Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco hornworm and their control by 20-hydroxyecdysone

Summary WhenManduca sexta larvae are allatectomized 5 h before head capsule slippage (HCS) in the final larval molt, the new larval cuticle contains granules that melanize 3 h before ecdysis when the ecdysteroid titer falls (Curtis et al. 1984). In both the epidermis and hemolymph of these allatectomized larvae dopamine was higher than dopa prior to and at the time of melanization. Dopamine also increased in the new cuticle as melanization began. Dopa decarboxylase (DDC) activity increased in the epidermis, cuticle, and fat body beginning 16 h after HCS, with a two-fold greater increase in the epidermis of allatectomized larvae. Both -MDH and -fluoromethyl-dopa inhibited epidermal DDC activity and inhibited melanization in vitro when dopa was used as a precursor. Addition of dopamine to the medium allowed melanization in the presence of the inhibitors. All these results indicate that dopamine is likely the primary precursor of cuticular melanin. The diphenoloxidase in the premelanin granules was activated in vivo between 19 and 21 h after HCS and was found to prefer dopamine to dopa and not to convert tyrosine to melanin. The activation of the prophenoloxidase was inhibited by 20-hydroxyecdysone (20-HE), both in vivo and in vitro, if hormone was given by 16 h after HCS. Infusion of 1.2 g/ml 20-HE into allatectomized larvae for 24 h from HCS prevented both the increase in DDC activity and the activation of the premelanin granules. Although the larvae ecdysed after a 15 h delay, melanization never occurred.Abbreviations -MDH L-3-(3,4 dihydroxyphenyl)-2-hydrazine-methylpropionic acid - -FM-dopa R-S--fluoromethyl-dopa - DCC dopa decarboxylase - 20-HE 20-hydroxyecdysone - JH juvenile hormone - HCS head capsule slippage     

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.     

Hormonal regulation of dopa decarboxylase during a larval molt

Cuticular sclerotization in insects requires dopamine derivatives and thus the presence of dopa decarboxylase (DDC), the enzyme which converts dopa to dopamine. During the last half of the larval molt of the tobacco hornworm, Manduca sexta, beginning at 16 hr after head capsule slippage, the epidermal DDC activity increased fourfold. By contrast, allatectomized larvae which were destined to produce a melanized cuticle showed a sevenfold increase. This increase in DDC activity was prevented by infusion of 20-hydroxyecdysone (20HE) into the larva, indicating that the fall of the ecdysteroid titer is necessary for the increase. In vitro 20HE also prevented the increase in a dose-dependent manner when the epidermis was explanted at 16 hr after head capsule slippage but had less effect on epidermis explanted 3 hr later. Both 5 micrograms/ml alpha-amanitin and 100 micrograms/ml cycloheximide also prevented the increase. Application of juvenile hormone I showed that the critical period for determination of the level of the later increase in DDC activity was about 4 hr after head capsule slippage at the peak of the ecdysteroid titer. Apparently then the rise and fall of ecdysteroid regulate different aspects of DDC synthesis, the rise determining its later appearance and the fall timing this appearance.     

Hormonal requirements for the larval-pupal transformation of the epidermis of Manduca sexta in vitro

In the tobacco hornworm, Manduca sexta, metamorphosis occurs in response to two releases of ecdysone that occur 2 days apart. Epidermis was explanted from feeding final-instar larvae before the first release of ecdysone and was cultured in Grace's medium. When exposed to 1 μg/ml of β-ecdysone for 24 hr and then to hormone-free medium for 24 hr, followed by 5 μg/ml of β-ecdysone for 4 days, the epidermis produced tanned pupal cuticle in vitro. During the first 24 hr of exposure to β-ecdysone, the epidermis first changed its cellular commitment to that for pupal cuticle formation (ET₅₀ = 14 hr), then later (by 22 hr) it became committed to tan that cuticle. Then, for most of the pupal cuticle to be tanned, at least a 12-hr period of culture in hormone-free medium was required before the cuticle synthesis was initiated. Consequently, some events prerequisite to sclerotization of pupal cuticle not only occur during the ecdysone-induced change in commitment but also during the ecdysone-free period. When the tissue was preincubated in 3 μg/ml of juvenile hormone (JH I or a mimic epoxygeranylsesamole) for 3 hr and then exposed to both ecdysone and juvenile hormone for 24 hr, it subsequently formed larval cuticle. The optimal conditions for this larval cuticle formation were exposure to 5 μg/ml of β-ecdysone in the presence of 3 μg/ml of epoxygeranylsesamole for 48 hr. When the epidermis was cultured in Grace's medium for 3 days and then exposed to 5 μg/ml of β-ecdysone for 4 days, 70% of the pieces formed pupal cuticle. By contrast, if both ecdysone and JH were added, 77% formed larval cuticle. Therefore, the change from larval to pupal commitment of the epidermal cells requires not only the absence of JH, but also exposure to ecdysone.     

Catecholamine metabolism and in vitro induction of premature cuticle melanization in wild type and pigmentation mutants of Drosophila melanogaster

The major pathway leading to adult cuticle melanization in Drosophila melanogaster has been investigated by a combination of biochemical and genetic approaches. By comparing catecholamine pools in newly emerged flies and in frass (excreta) collected 1 to 4 days after eclosion from wild type with those obtained from several pigmentation mutants, the major flow of catecholamines through the pathway to an unidentified final catabolite was determined. We also demonstrate that incubation with dopamine in vitro induces premature melanization in wild type unpigmented pharate adults several hours before the developmentally programmed onset of melanization, supporting the hypothesis that the availability of catecholamines may be the limiting factor determining the onset of melanization and that the major enzymatic activities that act downstream of dopa decarboxylase in the pathway are deposited into the cuticle before pigmentation begins. In vitro melanization studies with various pigmentation mutants that are associated with critical enzymatic steps in Drosophila catecholamine metabolism are consistent with their proposed function and suggest a central role of N-β-alanyldopamine in adult cuticle pigmentation. © 1996 Wiley-Liss, Inc.     

Regulation of dopa decarboxylase gene expression in the larval epidermis of the tobacco hornworm by 20-hydroxyecdysone and juvenile hormone

Dopa decarboxylase (DDC) which converts dopa to dopamine is important for cuticular melanization and sclerotization in insects. An antibody to Drosophila DDC was found to precipitate both DDC activity and a 49-kDa polypeptide synthesized by the epidermis of molting Manduca larvae. Using the Drosophila DDC gene, we isolated the Manduca DDC gene which on hybrid selection produced a 49-kDa translation product precipitable by the Drosophila DDC antibody. The 3.1-kb DDC mRNA appeared 12 hr after head capsule slippage (HCS) and reached maximal levels 7 hr later. Peak expression was twofold higher in melanizing allatectomized larvae and could be depressed to normal levels by application of 0.1 micrograms juvenile hormone I at HCS. Infusion of 1 microgram/hr 20-hydroxyecdysone (20-HE) for 18 hr beginning 2 hr after HCS or addition of 1 microgram/ml 20-HE to the culture medium for 24 hr prevented the normal increase in DDC mRNA. When Day 2 fourth instar epidermis was explanted before the molting ecdysteroid rise and cultured with 1-3 micrograms/ml 20-HE for 17 hr and then for 24 hr in hormone-free medium, DDC expression was three- to fourfold higher than that in epidermis cultured in the absence of hormone. Twelve or more hours of incubation with 20-HE was required for an increase in DDC mRNA, but continuous exposure to 20-HE prevented the increase. In all cultures an initial rapid increase in DDC mRNA was observed which decayed with time in vitro and apparently was associated with the wound response. Thus, ecdysteroid during a larval molt is necessary to program the later expression of DDC, but the subsequent decline of the ecdysteroid is required for this expression to occur.     

Temporal and spatial expression of the yellow gene in correlation with cuticle formation and dopa decarboxylase activity in Drosophila development

The yellow (y) gene of Drosophila is required for the formation of black melanin and its deposition in the cuticle. We have studied by immunohistochemical methods the temporal and spatial distribution of the protein product of the y gene during embryonic and pupal development and have correlated its expression with events of cuticle synthesis by the epidermal cells and with cuticle sclerotization. Except for expression in early embryos, the y protein is only found in the epidermal cells and may be secreted into the cuticle as it is being deposited. The amount of y protein in various regions of the embryo and pupa correlates directly with the intensity of melanization over any section of the epidermis. Expression of the y gene begins in the epidermal cells at 48 hr after pupariation and is well correlated with the beginning deposition of the adult cuticle. At this stage the adult cuticle is unsclerotized and unpigmented and dopa decarboxylase levels, a key enzyme in catecholamine metabolism which provides the crosslinking agents as well as the precursors for melanin, is low. As a separate event 26 hr after the onset of y gene expression, the first melanin deposition occurs in the head bristles and pigmentation continues in an anterior to posterior progression until eclosion. This melanization wave is correlated with elevated dopa decarboxylase activity. Crosslinking of the adult cuticle also occurs in a similar anterior to posterior progression at about the same time. We have shown by imaginal disc transplantation that timing of cuticle sclerotization depends on the position of the tissue along the anterior-posterior axis and that it is not an inherent feature of the discs themselves. We suggest that actual melanization and sclerotization of the cuticle by crosslinking are initiated at this time in pupal development by the availability of the catecholamine substrates which diffuse into the cuticle. Intensity of melanization and position of melanin pigment is determined by the presence or absence of the y protein in the cuticle, thus converting the y protein prepattern into the melanization pattern.     

Ecdysteroid regulation of the onset of cuticular melanization in allatectomized and Black mutant Manduca sexta larvae

The absence of juvenile hormone at the time of head cap slippage during the last-larval moult of the tabacco hornworm, Manduca sexta, causes deposition of premelanin granules into the outer regions of the newly forming endocuticle beginning 13 h later. These granules were found to contain an inactive phenoloxidase which becomes activated about 9 h later, 4 h before body melanization begins. The onset of melanization was not accelerated by melanization and reddish colouration hormone from Bombyx heads, extracts of pharate-adult corpora cardiaca or pharate-larval ventral nerve cords (sources of eclosion hormone), or extracts of pharate-larval suboesophageal ganglia or corpora cardiaca-corpora allata complexes. Instead the fall of the ecdysteroid titre to below 250 ng/ml 20-hydroxyecdysone equivalents appeared to be the cue that allowed melanization about 4.5 h later. Up to, but not after, this time both melanization and ecdysis could be delayed by exogenous 20-hydroxyecdysone in a dose-dependent fashion above 0.1 μg per larva. In vitro studies published elsewhere indicate that 20-hydroxyecdysone prevents the activation of the premelanin granules. Thus the granules can be deposited at the proper time in the newly forming endocuticle but their melanization is regulated by the declining ecdysteroid titre and it thus synchronized with other events occurring just before ecdysis.     

Granular phenoloxidase involved in cuticular melanization in the tobacco hornworm: regulation of its synthesis in the epidermis by juvenile hormone

The granular phenoloxidase (PO) that is responsible for cuticular melanization in Manduca sexta larva was purified and an antibody was prepared. This granular PO was found to consist of four isozymes of 90 kDa with isoelectric points ranging from 5.7 to 5.85. The enzyme was immunologically and electrophoretically distinct from the cuticular wound PO, a second cuticular PO common to all larval cuticle, and the hemolymph PO. Both [14C]mannose and [14C]sialic acid were incorporated into the granular PO, showing that this granular PO was a glycoprotein whose sugar moiety was a complex oligosaccharide. When no juvenile hormone (JH) was present at the head capsule slippage (HCS) stage, the epidermis began synthesizing PO 6 hr later. This epidermal synthesis was maximal 12 hr after HCS at which time the PO appeared in the cuticle, and then synthesis declined. When synthesis ceased about 23 hr after HCS, no further incorporation into the cuticle was observed. As melanization proceeded, immunologically detectable cuticular PO decreased. Application of 0.1 microgram JH I at the time of HCS inhibited synthesis of PO by the epidermis and thus prevented melanization. JH application after PO synthesis had begun (8 hr after HCS) prevented its subsequent synthesis, causing partial melanization. Thus, the absence of JH is necessary during the period of epidermal synthesis of the granular PO to allow complete melanization.     

Role of β-alanine in pupal cuticle coloration in the tobacco hornworm,Manduca sexta: Effects of β-alanine analogues on melanization and catecholamine levels

Hydrazino and aminooxy derivatives of β-alanine were found to cause blackening of Manduca sexta pupal cuticle when they were injected into pharate pupae at the onset of pre-ecdysial tanning. One of these compounds, ethyl hydrazinoacetate (EHA), was used for further study. It was effective if injected up to about 4 hr before pupal ecdysis. These melanized cuticles contained excessive amounts of dopamine and decreased amounts of N-β-alanyldopamine (NBAD) and N-acetyldopamine (NADA). Furthermore, EHA induced elevated dopamine and lowered β-alanine levels in the hemolymph. Similar blackening occurred when 20 mg/animal dopamine was injected. Injection of excess β-alanine rescued the normal brown color, irrespective of the concentration of EHA. Also, EHA caused melanization in vitro in the presence of dopamine, whereas the addition of β-alanine and NBAD allowed normal pupal coloration in vitro. These hydrazino and aminooxy compounds likely interfere with β-alanine synthesis or mobilization and thus with N-acylation of the catecholamines to form NBAD and N-β-alanylnorepinephrine.     

Effects of U.V. light and temperature on the melanization and the formation of daily growth layers of Sarcophaga falculata.

Adult Sarcophaga flies, immediately after eclosion, were subjected to different temperature régimes or irradiated with u.v. light. The effect of the treatment on cuticular melanization was studied by comparison with specimens of control series or by comparing the areas of cuticle on the thoracic phragma of the same specimen that were deposited at different times under different conditions. The cuticle of flies that were tanned at 15 or 31°C was less melanized than that of control flies at 26°C. Irradiation with long wave u.v. light suppressed mainly the melanization whereas both the melanization and sclerotization processes were inhibited by short wave u.v. A decrease in adult melanization was caused also by exposure to u.v. of the heads of pharate adults 24 hr before eclosion.     

The juvenile hormone analog pyriproxyfen affects ecdysteroid-dependent cuticle melanization and shifts the pupal ecdysteroid peak in the honey bee (Apis mellifera)

The control of the pupal melanization in the honey bee by ecdysteroids, and the modulation of these processes by a juvenile hormone analog were investigated by a combination of in vivo and in vitro experiments. Injection of 1-5 microg of 20-hydroxyecdysone (20E) into unpigmented pupae showed a dose- and stage-dependent effect. The higher the dose and the later the injection was performed, the more pronounced was the delay in cuticle pigmentation. This inhibition of cuticular melanization by artificially elevated ecdysteroid titers was corroborated by in vitro experiments, culturing integument from unpigmented, dark-eyed pupae for 1-4 days in the presence of 20E (2 or 5 microg/ml culture medium). Topical application (1 microg) of pyriproxyfen to unpigmented, white-eyed pupae had the opposite effect, leading to precocious and enhanced melanization of the pupal cuticle. In vitro incubation of integuments in the presence of this juvenile hormone analog (1 microg/ml) confirmed these results, showing that pyriproxyfen is apparently capable of triggering melanization. The in vivo mode of action of pyriproxyfen was further investigated by quantifying hemolymph ecdysteroids by radioimmunoassays. Topical application leads to a delay of the pupal ecdysteroid peak by 4 days. The pyriproxyfen-induced low ecdysteroid titers during early pupal development could account for precocious pigmentation by removing an inhibition on prophenoloxidase activation normally imposed by the elevated ecdysteroid titer during this phase.     

Chiasma-induction and tyrosine metabolism in locusts

The production of a gregarization pheromone has been postulated in locusts, with effects on melanization of the hopper cuticle and increased chiasma frequency during meiosis in the adult on crowding or gregarization. Lack of chiasma-inducing effect of the pheromone on albino strains is correlated with the absence or deficiency of some of the products of the metabolic pathways of tyrosine. Some of these products, commercially obtainable, are the amino acids phenylalanine and tyrosine leading to both the melanization and sclerotization pathways; dopamine formed from dopa in the lastnamed pathway; three products of dopamine i.e. protocatechuic acid, noradrenaline and adrenaline. The injection of solutions of these metabolites into the haemolymph of solitary hoppers has shown that only dopa to some extent but noradrenaline to a large extent are effective in raising chiasma frequency in solitarised individuals of normal-coloured strains of Locusta, while in two albino strains, which differ genetically, the injection of dopa, dopamine, protocatechuic acid and noradrenaline proved effective; phenylalanine was effective in only one of these albino strains, while adrenaline was effective in neither. The chiasma-inducing effect of noradrenaline, common to the three strains, is accompanied in the normal-coloured strain by a greater retention of dark coloration during solitarization and by some attainment of the crowded type of morphometric ratios which is a third physical criterion of gregarization. The genetic blocks to the physical criteria of gregaria in the albino strains lie at the immediate level of dopa production or previous to this reaction; it may be construed that such a block in the solitaria of normal-coloured strains also lies at this early level, in this case being induced by too low a pheromone concentration.     

Diflubenzuron-Induced alterations during in vitro development of Tenebrio molitor pupal integument

The effects of diflubenzuron (DFB) in Tenebrio molitor pupae were first investigated on cuticle secretion induced by 20-hydroxyecdysone in vitro. The sternal integuments were treated by DFB either 3 days before culture or during culture. DFB, when applied before culture, did not prevent the molting hormone from inducing a new cuticle deposition by integument explants in vitro. However, this cuticle showed several architectural alterations and a thickness reduction. When applied during the culture in the presence of 20-hydroxyecdysone, DFB at high dose (≥ 20 μg/ml) was able to inhibit cuticle secretion, but lower doses (? 10 μg/ml) resulted in epicuticle deposition. These observations confirm in vivo studies showing antagonistic effects of DFB and ecdysteroids at the level of epidermal cells. In another series of experiments, the DFB effects were analyzed without addition of exogenous molting hormone in vitro. Because it had been observed in previous studies that pupal epidermal explants of Tenebrio secrete low but significant amounts of ecdysteroids in the culture medium, this in vitro secretion was measured by radioimmunoassay after DFB treatment. It was observed that DFB, when applied either before or during culture, significantly reduced the hormonal secretion in vitro. This reduction, observed at the level of epidermal cells, could be homologous with the diminution of the endogenous ecdysteroid peak previously described after in vivo DFB treatment in Tenebrio pupae.     

A Diphenol Oxidase Gene Is Part of a Cluster of Genes Involved in Catecholamine Metabolism and Sclerotization in Drosophila. I. Identification of the Biochemical Defect in Dox-A2 [l(2)37Bf] Mutants

Phenol oxidase, a complex enzyme, plays a major role in the processes of sclerotization and melanization of cuticle in insects. Several loci have been reported to affect levels of phenol oxidase activity, but to date only one structural locus has been identified [Dox-3F (2-53.1+)]. Recently isolated Dox-A2 mutations (2-53.9) are recessive, early larval lethals, which as heterozygotes reduce phenol oxidase activity. A homozygous mutant escaper had weak, completely unpigmented cuticle and unpigmented bristles. Enzyme assays show that Dox-A2 heterozygotes have diphenol oxidase activity reduced to 47-79% of wild type, whereas monophenol oxidase activity, at 94-106% of wild type, is normal. Elevated pool sizes of the diphenol oxidase substrates DOPA, dopamine, and N-acetyldopamine are observed in the mutant, confirming the enzyme assay results. Separation of the three phenol oxidase A component activities on polyacrylamide gels shows that Dox-A2 mutations reduce the activity of only the A2 component. Dox-A2 may identify a structural locus for the A2 component of the diphenol oxidase enzyme system. The Dox-A2 locus is one of 18 loci in the dopa decarboxylase, Df (2L)TW130 region of the second chromosome, at least 14 of which affect the formation, melanization or sclerotization of cuticle in some way. These loci form an apparent cluster of functionally related genes.     

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.     

Role of beta-alanine in cuticular tanning,sclerotization, and temperature regulation in Drosophila melanogaster

Injection of 20 nl of 1.0 M beta-alanine, about the minimal amount needed to produce wild-type tanned phenomenocopies from newly eclosed mutant black Drosophila melanogaster, increases stiffness and puncture-resistance of the wing cuticle. Increasing the concentration of beta-alanine to 2.0 M increases puncture-resistance further. Injection of 1.0 M of the beta-alanine analogue beta-aminoisobutyric acid, does not induce tanning or puncture-resistance, nor does injection of 1.0 M dopamine. However, injection of 1.0 M beta-alanine and 1.0 M dopamine increases puncture-resistance more than an injection of 1.0 M beta-alanine, though not more than an injection of 2.0 M beta-alanine alone. Within 10 min after injection of [3-³H]beta-alanine into newly eclosed normal flies, ³H becomes 8.7 times more concentrated in the cuticle than in an equal area of underlying epidermis. ³H is excluded from the epidermis or cuticle of ebony strains. Ebony strains show a deficiency of cuticular electron-absorbing material, and the cuticular lamellae show a tendency to separate from each other. Compaction of the chitinous lamellae is induced in alkali-detanned pupal sheaths by exposure to nascent quinones of N-acetyldopamine or N-beta-alanyldopamine. Glucosamine, but not N-acetylglucosamine, reacts with such quinones in tanning reactions. Under an infrared beam, black cuticular pigmentation induces more rapid heating of haemocoel fluids than does tan pigmentation. A theory of pigmentation and sclerotization relative to environmental adaptation is presented.