Inhibition of the larval ecdysis and emergence behavior of the parasitoid Cotesia congregata by methoprene

Parasitism of the tobacco hornworm, Manducasexta, by the braconid wasp Cotesiacongregata, induces developmental arrest of the host in the larval stage. During the final instar of the host, its juvenile hormone (JH) titer is elevated, preventing host metamorphosis. This study investigated the effects of hormonal manipulation of the host on the parasitoid’s emergence behavior. The second larval ecdysis of the wasps coincides with their emergence from the host, and application of the juvenile hormone analogue methoprene to day 4 fifth instar hosts either delayed or totally suppressed the subsequent emergence of the wasps. Effects of methoprene were dose-dependent and no parasitoids emerged following treatment of host larvae with doses >50 μg. Parasitoids which failed to emerge eventually succumbed as unecydsed pharate third instar larvae in the hemocoel of the host. Effects of host methoprene treatment on parasitoid metamorphosis were also assessed, and metamorphic disruption occurred at much lower dosages compared with doses necessary to suppress parasitoid emergence behavior. The inhibitory effect of methoprene on parasitoid emergence behavior appears to be mediated by effects of this hormone on the synthesis or release of ecdysis-triggering hormone (ETH) in the parasitoid, the proximate endocrine cue which triggers ecdysis behavior in free-living insects. ETH accumulated in the epitracheal Inka cells of parasitoids developing in methoprene-treated hosts, suggestive of a lack of hormone release. Thus, the hormonal modulation of parasitoid emergence behavior appears to be complex, involving a suite of hormones including JH, ecdysteroid, and peptide hormones.     

Role of juvenile hormone in regulating tyrosine glucoside synthesis for pupal tanning in Manduca sexta (L.)

Tyrosine glucoside (α-d-glucopyranosyl-O-l-tyrosine) serves as a reservoir of tyrosine and glucose for pupal and adult cuticle formation, tanning, and pigmentation in several Lepidopteran insects. In the tobacco hornworm, Manduca sexta (L.), detectable quantities appear in the haemolymph 1–2 days after ecdysis of the fifth instar and very high concentrations accumulate between the fourth and eighth days of the stadium. If juvenile hormone II or a mimic (methoprene) is injected into fifth-instar larvae at 24-h intervals after ecdysis, tyrosine glucoside synthesis is almost completely suppressed. Temporary starvation of newly ecdysed larvae that results in the maintenance of a high endogenous juvenile hormone titre, also suppresses and delays the onset of tyrosine glucoside synthesis. The decrease and eventual disappearance of juvenile hormone after ecdysis of the last-larval instar appears to be a necessary prerequisite for the synthesis or activation of tyrosine glucoside synthetase along with the initiation of other metamorphic events.     

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.     

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.     

Lepidopteran anti-juvenile hormones: Effects on development of Apanteles congregatus in Manduca sexta

Parasitism by the braconid wasp Apanteles congregatus decreases the effectiveness of the anti-juvenile hormone agents ETB (ethyl 4-[2-{ittert-butyl carbonyloxy}bytoxy]benzoate) and fluoromevalonolactone (FMev) in inducing precocious metamorphosis of Manduca sexta larvae. Topical application of 1–200 μg ETB to parasitized third-instar larvae had no effect on either host or parasite development, whereas doses of 50μg or more ETB applied to unparasitized third-instar larvae caused formation of larval-pupal intermediates after the fourth instar. Parasitism also decreased the effectiveness of 100–200 μg FMev in causing metamorphosis at the moult following its application. In contrast to ETB, FMev disrupted development of the parasitoids. No wasps emerged when preterminal stage hosts were treated with FMev and the hosts formed larval-pupal intermediates. After treatment of terminal stage hosts with FMev, the number of emerging parasitoids was reduced by one-third. Precocene II (100 μg per larvae) had no effect on development of either M. sexta or A. congregatus.     

Inhibition of testicular growth and development in Manduca sexta larvae parasitized by the braconid wasp Cotesia congregata

Tobacco hornworm larvae parasitized by the gregarious larval endoparasitoid Cotesia congregata exhibited an inhibition in testicular growth and development, the extent of which was determined by the age and developmental stage of the host at the time of parasitization. The degree of parasitic castration, as assessed by measurements of testicular volume, was correlated with the stadium in which parasitization occurred. A mathematical formula requiring the measurement of testicular length, width and depth was used to calculate testicular volume. The use of the depth parameter revealed a negative correlation between host weight and testicular volume in parasitized larvae. Testicular volumes of fifth instar hosts, which had been parasitized in the first stadium, were significantly smaller than those originally parasitized as fourth or fifth instar larvae and were not correlated with parasitoid load. Effects of natural parasitism were not duplicated by injections of C. congregata polydnavirus and venom, topical treatment with the juvenile hormone analog methoprene, or starvation of nonparasitized larvae. Larvae receiving virus plus venom or methoprene grew larger due to delayed wandering and had larger testes than controls. Deleterious effects on host testes may be due to the effects of nutrient competition between the developing parasitoid progeny and the gonads, combined with the juvenilizing effects believed to be caused by the polydnavirus.     

Effects of methoprene and juvenile hormone on larval ecdysis,emergence, and metamorphosis of the endoparasitic wasp, Apanteles congregatus

Topical application of juvenile hormone I and III or the hormone analogue methoprene to parasitized Manduca sexta larvae inhibited subsequent emergence of the endoparasitic wasp Apanteles congregatus. Methoprene treatment inhibited wasp emergence in a dose-dependent manner, causing either a delay or total inhibition of emergence. These results were interpreted as reflecting inhibitory effects of juvenile hormone on the second-larval ecdysis of the parasitoid that normally occurs during emergence from the host larva. Parasitoid ecdysis was disrupted even when methoprene was applied to host larvae a few hours prior to the normal expected time of emergence. A correlation between the number of emerging parasitoids and the timing of emergence was seen in methoprene-treated hosts, and few parasitoids emerged after day 9 of the host's fifth-instar. Our findings suggest that the suppression of emergence by juvenile hormone analogues noted in previous studies may be due to a similar inhibitory effect on parasitoid ecdysis. We also observed that parasitoids emerging from hosts treated with a low dose of methoprene (1 μg) later pupated normally but then formed nonviable pupal-adult intermediates. Thus use of this insect growth regulator must be undertaken carefully to prevent possible adverse effects on natural parasitoid populations.     

Altered tyrosine metabolism and melanization complex formation underlie the developmental regulation of melanization in Manduca sexta

The study of hemolymph melanization in Lepidoptera has contributed greatly to our understanding of its role in insect immunity. Manduca sexta in particular has been an excellent model for identifying the myriad components of the phenoloxidase (PO) cascade and their activation through exposure to pathogen-associated molecular patterns (PAMPs). However, in a process that is not well characterized or understood, some insect species rapidly melanize upon wounding in the absence of added PAMPs. We sought to better understand this process by measuring wound-induced melanization in four insect species. Of these, only plasma from late 5th instar M. sexta was unable to melanize, even though each contained millimolar levels of the putative melanization substrate tyrosine (Tyr). Analysis of Tyr metabolism using substrate-free plasmas (SFPs) from late 5th instar larvae of each species showed that only M. sexta SFP failed to melanize with added Tyr. In contrast, early instar M. sexta larvae exhibited wound-induced melanization and Tyr metabolism, and SFPs prepared from these larvae melanized in the presence of Tyr. Early instar melanization in M. sexta was associated with the formation of a high mass protein complex that could be observed enzymatically in native gels or by PO-specific immunoblotting. Topical treatment of M. sexta larvae with the juvenile hormone (JH) analog methoprene delayed pupation and increased melanizing ability late in the instar, thus linking development with immunity. Our results demonstrate that melanization rates are highly variable in Lepidoptera, and that developmental stage can be an important factor for melanization within a species. More specifically, we show that the physiological substrate for melanization in M. sexta is Tyr, and that melanization is associated with the formation of a PO-containing protein complex.     

Hormonal control of uric acid storage in the fat body during last-larval instar of Manduca sexta

In the last-larval instar of the tobacco hornworm, Manduca sexta, a switch from excretion of uric acid to storage in the fat body occurs during transition from the feeding to the wandering stage. Neuroendocrine control of this change from excretion to storage was demonstrated by neck-ligation experiments with synchronously reared larvae. Results indicate that a neurohormone is released from the head 24–30 hr before the initiation of wandering and coincident with the first release of ecdysone that initiates metamorphosis. Direct involvement of the moulting hormone was shown by the effects of multiple injections of 20-hydroxyecdysone into the abdomen of larvae that had been ligated before the release of hormone. Urate levels in the fat body were 20- to 100-fold higher from hormone-injected larvae as from saline inject controls. Topically applied juvenile hormone or methoprene reversed the 20-hydroxyecdysone-induced storage of urate. Increased levels of uric acid in the haemolymph during pupal development result from the presence of juvenile hormone, and the abrupt decrease in uric acid concentration in the haemolymph just prior to pupal ecdysis results from a decreased titre of juvenile hormone. Applications of methoprene prevented the decrease in uric acid levels in the haemolymph.     

The activity of the corpora allata in the fourth and fifth larval instars of the migratory locust

The presence of juvenile hormone in the haemolymph of larvae of Locusta has been detected by a modified Galleria bioassay and these results are compared with indirect methods of estimating corpus allatum activity. Juvenile hormone is present in the haemolymph during the fourth larval instar except on the last day of the instar, and is absent from the haemolymph of the fifth and final larval instar except on the last day of the instar. Changes in the volumes of the corpora allata simply reflect changes in the growth of the whole insect and are of no value in predicting endocrine activity. Changes in the size of the cells of the corpora allata can be correlated with the presence of juvenile hormone in the haemolymph in the fourth larval instar, but similar changes in cell size occur in the fifth larval instar when no juvenile hormone is present in the haemolymph. The effects of the implantation of corpora allata are unreliable as estimates of corpus allatum activity as isolated corpora allata from fifth instar larvae release juvenile hormone. Indirect methods of measuring corpus allatum activity are thus shown to be unreliable. The R_f value of Locusta juvenile hormone as determined by thin-layer chromatography differs from that of Roeller's juvenile hormone, suggesting that the two hormones might be chemically distinct.     

A reinvestigation of the effects of cortisol on growth in insects

Incorporation of cortisol, or various derivatives and analogues of cortisol, into the diet had no effect on the rate of growth of Tenebrio molitor. Injections of cortisol, or its derivatives, into fourth instar larvae of Schistocerca americana gregaria, had no effect on their rate of growth. The moult to fifth instar was normal. Administration in the diet, or injections of cortisol had no effect on the rate of growth of fourth and fifth instar larvae of Manduca sexta, or on the insect's uptake and utilization of food. The development of larvae to pupae and adults was unaffected. It is concluded, contrary to the findings of two previously published reports, that the vertebrate hormone cortisol does not affect the growth and development of insects.     

Action of two juvenile hormone antagonists on lepidopteran epidermis

The juvenile hormone antagonist ETB (ethyl-4-2(t-butylcarbonyloxy)-butoxybenzoate) caused formation of precocious larval-pupal intermediates after the 4th (penultimate)-larval instar of the tobacco hornworm, Manduca sexta, when 50 μg were applied to any 3rd stage larvae or to 4th stage larvae within 12 hr after ecdysis. This dose was most effective within 12 hr after ecdysis to the 3rd stage. In the black mutant larval assay for juvenile hormone, ETB had activity, 0.75 μg per larva giving half-maximal score. In vitro ETB acted as a juvenile hormone to prevent the ecdysteroid-induced change in commitment at concentrations above 0.1 μg/ml with an ED₅₀ at 2.8 μg/ml and as a partial juvenile hormone antagonist to 0.1 μg/ml juvenile hormone I at concentrations between 10^?3 and 10^?2 μg/ml. By contrast, EMD (ethyl-E-3-methyl-2-dodecenoate) had little juvenile hormone-like activity in vitro up to its limits of solubility (100 μg/ml) and exhibited sporadic partial juvenile hormone antagonistic activity in vitro at concentrations between 1 and 100 μg/ml. Since these concentrations were 10–1000 times that of juvenile hormone I in the medium, EMD apparently is not an efficient competitor.     

Juvenile hormone modifications of gene expression in the fat body and posterior silk glands of Bombyx mori

The patterns of protein synthetic activities were determined in the fat body and silk glands of Bombyx mori larvae during the fifth instar. Comparisons were made between control and juvenile hormone or methoprene—treated animals. In normal larvae, the relative synthetic activities of a few proteins increase up to the time of spinning. In treated larvae, the presence of high levels of juvenile hormone or methoprene never results in an arrest of the expression of differentiation at the molecular level. The syntheses of the most abundant markers of development in both organs are reduced by the hormone, but total inhibition has never been observed. The transfer of a large amount of proteins (28–29 kd) from the fat body to the haemolymph is decreased.     

Induction of perfect superlarvae by the application of juvenile hormone analogue to starved larvae of the silkworm,Bombyx mori

Topical application of juvenile hormone analogue, methoprene, induced a supernumerary larval moult in the silkworm, Bombyx mori. The incidence changed greatly depending on developmental stages and physiological states of the methoprene-treated larvae. When methoprene was applied to feeding larvae, only those treatments from the middle of the 2nd instar until the middle of the 4th instar were effective. An 18-h starvation period from the beginning of the 4th instar and a dose of 1 μg of methoprene per larva were required for 100% incidence of the perfect superlarvae. Allatectomy had no effects on the induction of superlarvae by methoprene. The treated 4th-instar larvae ecdysed to the 5th instar without any delay compared to the controls, and underwent an additional larval ecdysis 4.5 days later. The induced 6th-instar larvae took 8.5 days until the onset of cocoon spinning. The induced superlarvae showed reduced growth rates but an increase of final mass due to prolonged feeding period. A sharp but reduced peak in ecdysteroid titre in the haemolymph appeared one and a half days prior to each larval ecdysis in the treated larvae, suggesting that methoprene provokes the extra larval moult through an additional release of ecdysteroids.     

Effects of anti-juvenile hormone “ETB” on the development and metamorphosis of the silkworm, Bombyx mori

As in the tobacco hornworm Manduca sexta, the synthetic juvenile hormone analogue ETB (ethyl 4-[2-(tert-buthylcarbonyloxy)butoxy]benzoate) showed both juvenile hormone-like and anti-juvenile hormone activities in the silkworm, Bombyx mori. When ETB was topically applied to allatectomized 4th-instar larvae, the compound counteracted the effects of allatectomy, such as induction of precocious metamorphosis and black pigmentation in the larval markings. Therefore, ETB had juvenile hormone activity, but it could neither induce brown pigmentation in the markings nor induce an extra-larval moult as can juvenile hormone.When intact 3rd-instar larvae were treated with the compound, the majority underwent precocious metamorphosis in the 4th-instar, and later formed fertile miniature adults. Some moulted into larval-pupal intermediates or 5th-instar larvae with darkened larval markings and/or with abnormality of specific regions of the silk-gland. The optimal dose for such anti-juvenile effects was about 1–10 μg/larva, and higher doses showed less activity. Such anti-juvenile hormone effects of ETB were counteracted by administration of the juvenile hormone analogue, methoprene, before a certain critical time in the 4th-instar. The corpora allata of treated larvae appeared cytologically normal, and the corpora allata from ETB-induced miniature moths secreted juvenile hormone when implanted into allatectomized 4th-instar larvae.     

Inhibitory effects of parasitism by the gregarious endoparasitoid Cotesia congregata on host testicular development

Cotesia congregata is a gregarious larval endoparasitoid of the tobacco hornworm, Manduca sexta. Parasitized larvae exhibit a variety of physiological and developmental aberrations, the most obvious of which is the induction of developmental arrest characterized by the absence of wandering behavior and suppression of pupation. This arrest appears attributable to continued maintenance of an elevated titer of juvenile hormone and reduced levels of hemolymph juvenile hormone esterase activity. Injection of the wasp's polydnavirus into nonparasitized larvae also causes arrest and the larvae eventually form larval-pupal intermediates instead of normal pupae, indicating the virus may be partially responsible. Aside from causing arrested host development, parasitism also inhibits the normal development and differentiation of testes in male host larvae, so that the testes atrophy instead of growing synchronously with other larval tissues. Here we report that parasitism has pronounced disruptive cytological effects on the developing reproductive organs of male hosts, in addition to causing them to atrophy. Parasitism results in a reduction in testicular volume attributable to a reduction in the number of developing germ cells. Microscopy revealed that the structural integrity of the sheaths surrounding the testicular follicles also is disrupted, so that the tissues appear grossly abnormal compared to those of nonparasitized larvae. Intrahemocoelic injection of purified C. congregata polydnavirus in combination with venom into nonparasitized fourth instar larvae, or topical application of 100 μg of methoprene to fourth instar larvae, also alters sheath integrity and reduces the numbers of developing germ cells, but not to the same degree as the pattern observed in truly parasitized hosts. The occurrence of cell death in the male gonad was documented using the vital dyes acridine orange and ethidium bromide. Arch. Insect Biochem. Physiol. 36:95–114, 1997. © 1997 Wiley-Liss, Inc.     

Antijuvenile influence of the precocene on the development of adult antennae in the large fruit-tree tortrix Archips podana Scop. (Lepidoptera: Tortricidae)

The influence of precocene II, an antijuvenile agent, on the development of adult antennae in the large fruit-tree tortrix A. podana Scop. was demonstrated. Treatment of the fifth instar larvae and prepupae with different doses of precocene proved to cause different sensitivity of the specimens to the juvenile hormone deficit. Treatment with 450 and 600 ??g precocene per specimen during the first days after ecdysis to the fifth instar caused the death of larvae. Treatment with 300, 450, and 600 ??g per specimen on the third day of the fifth instar larvae and prepupae caused a delay in the development of adult antennae. The results are discussed with respect to the role of the juvenile hormone in the development of imaginal structures during metamorphosis.     

Effects of the parasitization of a braconid, Apanteles, on the blood of its host, Pieris

Parasitization of a braconid wasp, Apanteles glomeratus, of larvae of a common cabbage butterfly, Pieris rapae crucivora, caused changes in differential haemocyte count (DHC), total haemocyte count (THC), and encapsulative capacity against dead eggs of Apanteles in the fourth and fifth instar host larvae.However, no correlation could be found between the number of Apanteles eggs deposited and THC of the middle fourth instar host larvae or between the number of parasitoid larvae and specific gravity of the haemolymph from the late fifth instar host larvae.From the changes in DHC and in THC of both non-parasitized and parasitized Pieris larvae, an increase in the number of plasmatocytes of non-parasitized Pieris larvae in the early fourth instar period was supposed to be due to transformation of prohaemocytes into plasmatocytes, and a low population of plasmatocytes of parasitized larvae in the comparable period was assumed to be due to a suppression of transformation of prohaemocytes by some factor released from the parasitoid eggs.Failure of the parasitized fourth instar Pieris larvae to encapsulate injected dead eggs of Apanteles indicated that the parasitoid embryos were, in some way, actively inhibiting the encapsulation reactions of the host.The increase in THC of the parasitized fifth instar larvae could not be ascribed to a decrease in the volume of host haemolymph. Rather it could be interpreted by a suppression of adhesive capacity of haemocytes in the host haemocoel to tissue surfaces.Reduced encapsulative capacity of the parasitized fifth instar larvae might be attributed either to a depression of the adhesive activity of plasmatocytes resulting from a depletion of energy source for haemocytes in the host haemolymph by parasitization, or from an active suppression of adhesiveness of the plasmatocytes by secretions from ‘giant cells’ (teratocytes) originated from the parasitoid.     

Juvenile hormone titres and metabolism during starvation-induced supernumerary larval moulting of the tobacco hornworm, Manduca sexta L.

When tobacco hornworm (manduca sexta) larvae are starved for 5 days immediately after ecdysis to the 5th instar, then fed normal diet, they undergo a supernumerary moult instead of metamorphosis. During starvation the titre of juvenile hormone in the haemolymph increased to a maximum of 3 ng juvenile hormone I equivalents/ml (determined by the black Manduca larval bioassay) on the fourth day of starvation, then began a decline which continued through the subsequent feeding period. The changes in juvenile hormone titre were not attributable to changes in haemolymph volume during starvation (only a 5% decrease) and subsequent feeding. During starvation the esterase activity of the haemolymph declined 4-fold with a 2-fold larger decrease in the DFP-insensitive, presumably juvenile hormone specific, esterase activity. Both the total and the juvenile hormone-specific esterase activity then increased as a function of larval weight during the subsequent feeding period. As growth was slow in the prolongedly starved larvae, sufficient juvenile hormone was present at the time of prothoracicotropic hormone (PTTH) and ecdysteroid release at the beginning of the fourth day of feeding to prevent metamorphosis.