Walter E. Bollenbacher

Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta.

Abstract The titer of ecdysone in whole animal extracts of Manduca sexta was determined by radioimmunoassay during the fifth (last) larval instar, pharate pupal development and pupation. A subtle peak in ecdysone concentration was noted at day 4 (just prior to the onset of the wandering stage) and a second and greater peak at day 8.5 (coincident with pharate pupal development). The titer fluctuations during development were a result of changes in tissue ecdysone and not of alterations in the ecdysone content of the gut. When prothoracic gland secretory activity was analyzed in vitro at the same stages, the most rapid rate of α-ecdysone secretion was shown to occur on day 7 (one day prior to the peak in whole-animal ecdysone concentration). An earlier peak in prothoracic gland activity may occur at day 4–5. Thin layer and gas-liquid chromatographic analyses revealed developmental changes in the ratio of β:α-ecdysone in hemolymph and whole-animal extracts. It is suggested that the steroid-hydroxylating capacity of the insect increases during the instar.

Changes in ecdysone content during postembryonic development of the wax moth, Galleria mellonella: The role of the ovary

Abstract The ecdysone titers of representative developmental stages during the life cycle of Galleria mellonella were determined by radioimmunoassay. During the last larval instar two ecdysone peaks are present, the first peak occurring relatively late in the instar concurrent with the initiation of wandering and the second separated from the first by only 1.5 days. The second increase in ecdysone titer marks larval-pupal apolysis and is only slightly larger than the first peak in contrast to studies on other Lepidoptera. Two days after pupation a large ecdysone peak occurs denoting the beginning of pharate adult development and the titer declines thereafter. In female pharate adults, but not in males, the ecdysone titer rises to the maximum for the life cycle at about the time of adult eclosion and then declines slowly during the next 4 days. When larvae are gonadectomized, the resulting females no longer yield the ecdysone peak found under normal conditions at about the time of pupal-adult ecdysis. Extraction of ovaries from late pharate adult G. mellonella and analyses of ovarian in vitro secretory kinetics demonstrate unequivocally that the developing ovary synthesizes the ecdysones characterizing the female at the time of adult emergence. Further studies revealed that most of the ovarian ecdysones are associated with mature oocytes and are probably synthesized by the nurse and/or follicle cells. The ovarian ecdysoones are a mixture of α- and β-ecdysone and another molecular species tentatively identified as 2-deoxy-α-ecdysone.

Ecdysone 20-monooxygenase: Characterization of an insect cytochrome P-450 dependent steroid hydroxylase ☆

Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone at C-20 of the side-chain to form ecdysterone, has been characterized in the fat body of early last instar larvae of the tobacco hornworm, Manduca sexta, using a radioenzymological assay. Ecdysterone was demonstrated to be the product of the enzyme system by high-pressure liquid chromatography, gas-liquid chromatography and mass spectrometry. Differential centrifugation, sucrose-gradient centrifugation, electron microscopy and organelle-marker enzyme analysis revealed that ecdysone 20-monooxygenase activity is associated with the mitochondria. The enzymatic properties of ecdysone 20-monooxygenase are that it is most active in a 0.05 M phosphate buffer, is inhibited by Mg2+ and exhibits pH and temperature optima at 7.5 and 30 degrees C, respectively. The enzyme complex has an apparent Km for ecdysone of 1.60 x 10(-7) M and is competitively inhibited by its product, ecdysterone, with an apparent Ki of 2.72 x 10(-5) M. The cytochrome P-450 nature of this insect steroid hydroxylase was initially suggested by its obligate requirement for NADPH and its inhibition by carbon monoxide, p-chloromercuribenzoate, metyrapone and p-aminoglutethimide but not by cyanide. Difference spectroscopy revealed the presence of cytochrome P-450 in the fat-body mitochondrial fraction. A photochemical action spectrum of ecdysone 20-monooxygenase activity confirmed the involvement of cytochrome P-450 in this monooxygenase system.

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

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

Hormones controlling insect metamorphosis.

Publisher Summary This chapter focuses on the control of postembryonic growth and metamorphosis. It highlights the chemistry, biochemistry, and regulation of prothoracicotropic hormone (PTTH), juvenile hormone, and molting hormone (20-hydroxyecdysone, ecdysterone) in the tobacco hornworm, Manduca sexta, and deals only in a cursory manner with hormone action. Under the controlled environmental conditions of the laboratory, almost synchronously developing insects can be obtained. As the larva grows and reaches the boundaries of its cuticular exoskeleton, its epidermal cells secrete a new and larger cuticle, and enzymes that partially digest the original cuticle, and the insect molts. This process is continued with the resulting second, third, and fourth instar larva until the insect is a fifth instar larva, almost identical in shape and form to the first instar animal, but several thousand times its mass. During the fifth instar, the animal is hormonally re-programed so that it undergoes metamorphosis to the pupa at the next molt. The relatively immobile and nonfeeding pupa ultimately molts into an adult hawk moth, at which time mating occurs, eggs are oviposited, and the cycle begins anew. The process of molting, which consists of retraction of the epidermis from the old cuticle, secretion of new cuticle, and ultimate shedding of the partially digested old cuticle, is controlled by hormones.

Correlations between epidermal DNA synthesis and haemolymph ecdysteroid titre during the last larval instar of the tobacco hornworm, Manduca sexta

Abstract During the fifth larval instar of Manduca sexta the commitment of the epidermis to the synthesis of pupal cuticle is presumably affected by a small increase in ecdysteroid titre when juvenile hormone levels are minimal. Two sequential rounds of DNA synthesis without an intervening mitosis occur at about this time, resulting in polyploidy of the epidermis. There is a definite temporal correlation between the first peak of ecdysone and the second round of DNA synthesis and indirect evidence has been presented which suggests that this small increase in ecdysteroid titre actually initiates the second period of DNA synthesis. Further, it appears that large doses of ecdysteroids do not elicit the same response as smaller doses at a specific developmental stage, indicating that the different physiological effects of ecdysteroids (reprogramming and apolysis) may be dependent upon the relative concentration of the hormone. Following mitosis which takes place on approximately day 6 of the last instar, the epidermis undergoes apolysis and secretes pupal cuticle, expressing the commitment made 4.5 days earlier. These results support the ‘quantal mitosis’ theory of cytodifferentiation since the covert differentiative event occurs during a period of DNA synthesis and since mitosis precedes the expression of that event.

Insect prothoracic glands: a role for cyclic AMP in the stimulation of α-ecdysone secretion ☆

The prothoracic glands of the tobacco hornworm, Manduca sexta, were studied to determine if cyclic AMP is involved in the regulation of α-ecdysone secretion. Culturing glands in the presence of the phosphodiesterase inhibitors, aminophylline and 1-methyl-3-isobutylxanthine, caused a greater than 2-fold stimulation of ecdysone secretion while cyclic AMP alone was ineffective. Based on a dose-response analysis, 1-methyl-3-isobutylxanthine was 200 times more potent than aminophylline. Measurements of endogenous prothoracic gland cyclic AMP during the fifth larval instar demonstrated that dramatically increased levels preceded the increase in in vitro ecdysone-secretory ability. The data suggest that cyclic AMP may act as a second messenger in the stimulation of prothoracic gland α-ecdysone secretion by the prothoracicotropic brain hormone.

Synthesis of juvenile hormone binding proteins by the fat body of Manduca sexta.

Abstract The hemolymph of fourth instar Manduca sexta larvae contains both a relatively low molecular weight protein and a large molecular weight lipoprotein that are capable of binding the C18 juvenile hormone (JH). The low molecular weight binding protein (BP) has a much greater affinity for JH than the lipoprotein. A variety of organs were incubated in vitro and both the media and homogenate supernatants were analyzed in order to determine the site of synthesis of the BP. By obtaining antibodies against the BP, immunoprecipitation and immunoelectrophoretic studies could be conducted along with polyacrylamide slab gel electrophoresis and double labeling experiments. The data strongly suggest that the fat body is the insects source of JH-BP and that the BP is rapidly released from the fat body. That is, the fat body synthesizes BP but does not store it in significant quantities. The same appears to be true of the specific lipoprotein capable of binding JH.

The in vitro synthesis and secretion of α-ecdysone by the ring glands of the fly,Sarcophaga bullata

The in vitro secretory product of larval Sarcophage bullata ring glands has been identified as 2beta, 3beta, 14alpha, 22R, 25-pentahydroxy-5beta-cholest-7-en-6-one (alpha-ecdysone). Mid to late 3rd instar larval ecdysones were isolated and identified as 2beta, 3beta, 14alpha, 20R, 22R, 25-hexahydroxy-5beta-cholest-7-en-6-one (beta-ecdysone) and alpha-ecdysone at a ratio of 27:1. The low level of alpha-ecdysone in vivo, relative to its exclusive in vitro synthesis and secretion by the ring glands, is a function of the very active C20 hydroxylation mechanism in tissues peripheral to the ring gland. The role of alpha-ecdysone as a prohormone in dipteran metamorphosis is discussed.

In vitro biosynthesis of juvenile hormone by the larval corpora allata of Manduca sexta: Quantification by radioimmunoassay ☆

An in vitro method has been developed for the investigation of the regulation of juvenile hormone biosynthesis by insect corpora allata. Glands were maintained in Marks medium 19AB and JH synthesis quantified by a modified radioimmunoassay for juvenile hormone I. The radioimmunoassay is specific for JH I and exhibits approximately 12.6% cross reactivity with JH II and no cross reactivity with JH III. The assay directly measures the JH present in culture medium and has a maximum sensitivity of 50 pg JH I equivalents. Corpora allata from day 5 last instar Manduca sexta larvae were used to define the kinetics parameters of the in vitro system, including a demonstration that small groups of right and left glands synthesize equivalent amounts of juvenile hormone. The juvenile hormones synthesized were identified as juvenile hormones I and II in a ratio of 1:4, respectively. Juvenile hormone III could not be excluded as a product of the corpora allata owing to the low cross reactivity of this homolog (1.7%) in the radioimmunoassay. Corpora allata from different developmental stages exhibited synthesis rates generally consistent with predicted activity based on in vivo hormone titers with the exception of day 5 of the last instar. The variation in gland activity relative to the control of juvenile hormone titer in vivo is discussed.