Lawrence I. Gilbert

The juvenile hormones: historical facts and speculations on future research directions

In all of endocrinology there is no more wondrous name for a hormone than the insect juvenile hormone (JH). Could V.B. Wigglesworth have predicted some six decades ago that his term “juvenile hormone” would offer promise of immortal youth to the aged, the expectation of a bloom of dollars to agrochemical concerns, and the hope of solutions to basic problems by developmental biologists and entomologists. The aged have been disappointed and the high expectations of commercial firms have not been met, but hope remains that JH can be used as a probe to ultimately solve basic questions in development. It has been more than two centuries since Lyonet (1762) described granulated vessels in the thorax of lepidopteran larvae that proved to be the prothoracic glands. By contrast, the corpora allata were not mentioned in the literature until Mu ̈ller (1828) described organs in the cockroach that he called pharyngeal bodies and which he thought innervated the dorsal vessel and esophagus. During the remainder of the 19th century, the corpora allata were described as sympathetic ganglia or other components of the nervous system, as indicated by the various descriptive terms given them, e.g. accessory ganglia, tracheal ganglia, lateral ganglion, lateral head ganglion, appendage of the pharyngeal ganglion, etc. In 1899, Heymons dubbed these organs the corpora allata and correctly described their embryological origin, but also believed that they were a pair of sympathetic ganglia concerned with the innervation of the digestive system.

Lipid Metabolism and Function in Insects

Publisher Summary One of the fundamental questions concerning the role of lipids in the physiology of the insect concerns the quantity of lipid contained upon, and interior to, the rigid exoskeleton. Lipids are of vital importance to many insects as substrates for embryogenesis, metamorphosis and flight. Although several problems of the function and metabolism of lipid in insects have been unraveled in recent years, this research area remains ripe for invasion by both the entomologist and biochemist. The task of assuring that insects utilize the same metabolic pathways as micro-organisms and vertebrates has not yet been completed. This chapter concentrates on developments within the past ten years but also discusses older literature when applicable to the historical development of the topic. In most cases, sophisticated experiments have been conducted on only a few species of “domesticated” insects and the result cannot be extrapolated to all insects. The multitude of different ecological niches and behavioral characteristics are no doubt reflected in a great number of metabolic variations on perhaps more than one basic theme.

Ecdysteroid titer during larval-pupal-adult development of the tobacco hornworm, Manduca sexta

Abstract The ecdysteroid titer during the fourth (penultimate) and fifth larval instars of Manduca sexta was characterized by large increases lasting approximately 24 and 60 hr, respectively, and these peaks occurred just before ecdysis. During the last larval instar, an earlier, smaller increase in the titer was also observed lasting approsimately 20 hr and is the increase presumed responsible for commitment to pupal development. After pupation, the ecdysteroid titer increases again, reaching a maximum between Days 7 and 9. At Day 10 postpupation, the titer declined dramatically and stabilized at ∼day 14. During pupal-adult development, there was no sexual dimorphism in the ecdysteroid titer. However, a difference in the ecdysteroid titer between males and females was observed after adult eclosion. Qualitative analysis of the ecdysteroid RIA activity revealed the presence of only ecdysone and 20-hydroxyecdysone at varying ratios depending on developmental stage. For the large peaks preceding larval and pupal ecdysis, 20-hydroxyecdysone was the predominant hormone, while ecdysone was the major component during pupal-adult development. The fifth instar commitment peak was comprised of approximately equal quantities of the two ecdysteroids.

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.

Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone

The steroid 20-hydroxyecdysone (20E) is the primary regulatory hormone that mediates developmental transitions in insects and other arthropods. 20E is produced from ecdysone (E) by the action of a P450 monooxygenase that hydroxylates E at carbon 20. The gene coding for this key enzyme of ecdysteroidogenesis has not been identified definitively in any insect. We show here that the Drosophila E-20-monooxygenase (E20MO) is the product of the shade (shd) locus (cytochrome p450, CYP314a1). When shd is transfected into Drosophila S2 cells, extensive conversion of E to 20E is observed, whereas in sorted homozygous shd embryos, no E20MO activity is apparent either in vivo or in vitro. Mutations in shd lead to severe disruptions in late embryonic morphogenesis and exhibit phenotypes identical to those seen in disembodied (dib) and shadow (sad) mutants, two other genes of the Halloween class that code for P450 enzymes that catalyze the final two steps in the synthesis of E from 2,22-dideoxyecdysone. Unlike dib and sad, shd is not expressed in the ring gland but is expressed in peripheral tissues such as the epidermis, midgut, Malpighian tubules, and fat body, i.e., tissues known to be major sites of E20MO activity in a variety of insects. However, the tissue in which shd is expressed does not appear to be important for developmental function because misexpression of shd in the embryonic mesoderm instead of the epidermis, the normal embryonic tissue in which shd is expressed, rescues embryonic lethality.

Ecdysone metabolism and distribution during the pupal-adult development of Manduca sexta

The metabolism and distribution of endogenous ecdysone and injected [3H]ecdysone were studied during the pupal-adult development of Manduca sexta. Well-characterized antisera were used to detect and quantify endogenous metabolites by radioimmunoassay (RIA) following their separation by ion-suppressed reverse phase, and normal phase, high performance liquid chromatography. Identical chromatographic procedures were employed to determine the metabolic fate of the [3H]ecdysone in the haemolymph pool. These studies revealed the sequential appearance in the haemolymph and gut of progressively oxidized metabolites of ecdysone—hydroxylation at C-20 was followed by hydroxylation at C-26. The data are suggestive of both the induction of the steroid hydroxylases (oxidases) by substrate or other effector substances and the possible coordination of developmental events by ecdysteroids other than 20-hydroxyecdysone. In the haemolymph, two highly-polar conjugates of ecdysone were observed together with conjugates of the other free ecdysteroids, especially those hydroxylated at C-26. In contrast, relatively little 20-hydroxycdysone conjugate was detected in the insect. As adult development proceeded, both endogenous and radiolabelled ecdysteroids were increasingly localized in the gut, so that just prior to eclosion most ecdysteroids were present in the meconium of the high gut (rectal pouch). The peak titres and the kinetics of appearance of ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone were similar for both haemolymph and gut (and for males and females), but considerably higher levels of C-26 oxidized (acid) metabolites of ecdysone and 20-hydroxyecdysone were localized in the gut. Although levels of highly-polar ecdysteroid conjugates found in the haemolymph and gut were similar, considerable amounts of three less polar ecdysone conjugates, of 3-α-epimers of ecdysone and 20-hydroxyecdysone, and of a substance tentatively identified as 2-deoxyecdysone were found only in the gut. Whether ionized, conjugated, or free, the gut ecdysteroids did not appear to equilibrate with the haemolymph compartment. Differences were observed in the metabolism kinetics of exogenously administered radiolabelled ecdysone when compared to the endogenous ecdysteroids; and some RIA positive gut metabolites did not become significantly radiolabelled. This suggests that injection of ecdysone may not simulate the endogenous secretion of ecdysone or its subsequent metabolism and distribution completely accurately.

Molecular and biochemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster

Five different enzymatic activities, catalyzed by both microsomal and mitochondrial cytochrome P450 monooxygenases (CYPs), are strongly implicated in the biosynthesis of ecdysone (E) from cholesterol. However, none of these enzymes have been characterized completely. The present data show that the wild-type genes of two members of the Halloween family of embryonic lethals, disembodied (dib) and shadow (sad), code for mitochondrial cytochromes P450 that mediate the last two hydroxylation reactions in the ecdysteroidogenic pathway in Drosophila, namely the C22- and C2-hydroxylases. When sad (CYP315A1) is transfected into Drosophila S2 cells, the cells metabolize 2-deoxyecdysone (2dE) to E and the [3H]ketotriol (2,22-dideoxyecdysone) to 22-deoxyecdysone. In contrast, dib (CYP302A1) is responsible for the conversion of the [3H]ketotriol to [3H]2dE. When cells are transfected with both dib and sad, they metabolize the [3H]ketotriol to [3H]E in high yield. The expression of sad and dib is concentrated within the individual segments of the developing epidermis when there is a surge of ecdysteroid midway through embryogenesis. This result occurs before the ring gland has developed and suggests that the embryonic epidermis is a site of ecdysteroid biosynthesis. This pattern then diminishes, and, during late embryogenesis, expression of both genes is concentrated in the prothoracic gland cells of the developing ring gland. Expression of dib and sad continues to be localized in this endocrine compartment during larval development, being maximal in both the late second and third instar larvae, about the time of the premolt peaks in the ecdysteroid titer.

Juvenile hormone esterase activity in precisely timed last instar larvae and pharate pupae of Manduca sexta

Juvenile hormone esterase (JHE) from fifth instar Manduca sexta haemolymph was purified 38 fold, treated with DFP and used to determine a Km. Assay parameters for JHE such as partitioning of substrate and product, effect of protein, and time course were determined. Animals staged to within 1.5 hr of ecdysis to the fifth instar were used to determine JHE activity during the instar and pharate pupal life. Two peaks of activity were observed, one just before wandering and the other just before larval-pupal ecdysis.

Halloween genes encode P450 enzymes that mediate steroid hormone biosynthesis in Drosophila melanogaster

Mutation of members of the Halloween gene family results in embryonic lethality. We have shown that two of these genes code for enzymes responsible for specific steps in the synthesis of ecdysone, a polyhydroxylated sterol that is the precursor of the major molting hormone of all arthropods, 20-hydroxyecdysone. These two mitochondrial P450 enzymes, coded for by disembodied (dib) (CYP302A1) and shadow (sad) (CYP315A1), are the C22 and C2 hydroxylases, respectively, as shown by transfection of the gene into S2 cells and subsequent biochemical analysis. These are the last two enzymes in the ecdysone biosynthetic pathway. A third enzyme, necessary for the critical conversion of ecdysone to 20-hydroxyecdysone, the 20-monooxygenase, is encoded by shade (shd) (CYP314A1). All three enzymes are mitochondrial although shade has motifs suggesting both mitochondrial and microsomal locations. By tagging these enzymes, their subcellular location has been confirmed by confocal microscopy. Shade is present in several tissues as expected while disembodied and shadow are restricted to the ring gland. The paradigm used should allow us to define the enzymes mediating the entire ecdysteroid biosynthetic pathway.

The Insect Neuropeptide PTTH Activates Receptor Tyrosine Kinase Torso to Initiate Metamorphosis

Metamorphosis Receptor Identified One of the challenges facing many multicellular organisms is when to change from the juvenile stage to the reproductively mature adult. In insects, this metamorphosis is activated by the brain-derived neuropeptide, prothoracicotropic hormone (PTTH), when larvae reach a characteristic weight. Almost a century after this brain hormone was discovered, Rewitz et al. (p. 1403) have identified the PTTH receptor and its signaling cascade. The PTTH receptor is Torso (a receptor tyrosine kinase that signals through Ras/Raf/Erk), which patterns the embryonic termini during early development in response to the distantly related PTTH factor, Trunk. The receptor of the Drosophila brain hormone that initiates metamorphosis is identified. Holometabolous insects undergo complete metamorphosis to become sexually mature adults. Metamorphosis is initiated by brain-derived prothoracicotropic hormone (PTTH), which stimulates the production of the molting hormone ecdysone via an incompletely defined signaling pathway. Here we demonstrate that Torso, a receptor tyrosine kinase that regulates embryonic terminal cell fate in Drosophila, is the PTTH receptor. Trunk, the embryonic Torso ligand, is related to PTTH, and ectopic expression of PTTH in the embryo partially rescues trunk mutants. In larvae, torso is expressed specifically in the prothoracic gland (PG), and its loss phenocopies the removal of PTTH. The activation of Torso by PTTH stimulates extracellular signal–regulated kinase (ERK) phosphorylation, and the loss of ERK in the PG phenocopies the loss of PTTH and Torso. We conclude that PTTH initiates metamorphosis by activation of the Torso/ERK pathway.