Biochemical interaction between chelonine wasps and their lepidopteran hosts: after a decade of research - the parasite is in control

A decade of research on the biochemical interaction between chelonine wasps and their lepidopteran hosts has yielded considerable data on the underlying basis for the developmental, immunological and reproductive effects that these parasites inflict upon their hosts. These egg-larval parasites induce their immunologically compromised host larvae to precociously initiate metamorphosis, followed by suppression of development of the precocious prepupa, in addition to castration of the host. The results from numerous laboratories have shown that the parasite egg that is normally injected by the adult female into the host along with venom, polydnavirus and calyx fluid proteins need not hatch or even be present for the host to exhibit each of these alterations. In addition to these aspects the parasite larva, when present, itself releases hormones and proteins into the hemolymph of the host. A review of the data amassed to date leads inexorably to the conclusion that it is the chelonine wasp that is the biochemically dominant partner. Thus, after 10 years of research, it still appears that in chelonine-lepidopteran parasite-host systems, the parasite is in control of specific points of the biochemistry and development of its host.     

Hemolymph concentrations of host ecdysteroids are strongly suppressed in precocious prepupae of Trichoplusia ni parasitized and pseudoparasitized by Chelonus near curvimaculatus.

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 metamorphosis. At the developmental stage at which the hemolymph of the unparasitized metamorphosing host has its maximum titer of prepupal ecdysteroids, the hemolymph of 4th instar "truly parasitized" hosts (hosts with a surviving endoparasite) had a strongly reduced ecdysteroid titer. However, during the photophase about 12 h later, just prior to emergence of the parasite larva, an ecdysteroid peak was observed in the host hemolymph. Fourth instar pseudoparasitized 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 ecdysteroids in normal, truly parasitized, and 4th or 5th instar pseudoparasitized prepupae showed that both ecdysone and 20-OH ecdysone* were suppressed in truly and pseudoparasitized prepupae, with ecdysteroid levels being lowest in pseudoparasitized hosts. These data, and those of Brown and Reed-Larsen (Biol Contr 1, 136 [1992]), 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 endoparasite, 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.     

Parasite regulation of host insect metamorphosis: a new form of regulation in pseudoparasitized larvae ofTrichoplusia ni

Summary When eggs ofTrichoplusia ni (Lepidoptera) are stung by a parasitic wasp,Chelonus sp., the developing host larvae precociously initiate metamorphosis ten days later. Precocious initiation of metamorphosis occurs even in ‘pseudoparasitized’ stung hosts which contain no living parasites at the time of symptoms of host regulation by the parasite. In feeding, penultimate instar, pseudoparasitized hosts, the corpora allata activity, hemolymph juvenile hormone esterase activity, in vivo rates of juvenile hormone metabolism and changes in hemolymph protein composition all follow the pattern of the normal last instar. This and other evidence suggests the entire developmental pattern of the last larval instar is precociously expressed in penultimate instar, pseudoparasitized hosts. The cause of precocious expression of the developmental program leading to metamorphosis is a significant decrease in the critical size parameter that, in normal larvae, signals attainment of the last instar. The induction, in preultimate instar larvae, of the entire feeding stage developmental program leading to metamorphic commitment, using either biochemical, surgical or parasitic experimental probes, has not been previously reported. The results have important implications for the study of host-parasite endocrine interaction, of normal insect metamorphosis and even of human puberty.     

Analysis of endoparasitoid-released proteins and their effects on host development in the system Chelonus inanitus (Braconidae)-Spodoptera littoralis (Noctuidae)

Having shown earlier that the larva of C. inanitus is essential in inducing the precocious onset of metamorphosis in polydnavirus/venom containing S. littoralis, we here analysed release of proteins by parasitoid larvae and their effects on host development. Parasitoid larvae released proteins in vivo and in vitro in a stage dependent manner. An approximately 212 kD protein was released from the mid 1st instar onwards and additional smaller proteins could be associated mainly with the 2nd and 3rd instar. When parasitoids were implanted into S. littoralis larvae, parasitoid-released proteins were seen 6 hr later. When parasitoids were removed from hosts, parasitoid-released proteins persisted in the host haemolymph for some time. Injection of antiserum against parasitoid-released proteins after removal of the parasitoid larva accelerated the disappearance of the 212 kD protein and reduced the number of larvae entering metamorphosis precociously. Repeated injections of concentrated parasitoid medium into polydnavirus/venom containing larvae caused a reduction of the head capsule width and formation of miniature 6th instar larvae; this effect was not seen in the absence of polydnavirus/venom. These observations suggest that proteins released by the parasitoid might play a role in modifying host metamorphosis in the presence of polydnavirus/venom, and the temporal appearance of the 212 kD protein makes it the most interesting candidate for being involved in such an effect.     

Interaction of calyx fluid and venom from Microplitis croceipes (Braconidae) on developmental disruption of the natural host,Heliocoverpa zea,and two atypical hosts,Galleria mellonella and Spodoptera exigua

Polydnaviruses of many braconid and ichneumonid endoparasitoids play an important role in the successful parasitism of their hosts. The host's development is altered and its immune response is also suppressed. In this study, we compared the effects of calyx fluid and venom on the development of the natural host, Helicoverpa zea, and two atypical hosts that the parasitoid does not normally attack in nature, Galleria mellonella and Spodoptera exigua. The levels of calyx fluid and\or venom injected was 0.05, 0.1 and 0.2 female equivalents (FE)/larva. In H. zea, calyx fluid significantly reduced larval growth on day 5 post injection. Venom alone did not affect larval growth but it synergized the action of calyx fluid by reducing growth earlier and for a longer period after injection. Other effects of calyx fluid on the host, either alone or in combination with venom, were an increase in developmental period, and a reduction in percent emergence and weight of adult moths. The percentage of H. zea larvae that pupated was not affected by calyx fluid or venom. In Galleria mellonella, venom alone reduced larval growth comparable to calyx fluid and both tissues induced the effects on day 1 post injection. Other effects caused by calyx fluid or venom alone or the combination were a reduction in percent pupation and emergence, and the average adult weight. In S. exigua, high mortality occurred when 4th instar larvae were injected. Although the injection of larger fifth instars reduced overall mortality, the sham-injected larvae only gained weight during the first 24 hours after injection (from day 0 to day 1). However, adults were produced at all doses of calyx fluid or venom. The effects of the virus on development in this species were a prolongation of the larval stage and reduction of adult weight by calyx fluid in combination with venom. In conclusion, injections of calyx fluid and venom of Microplitis croceipes can differentially affect the growth and development of its natural host H. zea, and atypical host, G. mellonella, but only a minimal effect was observed in S. exigua.     

Titres of juvenile hormone I,II and III in Spodoptera littoralis (Noctuidae) from the egg to the pupal moult and their modification by the egg-larval parasitoid Chelonus inanitus (Braconidae)

Physico-chemical analysis of juvenile hormones (JHs) of Spodoptera littoralis revealed highest quantities in the second half of embryonic development and in newly hatched 1st instar larvae. At these stages, mostly JH II, JH I and little JH III were found, while in later stages only JH II and JH III were found. Titres fluctuated in a similar manner in all larval instars, being lowest during the moults. In last (=6th) instar larvae, JHs disappeared in the late feeding-digging stage and again increased in the early prepupal stage. Parasitisation with Chelonus inanitus, a solitary egg-larval parasitoid which induces in its host the precocious onset of metamorphosis in the 5th instar, did not alter JH homologue composition but led to a disappearance of JHs in the 5th instar. Implantation of a parasitoid larva into early 5th instar larvae containing polydnavirus/venom caused a drop in the JH titre which indicates that the parasitoid larva plays an important role in the manipulation of the host's JH titre. In the parasitoid larva, only JH III was found; titres were highest in the 2nd larval instar, a stage when the host is in the 5th instar and contains almost no JHs. Thus, JHs of the parasitoid and the host fluctuate in an independent manner.     

Common and distinct roles of juvenile hormone signaling genes in metamorphosis of holometabolous and hemimetabolous insects

Insect larvae metamorphose to winged and reproductive adults either directly (hemimetaboly) or through an intermediary pupal stage (holometaboly). In either case juvenile hormone (JH) prevents metamorphosis until a larva has attained an appropriate phase of development. In holometabolous insects, JH acts through its putative receptor Methoprene-tolerant (Met) to regulate Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) genes. While Met and Kr-h1 prevent precocious metamorphosis in pre-final larval instars, BR-C specifies the pupal stage. How JH signaling operates in hemimetabolous insects is poorly understood. Here, we compare the function of Met, Kr-h1 and BR-C genes in the two types of insects. Using systemic RNAi in the hemimetabolous true bug, Pyrrhocoris apterus, we show that Met conveys the JH signal to prevent premature metamorphosis by maintaining high expression of Kr-h1. Knockdown of either Met or Kr-h1 (but not of BR-C) in penultimate-instar Pyrrhocoris larvae causes precocious development of adult color pattern, wings and genitalia. A natural fall of Kr-h1 expression in the last larval instar normally permits adult development, and treatment with an exogenous JH mimic methoprene at this time requires both Met and Kr-h1 to block the adult program and induce an extra larval instar. Met and Kr-h1 therefore serve as JH-dependent repressors of deleterious precocious metamorphic changes in both hemimetabolous and holometabolous juveniles, whereas BR-C has been recruited for a new role in specifying the holometabolous pupa. These results show that despite considerable evolutionary distance, insects with diverse developmental strategies employ a common-core JH signaling pathway to commit to adult morphogenesis.     

Physiological and endocrine changes associated with polydnavirus/venom in the parasitoid-host system Chelonus inanitus-Spodoptera littoralis

As shown earlier, parasitization by the egg-larval parasitoid C. inanitus causes in its host the precocious onset of metamorphosis in the 5th instar followed by developmental arrest in the prepupal stage. Polydnavirus/venom were shown to be responsible for the developmental arrest. We investigated how polydnavirus/venom affect growth of the host larvae and found that head capsule widths were smaller from the 4th to 6th stadium and weights were lower in the 6th stadium in polydnavirus/venom-containing larvae than in non-parasitized larvae. In an attempt to identify endocrine parameters that are modified by polydnavirus/venom and might be responsible for the developmental arrest in the prepupa, we compared juvenile hormones, juvenile hormone esterase and ecdysteroids between non-parasitized and polydnavirus/venom-containing larvae from the 4th instar until pupation or developmental arrest, respectively. Obvious differences became manifest only in the 6th instar at the pupal cell formation stage, i.e. 12 days after entry of polydnavirus/venom into the host egg. Then, prothoracic glands of polydnavirus/venom-containing larvae released less ecdysteroids and ecdysteroid titres were lower than in non-parasitized larvae; this was followed by a delayed, reduced and desynchronized increase in prepupal juvenile hormones and juvenile hormone esterase and a slightly modified metabolism of ecdysone. This indicates that polydnavirus/venom affects the endocrine system of the host only after pupal commitment and that inhibition of prothoracic gland activity is the first detectable effect.     

Analysis of precocious metamorphosis induced by application of an imidazole derivative to early third instar larvae of the silkworm,Bombyx mori

When an imidazole derivative (KK-42) was applied to day 1 third instar larvae of the silkworm, Bombyx mori, 100% underwent precocious metamorphosis at the end of the fourth instar. Thus, the fourth instar becomes the last instar in these KK-42–treated larvae. The endocrine systems underlying the precocious metamorphosis were analyzed in the present study. Hydroprene application during the prolonged third instar after KK-42 treatment can prevent precocious metamorphosis, and the results showed dose-dependent and stage-specific effects. From analysis of the developmental changes in ecdysteroid levels in both KK-42–treated larvae and KK-42– and hydroprene-treated larvae, we conclude that changes in JH levels during the third larval instar can modify the secretion pattern of prothoracic glands and that during the next larval instar, very low ecdysteroid levels during the early stages of the presumptive last (fourth) larval instar are directly related to precocious metamorphosis. Arch. Insect Biochem. Physiol. 36:349–361, 1997. © 1997 Wiley-Liss, Inc.     

Influence of the parasitoid Chelonus inanitus and its polydnavirus on host nutritional physiology and implications for parasitoid development

Chelonus inanitus is a solitary egg-larval endoparasitoid, which feeds on host haemolymph during its internal phase. Parasitization induces in the host Spodoptera littoralis a precocious onset of metamorphosis and a developmental arrest in the prepupal stage. At this stage the parasitoid larva emerges from the host and consumes it. We show here that parasitization and the co-injected polydnaviruses affect the nutritional physiology of the host mainly in the last larval instar. Polydnaviruses cause a reduced uptake of food and an increase in the concentration of free sugars in the haemolymph and of glycogen in whole body. The parasitoid larva, along with polydnaviruses, causes a reduction of proteins in the host's plasma and an accumulation of lipids in whole body. Dilution of host haemolymph led to a reduced concentration of lipid in parasitoid larvae and a reduced survival rate. Thus, a sufficient concentration of nutrients in the host's haemolymph appears to be crucial for successful parasitoid development. Altogether, the data show that the parasitoid and the polydnavirus differentially influence host nutritional physiology and that the accumulated lipids and glycogen are taken up by the parasitoid in its haematophagous stage as well as through the subsequent external host feeding.     

Resistance of Apanteles eggs to the haemocytic encapsulation by their habitual host, Pieris

The calyx fluid in the lateral oviduct of a gregarious parasitoid, Apanteles glomeratus contained ellipsoid particles of ca. 130 × 200 nm. These calyx fluid particles did not appear to be embedded in a fibrous outer layer on the surface of eggs in the lateral oviduct. They were not observed on the surfaces of the eggs 3 to 4 hr after being deposited into the host haemocoele. Oviposition experiments indicated that the occurrence of haemocytic defence reactions of the late 2nd instar larvae of the Pieris rapae crucivora against 1 st instar larvae of the parasitoid increased with a decreasing number of the parasitoid eggs introduced into a host, and that more than 5 to 9 parasitoid eggs were needed for suppressing the ability of the host to encapsulate its parasitoid larvae immediately after hatching. When eggs with calyx fluid obtained from egg reservoir were injected into the host, they were found to be encapsulated 1 to 2 days after the injection. They could not start their embryonic development. When calyx fluid-free 3-hr-old eggs were injected in a number of more than 5 eggs into a 5th instar larva of Pieris, 58% of 31 eggs injected had normally hatched without evoking encapsulation reactions by the host. Both electron microscopic observations of parasitoid eggs in the host haemocoele and the experimental results suggested that calyx fluid or calyx fluid particles of the parasitoid might not be involved in the encapsulation-inhibiting activity of the parasitoid eggs. Rather it was anticipated that a substance (or substances) might be secreted by the parasitoid eggs into the haemocoele of the host, which suppressed defence reactions of the host.     

菜蛾盘绒茧蜂多分DNA病毒的特性及其对小菜蛾幼虫的生理效应

对菜蛾盘绒茧蜂Cotesia plutellae多分DNA病毒的特性及其对寄主小菜蛾Plutella xylostella幼虫的生理效应进行了研究。结果表明：菜蛾盘绒茧蜂雌蜂输卵管萼中含有大量的多分DNA病毒（polydnavirus, PDV）;一个PDV内含多个核衣壳,最多可达16个;核衣壳长40～168 nm,直径39～40 nm;PDV仅在输卵管萼细胞内复制;雌蜂产卵时,随蜂卵将PDV注入寄主血腔,并扩散到寄主的许多组织中;PDV可能先通过脱膜再侵染寄主组织。雌蜂经Co⁶⁰辐射处理后再寄生（即假寄生）小菜蛾2龄、3龄和4龄初期的幼虫,被寄生后的寄主幼虫几乎全部不能化蛹,但末龄（即4龄）幼虫期显著延长,并在寄生后期,幼虫胸部有褐色的短翅芽出现;即将化蛹的4龄末小菜蛾幼虫被假寄生后,即使每头寄主被过寄生9次,依然能正常化蛹,但不能羽化。假寄生与正常寄生后寄主的脂肪体数量和形态结构有明显的不同,推测在正常寄生的情况下蜂卵孵化时释放的畸形细胞及随后的幼蜂可能对脂肪体的结构产生了作用。     

Changes in the haemolymph proteome of Spodoptera littoralis induced by the parasitoid Chelonus inanitus or its polydnavirus and physiological implications

The egg-larval parasitoid Chelonus inanitus induces in its host Spodoptera littoralis two major developmental effects, namely a precocious onset of metamorphosis followed by a developmental arrest in the prepupal stage. Along with each egg, the wasp injects polydnavirus and venom into the host egg. The polydnavirus has been shown to play a major role in inducing the developmental arrest while the parasitoid larva is instrumental in inducing the precocious onset of metamorphosis. Here we report that experimental dilution of haemolymph of polydnavirus-containing larvae can partially prevent the developmental arrest while injection of native, but not of heat-treated, haemolymph or plasma from polydnavirus-containing larvae into nonparasitized larvae could induce developmental arrest in 14-15% of the larvae. This illustrates that heat-labile factors present in haemolymph play a role in causing developmental arrest. Injection of parasitoid medium increased the proportion of larvae entering metamorphosis precociously while injection of antibodies against a parasitoid-released protein had the opposite effect; this indicates that this protein and possibly other parasitoid-released substances are involved in inducing the precocious onset of metamorphosis. Analysis of the plasma proteome of nonparasitized, parasitized and polydnavirus-containing larvae revealed that the developmental effects are associated with only minor differences: eleven low abundant viral or virus-induced proteins and five parasitoid-released proteins were seen at specific stages of the host.     

Stage dependent influences of polydnaviruses and the parasitoid larva on host ecdysteroids

In the solitary egg-larval parasitoid Chelonus inanitus (Braconidae) both polydnavirus and the parasitoid larva manipulate host development. Parasitization leads to a premature drop in juvenile hormone titre and a precocious onset of metamorphosis in the 5th larval instar. The C. inanitus bracovirus (CiBV) alone causes a reduction in host ecdysteroid titres at the pupal cell formation stage and prevents pupation. Here we report three new findings. (1) We show that parasitization causes a reduction in haemolymph ecdysteroid titre immediately after the moult to the 5th instar; similarly low values were seen in nonparasitized larvae after the moult to the 6th instar. These data along with parasitoid removal experiments indicate that the low ecdysteroid titre after the moult is a very early sign of the upcoming metamorphosis. (2) In vitro experiments with prothoracic glands and brain extracts showed that CiBV affects both prothoracic glands and prothoracicotropic hormone after the stage of pupal cell formation. (3) In the haemolymph of parasitized larvae the ecdysteroid titre increased in the late cell formation stage, i.e. immediately before egression of the parasitoid. In vitro experiments showed that late 2nd instar parasitoids release ecdysteroids and are thus very likely responsible for the rise in host ecdysteroids.     

Induction of metamorphosis in the marine gastropod Ilyanassa obsoleta: 5HT, NO and programmed cell death

The central nervous system (CNS) of a metamorphically competent larva of the caenogastropod Ilyanassa obsoleta contains a medial, unpaired apical ganglion (AG) of approximately 25 neurons that lies above the commissure connecting the paired cerebral ganglia. The AG, also known as the cephalic or apical sensory organ (ASO), contains numerous sensory neurons and innervates the ciliated velar lobes, the larval swimming and feeding structures. Before metamorphosis, the AG contains 5 serotonergic neurons and exogenous serotonin can induce metamorphosis in competent larvae. The AG appears to be a purely larval structure as it disappears within 3 days of metamorphic induction. In competent larvae, most neurons of the AG display nitric oxide synthase (NOS)-like immunoreactivity and inhibition of NOS activity can induce larval metamorphose. Because nitric oxide (NO) can prevent cells from undergoing apoptosis, a form of programmed cell death (PCD), we hypothesize that inhibition of NOS activity triggers the loss of the AG at the beginning of the metamorphic process. Within 24 hours of metamorphic induction, cellular changes that are typical of the early stages of PCD are visible in histological sections and results of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in metamorphosing larvae show AG nuclei containing fragmented DNA, supporting our hypothesis.     

Reprogramming in the absence of DNA synthesis in Galleria larval epidermis.

Larval epidermal cells from a day-1 penultimate instar Galleria larva on implantation into day-5 last instar larva metamorphose and deposit a pupal cuticle at the same time as the host pupates. DNA synthesis in the implanted larval cell was monitored with 3-H-thymidine. Various regimens of 3-H-thymidine application were used and under no conditions did the larval cells incorporate label during the period from implantation to deposition of pupal cuticle. This suggests that a wax moth larval ectoderm cell can reprogram its genome to secrete a pupal cuticle without a precedent cell division.     

Hormonal implication in Bracon-venom-induced paralysation of the host larva of Corcyra cephalonica (Lepidoptera: Pyralidae)

To facilitate oviposition, the ectoparasite Bracon hebetor, injects its venom, a paralysing toxin, to the host Corcyra larva that ultimately dies without showing any metamorphic change, even if allowed to remain unparasitised. At the initial stage of venom injection the rate of heartbeat of the host becomes abruptly high. This has been explained from the synergistic action of the substances of poison gland and calyx. The paralysed larvae subsequent to envenomization die within 240 hr. Application of hydroprene as single dose or with a booster dose after paralysation mostly increases the survival period considering heart beat as the index. The predicted value of survival period (714.4 hr), determined from a fitted equation obtained from the relationship between heart beat and survival period, indicates that a 100 microg treatment/larva with a booster dose of 50 microg/larva most effectively lengthens the period. It is concluded that the venom-induced physiological dysfunction of the immobilised larvae, as indicated in the rate of heart beat and survival period, though can be recovered to some extent after the application of juvenoids, there cannot occur any metamorphic change of these larvae. The parasitoid, therefore, succeeds in completing its development and metamorphosis by arresting the development of its host through an indirect hormonal suppression. The findings indicate an endocrine implication in host-parasite relationship in insect.     

Variation in growth and instar number in field and laboratory Manduca sexta

The tobacco hornworm Manduca sexta has been an important model system for understanding physiological control of growth, development and metamorphosis of insects for more than half a century. Like all Manduca, M. sexta typically has five larval instars, with developmental commitment to metamorphosis occurring early in the 5th (final) instar. Here we show that M. sexta from a field population in North Carolina (USA) shows substantial intraspecific variation in the number of larval instars when feeding on a modified artificial diet. Individuals with six instars consistently exhibited slower growth rates during early larval development than individuals with five instars. The frequency of individuals with six instars decreased with increased rearing temperature. In contrast, M. sexta from a laboratory colony consistently had five instars, and had more rapid larval growth rates than M. sexta from the field. We identify a threshold body size at the start of the 5th instar that predicts whether an individual will have five (greater than 600mg) or six instars (less than 600mg). Variation in field populations in Manduca provides an important resource for understanding physiological control, developmental plasticity and evolution of growth rate, body size and instar number.     

Synthesis and Anti-juvenile Hormone Activity of 1-Citronellyl-5-substituted Imidazoles

A number of 1-citronellyl-5-substituted imidazoles were synthesized and bioassayed on the silkworm, Bombyx mori, in order to assess their anti-juvenile hormone activity. Most of the 1-citronellyl-5-substituted imidazoles induced precocious metamorphosis in the 3rd and 4th instar larvae of B. mori by topical application. The percentage of precocious metamorphosis correlated well with the dosage. Among the compounds tested, l-citronellyl-5-(2-chlorophenyl)imidazole (8) and the 2-methylphenyl analog 10 showed the highest activity. When compounds 8 and 10 were applied to the 4th instar larvae at 10 μg/larva, precocious pupation was induced in 100% without any lethal effects.