4th (penultimate) stadium of its host Trichoplusia ni, emerges from the prepupa, and then feeds on it. Qualitative and quantitative changes in ecdysteroids and juvenile hormone were measured. Hemolymph of 3rd-to 4th-instar host larvae and the parasitoids they contained, as well as nonparasitized and parasitized eggs, were analyzed. In the host hemolymph a broad peak of ecdysteroids during molting into the 4th stadium and a continuous increase from day 2 (onset of precocious wandering) until day 4 (emergence of parasitoid) were observed; 20-hydroxyecdysone and 20,26-dihydroxyecdysone were predominant. The juvenile hormone titer fluctuated in the 3rd and early 4th stadium and fell to undetectable levels shortly before the precocious onset of wandering. The parasitoid's ecdysteroids started to increase on the molt to the 2nd instar (= early 4th instar of the host) and thereafter fluctuated on a high level, 20-hydroxyecdysone, 20,26-dihydroxy-ecdysone, and ecdysone being predominant. The juvenile hormone titer was high in late 1st-instar parasitoids, decreased to low levels at ecdysis into the 2nd instar, and increased again to high levels in the 2nd-instar larvae at the time when their shape changed from flat to cylindrical. After ecdysis to the 3rd instar the juvenile hormone titer fell. A comparison revealed that both ecdysteroids and juvenile hormone fluctuate independently in parasitoid and host at most stages, suggesting that the parasitoid produces its own hormones. The first data on ecdysteroids and juvenile hormones in the egg stage of a parasitoid/host system are reported. At the stage of eye pigmentation parasitized eggs contained more immunoreactive midpolar ecdysteroids than non-parasitized ones. 20-Hydroxyecdysone and 20,26-dihydroxyecdysone were the predominant ecdysteroids in both nonparasitized and parasitized eggs, but the latter contained several additional ecdysteroids which were not seen in nonparasitized eggs. The titer of juvenile hormone was similar in both. Shortly before hatching the ecdysteroids were low in parasitized and nonparasitized eggs, but the content of juvenile hormone was much higher in the former. At this stage the majority of parasitoids have already eclosed and teratocytes are released. The results of HPLC analysis indicated the presence of juvenile hormone III together with juvenile hormones I and II in parasitized eggs, but only juvenile hormones I and II in nonparasitized eggs.