In vitro differentiation ofPieris brassicae imaginal wing discs: Effects and metabolism of ecdysone and ecdysterone

Summary Imaginal wing discs ofPieris brassicae can be cultured in vitro for extended periods. Their ultrastructural development was investigated after culture in the presence of various concentrations of ecdysone and ecdysterone. When ecdysone or low concentrations of ecdysterone (2×10^–7 M) were added to culture media, larval discs secreted a pupal cuticle and they subsequently differentiated scales; prepupal discs completed their imaginal development. With a higher concentration of ecdysterone (2×10^–6 M), all discs produced abundant but fragmentary cuticular material.Prepupal discs were able to metabolize both hormones in vitro. Ecdysterone was mainly converted into a polar compound detectable after a short period of incubation. Ecdysone was transformed, at a slower rate, forming a polar compound and 26-hydroxyecdysone but no ecdysterone.     

The role of nutrition in creation of the eye imaginal disc and initiation of metamorphosis in Manduca sexta

With the exception of the wing imaginal discs, the imaginal discs of Manduca sexta are not formed until early in the final larval instar. An early step in the development of these late-forming imaginal discs from the imaginal primordia appears to be an irreversible commitment to form pupal cuticle at the next molt. Similar to pupal commitment in other tissues at later stages, activation of broad expression is correlated with pupal commitment in the adult eye primordia. Feeding is required during the final larval instar for activation of broad expression in the eye primordia, and dietary sugar is the specific nutritional cue required. Dietary protein is also necessary during this time to initiate the proliferative program and growth of the eye imaginal disc. Although the hemolymph titer of juvenile hormone normally decreases to low levels early in the final larval instar, eye disc development begins even if the juvenile hormone titer is artificially maintained at high levels. Instead, creation of the late-forming imaginal discs in Manduca appears to be controlled by unidentified endocrine factors whose activation is regulated by the nutritional state of the animal.     

Chitin biosynthesis in imaginal discs cultured in vitro

Summary We have investigated the stimulation of cuticle production by imaginal discs ofPlodia interpunctella in tissue culture. We turned to biochemical methods to assess the quantitative effects of beta-ecdysone on chitin biosynthesis in wing discs incubated with 0.5 C of C¹⁴-glucosamine for the final 24 h of culture.We demonstrated that imaginal discs ofP. interpunctella respond to increasing concentrations of -ecdysone with increased synthesis. The threshold is between 0.01 and 0.1 g/ml of hormone (2×10^–8 M to 2×10^–7 M). These data represent the first demonstration of quantitative biosynthesis of chitin by a developing tissue in vitro in relation to varying amounts of hormone. Additionally, protein synthesis during the -ecdysone-dependent period was necessary for chitin synthesis. This system thus lends itself to a detailed investigation of the control of chitin biosynthesis.We wish to dedicate this paper to the memory of a fine colleague and friend, Dr. Andrzej Dutkowski     

Juvenile hormone acid methyltransferase activity in imaginal discs of Manduca sexta prepupae

The occurrence of a peak of juvenile hormone (JH) during the prepupal period has been noted in several lepidopterans. In Manduca sexta and Hyalophora cecropia this peak is known to prevent the precocious onset of adult differentiation in imaginal tissues. However, it has previously been observed in our laboratory that corpora allata (CA) of this age are incapable of making JH owing to a lack of the terminal synthetic enzyme, juvenile hormone acid methyltransferase (JHAMT). Since the CA are required for normal pupation, it is likely that JH acid is the product released by the prepupal CA. Therefore, we analyzed whether JH acid treatment would prevent precocious adultoid differentiation in allatectomized M. sexta larvae. JH acid injections were found to be as effective as JH in normalizing pupation, and acted in a time- and dose-dependent manner. This finding led to a question of whether injected or endogenous JH acid could be methylated to JH. Homogenates of several tissues from prepupae were assayed for the presence of JHAMT. Of the tissues assayed, only imaginal discs possessed significant levels of the enzyme. These results support our previously proposed mechanism for production of the prepupal JH peak in M. sexta.     

Juvenile hormone and ovarian growth in Manduca sexta

In Manduca sexta the major size increase of ovarian follicles is accomplished by two processes: (1) vitellogenesis in which follicular volume and dry weight increase simultaneously, and (2) hydration in which absorption of water by the oocyte accounts for an 80% increase in volume prior to chorion formation. Vitellogenic growth occurs in both a slow and rapid phase. Rapid vitellogenic growth is initiated only by follicles of a threshold size (1 mm) and is a juvenile hormone (JH)-dependent event. In the absence of JH follicles grow to 1 mm and then degenerate.     

Sexual differentiation in the CNS of the moth,Manduca sexta. I. Sex and segment-specificity in production,differentiation, and survival of the imaginal midline neurons

We analyzed the development of several sets of postembryonic sex-specific motoneurons in Manduca sexta which belong to a group of homologous lineage of neurons called the imaginal midline neurons (IMNs). Adult female oviduct motoneurons and male sperm duct motoneurons are IMNs that show similar anatomical features and differentiate during metamorphosis, despite appearing in different segments: A7 for oviduct neurons, A9 for sperm duct neurons. These cells are born at the same time and, initially, similar sets are found in A7 and A9 ganglia of larvae of both sexes. The dimorphic adult pattern is generated by sex-specific production and cell death. A7 IMNs differentiate in both sexes through early pupal stages, whereupon they disappear in the male and become the oviduct motoneurons in the female. A9 IMNs are overproduced in the male, and subsequent cell death reduces male cell number and eliminates the small complement of female cells; the surviving male cells develop into the sperm duct motoneurons. Similar IMN arrays are generated in nongenital ganglia, but show non-sex-specific fates. This suggests that both the sex of these cells and their segment of residence play major roles in their subsequent differentiation. 1994 John Wiley & Sons, Inc.     

Juvenile hormone modulates 20-hydroxyecdysone-inducible ecdysone receptor and ultraspiracle gene expression in the tobacco hornworm, Manduca sexta

 Insect molting and metamorphosis are orchestrated by ecdysteroids with juvenile hormone (JH) preventing the actions of ecdysteroids necessary for metamorphosis. During the molt and metamorphosis of the dorsal abdominal epidermis of the tobacco hornworm, Manduca sexta, the isoforms involved in the ecdysone receptor (EcR)/Ultraspiracle (USP) complex change with the most dramatic switch being the loss of USP-1 and the appearance of USP-2 during the larval and pupal molts. We show here that this switch in USP isoforms is mediated by high 20-hydroxyecdysone (20E) and that the presence of JH is necessary for the down-regulation of USP-1 mRNA. The decrease of USP-1 mRNA in day 2 fourth instar larval epidermis in vitro required exposure to a high concentration (10^–5 M) of 20E equivalent to the peak ecdysteroid concentration in vivo, whereas the increase of USP-2 mRNA occurred at lower concentrations (effective concentrations, EC₅₀=6.3×10^–7 M). During the pupal molt of allatectomized larvae which lack JH, USP-2 mRNA increased normally with the increasing ecdysteroid titer, whereas USP-1 mRNA remained high until pupation. When day 2 fifth instar larval epidermis was exposed to 500 ng/ml 20E in the absence of JH to cause pupal commitment of the cells by 24 h, USP-1 RNA remained at its high preculture level for 12 h, then increased two- to threefold by 24 h. The increase was prevented by the presence of 1 μg/ml JH I which also prevents the pupal commitment of the cells. By contrast, USP-2 mRNA increased steadily with the same EC₅₀ as in fourth stage epidermis, irrespective of the presence or absence of JH. Under the same conditions, mRNAs for both EcR-B1 and EcR-A isoforms were up-regulated by 20E, each in its own time-dependent manner, similar to that seen in vivo. These initial mRNA increases were unaffected by the presence of JH I, but those seen after 12 h exposure to 20E were prevented by JH, indicating a difference in response between larvally and pupally committed cells. The presence of JH which maintained larval commitment of the cells also prolonged the half-life of the EcR proteins in these cells. These results indicate that both EcR and USP RNAs are regulated by 20E and can be modulated by JH in a complex manner with only that of USP-2 apparently unaffected. Received: 16 July 1998 / Accepted: 5 August 1998     

Sexual differentiation in the CNS of the moth,Manduca sexta. II. Target dependence for the survival of the imaginal midline neurons

While majority of the neurons in the adult nervous system of the moth Manduca sexta are produced postembryonically, little is known about how these cells interact with their targets during development. Few of these cells are motor neurons; most of Manduca's adult motor neurons are respecified larval motor neurons do develop postembryonically, including a large class of mixed neurosecretory and motor neurons called the imaginal midline neurons (IMNs). A subset of these cells show an unusual pattern of sex-specific development and survival (Thorn and Truman, 1994, J. Neurobiol. in press), which led us to suspect that factors extrinsic to the cells were controlling their fates. We analyzed one such potential factor by altering the contacts between a subset of these developing IMNs and their adult-specific target, the male sperm duct. When we transected the nerve that innervated the sperm duct in the pupa, we observed a loss of many sperm duct IMNs. In contrast, a transection of the same nerve in larvae showed no neuron loss. Immunocytochemistry showed that the pupal nerve transections were accompanied by a loss of axon endings on the sperm duct, while the larval nerve transections showed no such loss. Using local hormone application to slow the development of the sperm duct while leaving the nerve intact still resulted in a loss of IMNs. These results suggest that these IMNs need contact with a robust developing target in the pupa to survive metamorphosis. 1994 John Wiley & Sons, Inc.     

Steroid control of muscle remodeling during metamorphosis in Manduca sexta

During metamorphosis in the tobacco hornworm, Manduca sexta, the abdominal body-wall muscle DEO1 is remodeled to form the adult muscle DE5. The degeneration of muscle DEO1 involves the dismantling of its contractile apparatus followed by the degeneration of muscle nuclei. As some nuclei are degenerating, others begin to incorporate 5-bromodeoxyuridine (BrdU), indicating the onset of nuclear proliferation. This proliferation is initially most evident at the site where the motoneuron contacts the muscle remnant. The developmental events involved in muscle remodeling are under the control of the steroid hormones, the ecdysteroids. The loss of the contractile elements of the larval muscle requires the rise and fall of the prepupal peak of ecdysteroids, whereas the subsequent loss of muscle nuclei is influenced by the slight rise in ecdysteroids seen after pupal ecdysis. Incorporation of BrdU by muscle nuclei depends on both the adult peak of the ecdysteroids and contact with the motoneuron. Unilateral axotomy blocks proliferation within the rudiment, but it does not block its subsequent differentiation into a very thin muscle in the adult. © 1996 John Wiley & Sons, Inc.     

PTTH-stimulated ecdysone secretion is dependent upon tyrosine phosphorylation in the prothoracic glands of Manduca sexta

PTTH stimulates ecdysteroid secretion by the insect prothoracic glands. The peptide activates cAMP synthesis in a calcium-dependent manner, ultimately enhancing ecdysteroid synthesis. We have found that PTTH stimulates a rapid increase in tyrosine phosphorylation of at least four proteins in the prothoracic glands of larval Manduca sexta, as seen on Western blots of glandular lysates probed with antibody directed against phosphotyrosine. PTTH-stimulated tyrosine phosphorylation is blocked by an inhibitor of Src family tyrosine kinases, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1). The inhibitor also blocks PTTH-stimulated ecdysone secretion, as well as PTTH-stimulated cAMP synthesis. Direct activation of the catalytic subunit of adenylyl cyclase by forskolin is not affected by PP1. In addition, ecdysteroid secretion stimulated by the cAMP analog dbcAMP is not blocked by PP1. These findings point to an important role for a Src-family tyrosine kinase at a very early step in the PTTH signaling pathway, prior to the activation of adenylyl cyclase.     

Wound healing and regeneration in the imaginal wing disc ofDrosophila

Summary When complementary fragments of an imaginal disc ofDrosophila are cultured for several days prior to metamorphosis, usually one fragment will regenerate while the other will duplicate. It has been proposed that wound healing plays an important part in disc regulation (French et al. 1976; Reinhardt et al. 1977) by initiating cell proliferation and determining the mode of regulation. We tried to delay the wound healing process by leaving a region of dead cells between the wound edges. In 06 fragments (Bryant 1975a) wound healing has occurred after 1–2 days of culture and the regeneration of missing structures after 2–4 days of culture. We observed that leaving a region of dead cells between the wound edges delays both wound healing and the regeneration of missing structures by 2 days.When disc fragments are cultured in female abdomens and then exposed to³H-thymidine to label replicating cells, then the label is found to be localised around the wound. We observed that delaying wound healing does not delay this localisation of labelled nuclei indicating that wound healing may not be required to initiate DNA replication.     

The coordination of growth among Drosophila organs in response to localized growth-perturbation

The developmental mechanisms by which growth is coordinated among developing organs are largely unknown and yet are essential to generate a correctly proportioned adult. In particular, such coordinating mechanisms must be able to accommodate perturbations in the growth of individual organs caused by environmental or developmental stress. By autonomously slowing the growth of the developing wing discs within Drosophila larvae, we show that growing organs are able to signal localized growth perturbation to the other organs in the body and slow their growth also. Growth rate is so tightly coordinated among organs that they all show approximately the same reduction in growth rate as the developing wings, thereby maintaining their correct size relationship relative to one another throughout development. Further, we show that the systemic growth effects of localized growth-perturbation are mediated by ecdysone. Application of ecdysone to larvae with growth-perturbed wing discs rescues the growth rate of other organs in the body, indicating that ecdysone is limiting for their growth, and disrupts the coordination of their growth with growth of the wing discs. Collectively our data demonstrate the existence of a novel growth-coordinating mechanism in Drosophila that synchronizes growth among organs in response to localized growth perturbation.     

The effect of fat body on the differentiation in vitro of wing imaginal discs ofDrosophila melanogaster

Summary The effect of suboptimal levels of -ecdysone on the differentiation in vitro ofDrosophila melanogaster wing discs was enhanced by the addition of larval fat body to the cultures. However, similar experiments with -ecdysome showed no enhancement. It is suggested that a partial conversion of -ecdysone to -ecdysone by the fat body may well account for these results.     

Effects of dietary variation on growth,composition, and maturation of Manduca sexta (Sphingidae: Lepidoptera)

Most studies linking dietary variation with insect fitness focus on a single dietary component and late larval growth. We examined the effects of variation in multiple dietary factors over most life stages of the sphingid moth, Manduca sexta. Larvae received artificial diets in which protein, sucrose, and water content were varied. The relationship between larval size, growth and consumption rates differed significantly across diets. Larvae on control and low-sucrose diets grew most rapidly and attained the largest pupal and adult sizes. Conversely, larvae on low-water and low-protein diets initially grew slowly, but accelerated in the fifth instar and became pupae and adults comparable to control animals in size. There were no fundamental differences in protein:carbohydrate consumption patterns or strategies among experimental diets and larval instars. However, inadequate dietary water appeared to be more important for early than late instar larvae. Larvae on all artificial diets showed increasing fat content throughout all stages, including wandering and metamorphosis. Compensatory feeding among low-water and low-protein larvae was correlated with significantly higher fat content in larvae, pupae and adults, whereas low-sucrose animals were substantially leaner than those on the control diet. These differences may have strong effects on adult physiology, reproduction, and foraging patterns.     

Absorption and storage of phosphorus by larval Manduca sexta

The role of phosphorus (P) in numerous important biological structures, coupled with the observation that P-content of many insect foods is disproportionately low, suggests that P may be a critical nutrient for growing insects — however, the few studies examining the effects of dietary P on insect performance have generally found only weak relationships. This mismatch may be reconciled by understanding the physiological mechanisms by which insects handle P. Here we describe P processing by larvae of Manduca sexta. When given un-manipulated leaves of a common host plant, Datura wrightii, fifth-instar larvae retained about 85% of P consumed; when given P-enriched leaves larvae retained only 25% of P consumed. Analysis of gut concentrations of P at four sites along the digestive tract, and in leaves and feces, indicates that the rectum is the primary site of P transport between the gut and body and that differences in P retention may be accounted for by differential rates of rectal P transport. Larvae given P-enriched leaves also showed an eightfold increase in the concentration of P in the hemolymph, primarily as α-glycerophosphate — but only a 12% increase in the concentration of P in body tissues, suggesting that hemolymph plays a central role in storage and buffering of P.     

Absorption and tissue distribution of cholesterol in Manduca sexta

In Manduca sexta larvae, radioactive free cholesterol is absorbed directly from the midgut into mucosal cells where it is stored both in the free form (87% in males and 93% in females) and esterified form (13% in males and 7% in females). Subsequently, cholesterol is transported to fat body via lipophorin in the hemolymph exclusively in the free form. In fat body, the distribution of cholesterol between the free and esterified form varied significantly between genders and developmental stages. Except for the larval stage, males and females were able to store cholesterol in both free and esterified forms in the fat body and in the adult stage cholesterol ester accounted for more than 75% of the stored cholesterol. Placement of radioactive cholesterol at different locations in the gut-foregut, midgut, and hindgut-demonstrated that the midgut is the site where cholesterol is absorbed and released into the hemolymph. Cholesterol-labeled lipophorin injected into larval hemolymph was cleared from the hemolymph with a half-life of 10.2 h. After 17 h, most of the cleared radioactivity was recovered in the fat body (38%). Arch.     

Physiological mechanisms underlying the control of meal size in Manduca sexta larvae

Abstract. Fifth stadium larvae of the tobacco hornworm, Manduca sexta (L.), ate larger meals than usual when they had been deprived of food for periods of time longer than the usual intermeal interval (c. 45 min). Meal size increased with time since the last meal until 180 min, when it was about 3 times normal. There was no evidence of a role for volumetric feedback from the gut in controlling meal size. Injections of a paraffin oil/wax mixture, or of petroleum jelly (Vaseline) into the foregut, midgut or rectum failed to decrease meal size. Cutting the recurrent nerve failed to alter meal size compared to sham-operated controls (although both groups took smaller meals than unoperated controls). By contrast, injections of an extract of soluble nutrients from the diet into the midgut inhibited feeding in some insects and reduced subsequent meal size in others. Appropriate controls showed that these effects were not due to the volumetric or osmotic effects of the injections. These results imply that nutrient feedback plays an important role in controlling meal size in Manduca caterpillars, while volumetric feedback is probably unimportant.     

Flow visualization and unsteady aerodynamics in the flight of the hawkmoth,Manduca sexta

The aerodynamic mechanisms employed durng the flight of the hawkmoth, Manduca sexta, have been investigated through smoke visualization studies with tethered moths. Details of the flow around the wings and of the overall wake structure were recorded as stereophotographs and high-speed video sequences. The changes in flow which accompanied increases in flight speed from 0.4 to 5.7 m s^-1 were analysed. The wake consists of an alternating series of horizontal and vertical vortex rings which are generated by successive down- and upstrokes, respectively. The downstroke produces significantly more lift than the upstroke due to a leading-edge vortex which is stabilized by a radia flow moving out towards the wingtip. The leading-edge vortex grew in size with increasing forward flight velocity. Such a phenomenon is proposed as a likely mechanism for lift enhancement in many insect groups. During supination, vorticity is shed from the leading edge as postulated in the ''flex'' mechanism. This vorticity would enhance upstroke lift if it was recaptured diring subsequent translation, but it is not. Instead, the vorticity is left behind and the upstroke circulation builds up slowly. A small jet provides additional thrust as the trailing edges approach at the end of the upstroke. The stereophotographs also suggest that the bound circulation may not be reversed between half strokes at the fastest flight speeds.     

Hormonal control of ventral diaphragm myogenesis during metamorphosis of the moth, Manduca sexta

 Both the proliferation and differentiation of ventral diaphragm myoblasts are controlled by ecdysteroid during metamorphosis of the moth, Manduca sexta, but the responses have different hormonal requirements. Tonic exposure to moderate levels of ecdysteroid are required to stimulate myoblast proliferation. This is due to the presence of an ecdysteroid-dependent control point in the G₂ phase of the cell cycle. As a result, proliferation can be repeatedly turned on or off simply by adjusting the concentration of ecdysteroid to be above or below a critical threshold concentration. In contrast, high levels of ecdysteroid trigger irreversible proliferative arrest and differentiation of myofibers. Myoblast proliferation and differentiation also differ in their response to the juvenile hormone mimic, methoprene. Ecdysteroid-dependent proliferative arrest and differentiation are blocked by coculture with methoprene but methoprene has no effect on ecdysteroid-dependent proliferation. In the animal, premature exposure to high levels of ecdysteroid in the absence of juvenile hormone triggers precocious differentiation of the myoblasts, resulting in the formation of several thin bands of muscle rather than a complete diaphragm. Thus, ecdysteroid and juvenile hormone collaborate to determine the size and shape of the adult musculature. Received: 12 November 1998 / Accepted: 23 December 1998