Tanning Of Cuticle Research Articles

Biochemical and biological mode of action of ecdysone agonists on the spruce budworm

The mode of action of RH-5992 (tebufenozide), a non-steroidal ecdysone agonist, on the eastern spruce budworm, Choristoneura fumiferana, was investigated. This diacyl hydrazine compound, upon ingestion, initiates a precocious incomplete molt that is lethal in most lepidopteran larvae including the spruce budworm. This was found to be induced when the larvae ingested the compound early in the stadium prior to the appearance of the ecdysone peak in the hemolymph. The larvae stopped feeding within 8h post feeding (PF) and remained quiescent just as they do in preparation for a normal molt. Head capsule slippage started at 12h PF, became pronounced by 24h, and by 48h an untanned new head capsule was visible behind the old one. The lack of tanning of the new cuticle was due to the failure of dopadecarboxylase gene expression. Although the old cuticle was loose around the entire body, indicating that apolysis had occurred, there was no evidence of ecdysis of the old cuticle, suggesting that eclosion hormone was probably not released. The transcription factor, Choristoneura hormone receptor 3 (CHR3), which is normally expressed at the onset of the hemolymph ecdysone peak, was expressed in the epidermis 1h PF of RH-5992 confirming that this analogue acts through the ecdysone receptor system. This unique mode of action at the molecular level of this ecdysone agonist and its effectiveness as an environmentally benign control agent for the spruce budworm are described.

Tyrosine and catecholamine levels in the hemolymph of tobacco hornworm larvae,Manduca sexta, parasitized by the braconid wasp,Cotesia congregata, and in the developing parasitoids

Parasitism of fifth instar Manduca sexta larvae by the gregarious parasitoid Cotesia congregata prevented normal storage of tyrosine in the hemolymph, whereas total tyrosine levels increased over eight times in the hemolymph of unparasitized larvae by day 4. Tyrosine glucoside, the hemolymph storage form of tyrosine and the precursor for pupal cuticle sclerotizing agents, was found only in trace amounts in parasitized larvae at the time of parasitoid emergence, but had increased to over 6 mM in hemolymph of unparasitized larvae. Concentrations of dopamine and N-β-alanyldopamine (NBAD), precursors for melanization and sclerotization of cuticle, respectively, had approximately doubled in the hemolymph of parasitized larvae by the day of parasitoid emergence, but not in unparasitized larvae. Catecholamine biosynthesis may be transiently stimulated for wound-healing, as black melanic pigmentation appeared around the wasp emergence holes in the host integument. C. congregata larvae accumulate tyrosine, dopamine, and NBAD by the time of emergence and cocoon spinning, either by direct uptake or by synthesis from precursors obtained from the host. NBAD increased in parasitoid larvae close to pupation, suggesting it functions as the main precursor for pupal cuticle tanning. Both dopamine and NBAD increased dramatically in pharate adult wasps just before eclosion and N-acetyldopamine (NADA) appeared for the first time. Dopamine was highest in concentration and total amount, and it can serve both as a precursor for black melanic pigmentation of adult wasp cuticle and for synthesis of NADA and NBAD, the precursors for cuticle sclerotization. Arch. Insect Biochem. Physiol. 38:193–201, 1998. © 1998 Wiley-Liss, Inc.

EFFECT OF RH-5992, A NONSTEROIDAL ECDYSONE AGONIST, ON THE SPRUCE BUDWORM,CHORISTONEURA FUMIFERANA(LEPIDOPTERA: TORTRICIDAE): LABORATORY, GREENHOUSE, AND GROUND SPRAY TRIALS

AbstractIngestion of 0.1 μg of RH-5992, tebufenozide, by early 6th instar larvae of the spruce budworm,Choristoneura fumiferana(Clem.), prior to the appearance of the ecdysone peak in the hemolymph, resulted in the induction of a precocious incomplete moult that was lethal. The larvae stopped feeding within 8 h post ingestion and remained quiescent just as they do in preparation for a normal moult. Head capsule slippage started at 12 h post ingestion, became pronounced by 24 h, and by 48 h an untanned new head capsule was visible behind the old one. The lack of tanning of the new cuticle was due to the failure of dopadecarboxylase gene expression. Although the old cuticle was loose around the entire body, indicating that apolysis had occurred, there was no evidence of ecdysis of the old cuticle, suggesting that eclosion hormone was probably not released. Earlier instars required a lower dose than the later ones to elicit an "all or none" type of moulting response. The most effective routes of entry were by intrahemocoelic injection, followed by ingestion. Topical application was effective only when nonaqueous carriers such as acetone or dimethyl sulfoxide were used. The larvae were unable to discriminate between treated and untreated diet over a 48-h period. The transcription factor,Choristoneurahormone receptor 3, which is normally expressed at the onset of the hemolymph ecdysone peak, was expressed in the epidermis 1 h post ingestion of RH-5992, reached a peak level by 3 h, and became undetectable by 24 h, confirming that this analogue acts through the ecdysone receptor system. Greenhouse tests using potted white spruce trees sprayed with RH-5992 and colonized with 4th-instar spruce budworm indicated that field dosages of 35, 70, 140, and 280 g/ha would all be effective. Ground spray trials conducted in a spruce budworm infested white spruce stand in Zee Casault, Gaspé, Quebec, using a backpack sprayer showed that ≥ 70 g/ha of RH-5992 reduced the insect population by 100% with very little defoliation and was better than Chlorfluazuron®(an analogue of the chitin synthesis inhibitor, diflubenzuron or Dimilin®) treatment, which was used as a positive control. The unique mode of action of this ecdysone agonist and its effectiveness as an environmentally benign control agent for the spruce budworm are discussed.

Sclerotization of Insect Cuticle in a Cell-Free System

Abstract Incubation of deproteinized larval cuticle (chitin flakes) with purified arylphorin (calliphorin) or larval haemolymph of Calliphora vicina resulted in the formation of a chitin-protein complex. Enzymatic oxidation of N-β-alanyldopam ine (NDAB) in the presence of chitin flakes or the chitin-protein complex, resulted in various degrees of cross-linking of NBAD-quinone formed with chitin. This study confirms the involvement of arylphorin in the process of quinone tanning of insect cuticle.

N- β-Alanyldopamine levels and synthesis in integument and other tissues of Manduca sexta (L.) during the larval-pupal transformation

N- β-Alanyldopamine (NBAD) and other diphenols in tissues of the fifth larval instar of the tobacco hornworm, Manduca sexta (L.), were analyzed by HPLC with electrochemical detection. NBAD accumulated in the integument during the intermolt feeding period, with maximal levels in the wandering stage (6 mmol/g). It then declined to a low level during apolysis and endocuticle digestion, while hemolymph NBAD increased during the same interval to a peak concentration (3 mM) shortly before pupal ecdysis. Trachea and foregut contained lesser amounts of NBAD (0.5 mmol/g), perhaps associated with cuticle, whereas fat body, muscle, midgut and hindgut had 0.1 mmol/g or less. Dopamine (DA), N-acetyldopamine and 3,4-dihydroxyphenylalanine (DOPA) were at least 10-fold less abundant than NBAD in the integument. NBAD synthase, which catalyzes the formation of NBAD from DA and β-alanine, was assayed in both integument and fat body. Highest activity was detected in the integument, where two peaks were observed, one at day 3 near the end of larval feeding and the other at day 9 as pupal cuticle tanning was initiated. Fat body enzyme was substantially less and was detected only in the pharate pupa. Maximal NBAD synthesis by integument cultured in vitro was dependent upon DA supplementation of at least 1.4 mM. 20-Hydroxyecdysone did not alter NBAD synthesis in vitro in either the integument or the fat body, even though injection of this hormone into isolated larval abdomens induced synthesis and/or transport of integumental NBAD back into the hemolymph. The rate-limiting steps in the NBAD biosynthetic pathway appear to be the production of DOPA and DA, because β-alanine occurs in the hemolymph at relatively high levels throughout larval-pupal development.

Diphenols in hemolymph and cuticle during development and cuticle tanning of Periplaneta americana (L.) and other cockroach species

AbstractDiphenolic compounds in cockroach hemolymph and cuticle were extracted with 1.2 N HCI, partially purified by alumina adsorption, and analyzed by liquid chromatography. Dopamine (DA) is the major catecholamine in hemolymph of Periplaneta americana, Blatta orientalis, Blattella germanica, Gromphadorhina portentosa, and Blaberus craniifer at adult ecdysis, while N‐acetyldopamine (NADA) predominates in hemolymph of Leucophaea maderae. In P. americana, NADA is the second most abundant catecholamine, while N‐β‐alanyldopamine (NBAD), norepinephrine (NE), 3,4‐dihydroxyphenylalanine, 3,4‐dihydroxyphenylethanol, 3,4‐dihydroxyphenylacetic acid, and 3,4‐dihydroxybenzoic acid occur in lesser quantities. Catecholamines occur mainly as acid labile conjugates in hemolymph. Dopamine, conjugated primarily as the 3‐sulfate ester, increases in hemolymph from 0.1 to 0.8 mM during the last instar. Concentrations decrease by 75% in pharate adults, partially because of an increase in hemolymph volume. A second smaller peak of DA sulfate occurs after ecdysis followed by a rapid disappearance as the cuticle tans. A conjugate of catechol (o‐dihydroxybenzene) is also present in relatively high concentrations at all ages examined. In cuticle, N‐β‐alanylnorepinephrine accumulates during the early period of adult tanning, while NBAD, NADA, N‐acetylnorepinephrine, and DA increase more slowly. The N‐β‐alanyl and N‐acetyl derivatives of DA and NE occure in relatively high concentrations in tanned cutical of P. americana and probably play an important role in the stablization process.

Tyrosine metabolism for insect cuticle tanning

AbstractInsects have become one of the most successful animal groups in diversity and numbers through the development of a multifunctional exoskeleton and skin, which must be shed periodically in order for them to grow and develop into adults. The evolutionary choice of certain structural materials for the assembly and stabilization of a cuticle with remarkable mechanical and chemical properties has allowed insects to invade terrestrial environments and to evolve flight mechanics for dispersion relatively early in geological history. Diphenolic compounds derived from tyrosine play a central role in sclerotization or tanning of the new cuticle. The phenolic amino acid is stored during larval feeding, and it is mobilized for the production of both structural proteins and diphenolic tanning precursors that are transported into the cuticle. The latter compounds permeate the cuticle and serve as precursors for quinonoid derivatives that both sclerotize and pigment the exoskeleton. This report focuses on how tyrosine and derived diphenolic structures are stored as inactive molecules in preecdysial stages, and how they are released and metabolized to tanning chemicals that stabilize the new cuticle.

Tyrosinase catalyzed protein polymerization as an in vitro model for quinone tanning of insect cuticle

AbstractThe validity of Pryor's widely accepted quinone tanning hypothesis for the sclerotization of insect cuticle was examined using an in vitro model system. Quinones generated in situ by the oxidation of catechols with mushroom tyrosinase and molecular oxygen readily reacted with test proteins such as lysozyme, ribonuclease and cytochrome‐c, producing dimers, trimers, and higher oligomers. With the exception of 3,4‐dihydroxyphenylalanine, dopamine, and norepinephrine, most other catechols tested participated in protein polymerization. The inability of these three compounds to support oligomerization of test protein was attributed to their high rate of intramolecular cyclization reaction. Radioactive incorporation studies reveal the formation of catechol‐protomer adducts, as well as aryl‐protein crosslinks in the reaction mixture. The above results strongly support the quinone tanning hypothesis. Based on these findings, the mechanism of cuticular sclerotization is discussed.

Ecdysteroid influence on the development of the host Heliothis virescens and its endoparasite Microplitis croceipes

Ecdysteroid injections into H. virescens larvae parasitized by M. croceipes resulted in an array of effects dependent on the developmental phase of injected larvae. Injections into early 5th instars had little or no effect on either the parasite or the host. Injections into older pre-wandering 5th instars initiated both premature parasite emergence and premature entry of the host into the wandering phase. Injections of ecdysteroid into post-wandering, pre-parasite emergence larvae resulted in both a dose-dependent inhibition of parasite emergence and formation and tanning of host pupal cuticle. Injections into post-wandering, post-parasite-emergence larvae resulted in tanning of the host cuticle. A temperature effect was observed in which effects of the ecdysteroid injections administered at wandering and post-wandering phases were enhanced at the higher rearing temperature of 32°C. RIA of haemolymph from unparasitized larvae showed an early 8-fold increase in ecdysteroid titre 2 days before pupation. This increase was not observed in parasitized larvae of the same age.

Effects of catecholamines and β-alanine on tanning of pupal cuticle of Manduca sexta in vitro

When epidermis from wandering stage tobacco hornworm ( Manduca sexta) larvae was exposed to 5 μg/ml 20-hydroxyecdysone for 3 days, then exposed to hormone-free Grace's medium, the newly formed pupal cuticle tanned slowly up to 35% of its area by day 12. The addition of 1.3 mM dopamine on either day 4 or 5 slightly increased the area tanned and addition of β-alanine (to 11.2 mM) on days 3–5 enhanced tanning 2–2.5-fold by day 8. Later addition had no effect. When pharate pupal cuticle about 24 h before ecdysis was explanted to Grace's medium, little tanning occurred in 24 h unless dopa or dopamine or their derivatives were added; β-alanine up to 4.4 mM had no effect. Partial tanning occurred in 10 mM dopa or dopamine. More effective were N-β-alanylnorepinephrine and N-β-alanyldopamine which produced nearly maximal tanning at 1 and 5 mM respectively. Up to 10 mM N-β-acetylnorepinephrine had little effect. Thus, dopamine and β-alanine are important to cuticular tanning in vitro and apparently need to be incorporated into the exocuticle during its synthesis. Maximal tanning of this exocuticle then requires further incorporation of β-alanyl conjugates.

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.

Bursicon in the mealworm, Tenebrio molitor L. and its role in the control of postecdysial tanning

Using the adult Calliphora bioassay, we found that the tanning hormone, bursicon, is present in the blood of pupal and adult Tenebrio only at the time of ecdysis, when it is released massively from the thoracic and abdominal central nervous system. The hormone's half life in the blood is short (about 1–2 h). Contrary to the findings of other workers, we could find no evidence for the presence of the hormone in the haemolymph during pharate adult development, before ecdysis begins. When newly ecdysed pupae were ligated about the neck, adult development of the thorax and abdomen proceeded normally, but postecdysial tanning of the adult cuticle was almost completely prevented. This failure to tan was not due to lack of bursicon as the hormone was released normally in the ligated animals at the time of ecdysis. This suggests that a pre-ecdysial signal may be required for the development of epidermal competence to respond to bursicon.

Tyrosine glucoside hydrolase activity in tissues of Manduca sexta (L.): Effect of 20-hydroxyecdysone

When 20-hydroxyecdysone was injected into ligated abdomens from wandering larvae of the tobacco hornworm, Manduca sexta (L.), the titres of tyrosine glucoside and tyrosine in the haemolymph decreased and increased, respectively as a result of increased tyrosine glucoside hydrolase activity. Apolysis also occurred and the underlying pupal cuticle became tanned, while in control abdomens none of these events occurred. Tyrosine glucoside hydrolase is found primarily in the fat body and in highest activity in animals undergoing pupal ecdysis. The results support the hypothesis that 20-hydroxyecdysone regulates the build up of free tyrosine by inducing or activating tyrosine glucoside hydroxylase in the fat body. Tyrosine is then released back into the haemolymph for subsequent utilization in pupal cuticle tanning.

Hormonal requirements for the larval-pupal ecdysis induced in the cultured integument of Chilo suppressalis

The switchover from a larval to a pupal epidermal commitment was studied on integument tissue fragments from early last-instar larvae (1–2 days after ecdysis) of Chilo suppressalis cultured in Grace's medium containing 0.01–0.5 μg/ml 20-hydroxyecdysone for 24–72 hr. Fragments were subsequently cultured in medium containing 1 μg/ml 20-hydroxyecdysone for 24 hr and maintained in hormone-free media for 6 additional days. The degree of switchover induction was measured as the ratio of the number of tissue fragments showing pupal characteristics to the total number of fragments used. The degree of switchover increased with the duration of culture, as well as with the concentration of the hormone (up to 0.1 μg/ml), in the first hormonal treatment. Above this concentration, apolysis and new cuticle formation were induced without change in the epidermal commitment. Cultured integument fragments from larvae in the diapause stage, 40–50 days after hatching, and from those in the penultimate stage, showed the switchover under almost the same hormonal conditions as those used with tissue from the early last-instar larvae. After the first hormone treatment, culture in hormone-free medium was unnecessary for cuticle tanning. Juvenile hormone II added to the medium (3 ng/ml) in the first hormonal treatment completely inhibited the switchover induced by 20-hydroxyecdysone. The potential use of the C. suppressalis integument as a bioassay system for juvenoids is discussed.

Interaction Between Ecdysteroid, Eclosion Hormone, and Bursicon Titers inManduca sexta

SYNOPSIS. The end of the molting process in the tobacco hornworm includes the rapid digestion of the old cuticle, molting fluid resorption, ecdysis of the old cuticle, and expansion and hardening of the new cuticle. The coordination of these processes is accomplished by three hormones. Each ecdysis during the life of Manduca appears to be triggered by eclosion hormone. Depending on developmental stage, the hormone comes either from the brain-corpora cardiaca complex or from the chain of ventral ganglia. The neural programs triggered by eclosion hormone include a neuroendocrine event, the release of the tanning hormone, bursicon, thereby ensuring that tanning of the new cuticle must follow ecdysis. Ecdysis, itself, appears to be controlled by the ecdysteroid levels since ecdysteroid injections delay ecdysis at physiological concentrations and in a dose dependent fashion. This delay is due to inhibition of eclosion hormone secretion and to the retardation of the terminal phases of the molt including the digestion of the old cuticle and the onset of sensitivity to eclosion hormone. Thus, eclosion hormone secretion and the ecdysis it triggers are coordinated with the end of development because both are influenced by the same endocrine signal—the decline in the ecdysteroid titer.

Tyrosine metabolism for cuticle tanning in the tobacco hornworm, Manduca sexta (L.) and other lepidoptera: Identification of β- d-glucopyranosyl- O- l-tyrosine and other metabolites

When [ 14C ]tyrosine and [ 14C ]glucose were fed or injected into feeding fifth-instar larvae of the tobacco hornworm, Manduca sexta (L.), they were incorporated into a conjugate identified in hemolymph and carcass extracts as β- d-glucopyranosyl- O- l-tyrosine. In wandering larvae and pupae, the conjugate was hydrolyzed, and tyrosine was hydroxylated and decarboxylated to dihydroxyphenylalanine and 2-(dihydroxyphenyl)ethylamine. None of these metabolites were formed in fourth-instar larvae or in adults. [ 14C ]Phenylalanine was hydroxylated to tyrosine in all stages of insect development. β- d-Glucopyranosyl- O- l-tyrosine was also detected in 18 other species of Lepidoptera but not in species from other insect orders. This conjugate appears to be the major tyrosine storage metabolite for production of tanning diphenol substrates in Lepidoptera.

Metamorphic changes of wild type and mutant Drosophila tissues induced by 20-hydroxy ecdysone in vitro

Wild type (Oregon R) and non-pupariating as well as late-pupariating mutant larval tissues were cultured in vitro up to 5 weeks with and without 20-hydroxy ecdysone (1 μg/ml). The following responses were elicited by the hormone: in the case of wild type tissues detachment of the larval epidermis and muscles from the cuticle; puparial tanning and sclerotization of the larval cuticle; dissociation of the fat body into single cells; inhibition of the movement of the hind intestine. Most of these responses developed within 1 week of culturing. Of the 4 mutants tested, 3 behaved like the wild type. In cultures of ℓ(1) npr-1, however, puparial tanning, disc evagination, and inhibition of the movement of the hind intestine was abnormally weak and the dissociation of fat body was not observed at all. Detachment of the epidermis and muscles as well as formation of the pupal cuticle by disc tissue occurred normally. The results are discussed with respect to the ecdysteroid-induced metamorphosis of the tissues and the autonomy of mutant gene action.

The tanning of insect cuticle—A critical review and a revised mechanism

Two mechanisms to account for the stiffening of cuticle at tanning were proposed in 1940. The quinone tanning theory has been almost universally accepted; that of dehydration almost universally neglected. Calculations and tests on the mechanical properties of cuticle under differing conditions suggest that covalent cross-linking, even if it exists, is insufficient to account for the degree of stiffening of cuticle at sclerotisation. Dehydration will induce sufficient secondary bonds to account for the stiffness and insolubility of ‘tanned’ cuticle in the complete absence of covalent cross-links. It is suggested that the mechanism of sclerotisation is driven by quinones and other chemicals which are secreted into the cuticle at sclerotisation and cause highly controlled dehydration. Covalent cross-linking may still occur, but must be considered to be of secondary importance and unproven in all cuticles other than resilin.

Tyrosine and phenylalanine concentrations in the cockroaches Leucophaea maderae (F.) and Periplaneta americana (L.) in relation to cuticle formation and ecdysis

1. 1. Tyrosine concentrations in L. maderae and P. americana followed a cyclical pattern, remaining low during the interecdysial period, increasing rapidly in the pharate insect with a peak near ecdysis, and declining as tanning of the new cuticle proceeds. 2. 2. Tyrosine levels in integument, haemolymph and fat body reflected the cyclical patterns of the whole insect. 3. 3. Total free tyrosine released for tanning was proportional to body weight so that whole body concentrations were nearly equal in both species at ecdysis. L. maderae maintained higher post-ecdysial levels than P. americana. 4. 4. Phenylalanine concentrations remained very low in both species with only slight increases at ecdysis. 5. 5. Tyrosine and/or phenylalanine are apparently sequestered in a storage form during the larval feeding period.

Pupal cuticle formation by Manduca sexta epidermis in vitro: Patterns of ecdysone sensitivity

During the larval-pupal transformation, various regions of the epidermis of Manduca sexta larvae have previously been found to require different lengths of exposure to the prothoracic glands in order to form pupal cuticle. To distinguish between requirements for differing threshold concentrations of ecdysone and those for differing durations of exposure to ecdysone, wandering stage larval epidermis was cultured in Grace's medium. When most of the thick larval cuticle was removed, the epidermis responded to concentrations of β-ecdysone of 1.0 μ/ml or greater for 4 days by forming cysts which later formed tanned pupal cuticle. No fat body or protein supplement was required. When the larval integument was explanted intact, similar requirements for cuticle formation and for tanning were found. All regions of the fifth abdominal segment required similar concentrations of β-ecdysone (0.4–0.6 μg/ml) for 4 days for 50% to form pupal cuticle, but gin trap epidermis required the least exposure to a threshold concentration of ecdysone (1.5 days in 0.9 μg/ml). The anterior dorsal intersegmental region required about 0.5 day longer, followed by the posterior intersegmental and the dorsal intrasegmental regions. Thus, the duration of exposure seemed more important. About 1 day longer of exposure to ecdysone was required for subsequent tanning of the new cuticle than for cuticle formation, yet tanning of the cuticle did not occur with prolonged exposure to ecdysone.