An in vitro analysis of ecdysteroid-elicited cell death in the prothoracic gland of Manduca sexta

The prothoracic glands of the tobacco hornworm, Manduca sexta, secrete the precursor of the insect molting hormone and normally undergo programmed cell death (PCD) during pupal-adult metamorphosis, between days 5 and 6 after pupation. This phenomenon can be elicited prematurely in vitro by the addition of 20-hydroxyecdysone (20E) to the gland cultures. To induce nuclear condensation in vitro in the glands from day-1 pupae, the effective dose range of 20E is 0.7-7 micrograms/ml and the minimum exposure period is 24 h. Prothoracic glands from different stages of pupal-adult development express different responsiveness to exogenous ecdysteroids. By utilizing terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) and the apoptotic DNA laddering method together with transmission electron microscopy, it has been demonstrated that the ecdysteroid-induced cell death of the prothoracic glands occurs via not only apoptosis but also autophagy, i.e., the induced dying cells show both severe nuclear fragmentation and autophagic vacuole formation, characteristics typical of apoptotic and autophagic cell death. The composite data indicate that ecdysteroids regulate directly both apoptotic and autophagic mechanisms of PCD of the prothoracic glands.     

Neuropeptides, second messengers and insect molting

Insect molting is elicited by a class of polyhydroxylated steroids, ecdysteroids, that originate in the prothoracic glands. Ecdysteroid synthesis in the prothoracic glands is controlled in large measure by a peptide hormone from the brain, prothoracicotropic hormone (PTTH), which exists in two forms and is released into the general circulation as a result of environmental and developmental cues. The means by which PTTH activates the prothoracic glands has been examined at the cellular level and the data reveal the involvement of cAMP, calcium, calmodulin, cAMP-dependent protein kinase and the ultimate phosphorylation of a 34 kDa protein tentatively identified as ribosomal protein S6.     

Ultrastructure of prothoracic glands during larval-pupal development of the tobacco hornworm,Manduca sexta: A reappraisal

The structure of Manduca sexta prothoracic glands was investigated using a protocol that preserves membranes. During the last larval stadium, prothoracic gland cells increase in diameter, volume, protein content, and perhaps number, enhancing their capacity to produce ecdysteroids. The glands' strand-of-cells morphology, their in situ location, the presence of gap junctions between cells, and junctional foot-like structures within cells support previous findings that prothoracicotropic hormone stimulates ecdysteroidogenesis via Ca²⁺-induced Ca²⁺ release. A different method of tissue fixation from that previously used to investigate the ultrastructure of Manduca sexta prothoracic glands has revealed a significantly different ultrastructure. These new findings begin to define roles for endoplasmic reticulum and mitochondria in ecdysteroid synthesis and support the hypothesis that the glands secrete the steroid hormone via exocytosis. The structural dynamics of the glands are discussed in the context of the glands' function during Manduca sexta larvalpupal development. © 1993 Wiley-Liss, Inc.     

Degeneration of moulting glands in male crickets

The degeneration of the prothoracic glands of the male cricket, Gryllus bimaculatus, was analyzed by using an in vitro assay for ecdysteroid release from the moulting glands in last instar nymphs as well as in adult animals, and correlated with light and transmission electron microscopy. Apoptosis was examined by the TUNEL-reaction. The ability to synthesize ecdysteroids reached a peak at the 8th day of the last larval instar, identified as the moulting peak. After adult ecdysis it decreased to barely measurable values. Prothoracic gland degeneration was initiated at the time of the moulting peak, characterized by TUNEL positive reactions, nuclear and cytoplasmatic condensation, a striking abundance of residual basal laminae; besides a great amount of autophagic vacuoles are observed. The results reveal that apoptosis and autophagy are the basic mechanisms for programmed cell death in the prothoracic gland of Gryllus bimaculatus.     

A photosensitive circadian oscillator in an insect endocrine gland: photic induction of rhythmic steroidogenesis in vitro

The paired prothoracic glands of the insect Rhodnius prolixus each comprise a group of about 200 structurally identical cells. The synthesis (and release) of steroid moulting hormones (ecdysteroids) by these glands is under circadian control in vivo. We monitored ecdysteroid synthesis by single glands during long-term incubations in vitro. Synthesis is rhythmic in vitro and persists in continuous darkness. Glands which are arrhythmic (from prolonged continuous light) respond to transfer to darkness in vitro with the initiation of a free-running circadian rhythm of ecdysteroid synthesis. Therefore, the glands possess a light-sensitive circadian oscillator. These properties are conventionally associated with nervous tissue of animals. It is suggested that rhythmicity is synchronized within the gland by the known structural and electrical coupling between its component cells. The glands share properties with known pacemakers such as the avian pineal. However, the glands in vivo receive input from both light cues and the cerebral neuropeptide, prothoracicotropic hormone. Rhythmic release of this neuropeptide is controlled by a second oscillator located in the brain. We conclude that the pacemaker in the endocrine system of R. prolixus comprises at least three oscillators, one in each prothoracic gland and one in the brain, which are coupled hormonally. We conclude that the prothoracic gland is an important component of the circadian system controlling development in R. prolixus and that peripheral endocrine glands may play a more active role in the generation of animal circadian organization than has been thought. Accepted: 30 August 1997     

An ultrastructural analysis of the ecdysoneless ((l(3)ecd 1ts) ring gland during the third larval instar of Drosophila melanogaster

Summary In the late third larval instar of Drosophila melanogaster, the prothoracic gland, an endocrine portion of the ring gland, synthesizes ecdysteroids at an accelerated rate. The resultant ecdysteroid titer peak initiates the events associated with metamorphosis. The normal prothoracic gland displays several ultrastructural features at this developmental stage that reflect increased steroidogenic activity, including extensive infoldings of the plasma membrane (membrane invaginations) and an increase in both the concentration of smooth endoplasmic reticulum (SER) (or transitional ER) and elongated mitochondria. By contrast, the prothoracic glands of larvae homozygous for a conditional larval lethal mutation, l(3)ecd ^1ts, not only fail to produce ecdysteroids at normal levels at the restrictive temperature (29° C), but also acquire abnormal morphological features that reflect the disruptive effects of the mutation. These abnormalities include an accumulation of lipid droplets presumed to contain sterol precursors of ecdysteroids, a disappearance of SER and a drastic reduction of membrane invaginations in the peripheral area of the cell. These morphological defects are observed in prothoracic glands dissected from larvae transferred from 18° C to 29° C approximately 24 h before observation and also within 4 h of an in vitro transfer to 29° C following dissection from wandering third instar larvae reared at 18° C. No ultrastructural abnormalities were noted in the corpus allatum portion of mutant ring glands. These observations further indicate the direct involvement of the ecd gene product in ecdysteroid synthesis and suggest a role for the gene in the proper transport of precursors to the site where they can be utilized in ecdysteroid biosynthesis.     

Physiological investigation on the role of the innervation in the regulation of the prothoracic gland in Periplaneta americana

Under in vitro conditions the prothoracic gland nerve of the last larval instar of Periplaneta americana shows the same efferent nervous activity as under in situ conditions–ie, low activity at the 9th day and high activity at the 20th day of the molting interval. Isolation of the prothoracic ganglion from the subesophageal ganglion provokes an increase in this nerve activity, suggesting an inhibitory effect of the subesophageal ganglion on prothoracic gland nerve activity in vivo. Only in 20-day-old larvae does electrical stimulation of isolated prothoracic glands in vitro via the gland nerve result in a slightly increased release of ecdysteroids from the gland. This effect could not be influenced by different lengths of stimulation periods. Denervation of the prothoracic gland by transection of the gland nerve on the 13th day of the molting interval results in a complete abolition of the first peak of ecdysteroid production in the gland but has no influence on the occurrence and the amount of the main ecdysteroid peak just before the molt. The results suggest the participation of nervous activity in special periods of prothoracic gland regulation in the cockroach.     

Stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis by the prothoracic glands during the last larval instar of the silkworm, Bombyx mori

The stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis of the prothoracic glands during the last larval instar of the silkworm, Bombyx mori, were studied in the present study. When last instar larvae were starved beginning on day 1 of that instar, all larvae died between days 5 and 7 of the instar. Although the prothoracicotropic hormone (PTTH) release from the brain-corpus cardiacum-corpus allatum (BR-CC-CA) did not significantly change during starvation, a deficiency in PTTH signal transduction was maintained, which led to very low levels of hemolymph ecdysteroids after the beginning of starvation. However, when starvation began on day 3 of the last larval instar, the major hemolymph ecdysteroid peak, preceding larval-pupal transformation, occurred 1 day earlier than that in control larvae. Protein content of the prothoracic glands in day 3-starved larvae was maintained at a low level as compared to that of control larvae. The secretory activity of the prothoracic glands in day 3-starved larvae was maintained at a level similar to that of control larvae. However, the rate of ecdysteroidogenesis, expressed per microgram of glandular protein, was greatly enhanced in these starved larvae, indicating that upon starvation, larvae increased the ecdysteroid production rate to enhance the rate of survival.     

Development of an in vitro assay for prothoracicotropic hormone of the gypsy moth,Lymantria dispar (L.) following studies on identification,titers and synthesis of ecdysteroids in last-instar females

Summary Hemolymph ecdysteroid titers and in vitro prothoracic gland ecdysteroid synthesis have been examined in last-instar larval (5th instar) females of Lymantria dispar. Ecdysteroids were quantified by radioimmunoassay and characterized by co-elution with known standards of ecdysteroids on reverse-phase high-performance liquid chromatography. Analysis of hemolymph yielded ecdysone and 20-OH-ecdysone in ratios of 1:1 (day 6, shortly after attainment of maximum weight) and 1:28 (day 10, molting peak). Analysis of in vitro culture media from glands challenged with extracts of brains or retrocerebral complexes, or left unchallenged, revealed only immunoreactive material co-eluting with a known standard of ecdysone. Time-course studies of in vitro prothoracic gland ecdysone secretion demonstrated a major peak on day 10, 1–2 days prior to pupal ecdysis, and a small elevation on days 5–6. On days 5 and 6, 2.29±0.41 and 2.65±0.72 ng ecdysone per gland, respectively, were secreted in 6-h cultures. On day 10, 25.69±4.36 ng was secreted in 6-h culture. The ability of prothoracic glands of various ages to respond to brain extracts containing prothoracicotropic hormone activity was tested by determining an activation ratio for each day of the instar. The activation ratio was determined over a 90-min period by dividing the amount of ecdysone secreted by one member of a pair of prothoracic glands in the presence of brain extract by that of its contralateral control gland in Grace's medium. Prior to the addition of brain extract, the activity of the glands was allowed to subside to basal level for 180 min in Grace's medium. The activition ratio was highest on days 3–7 and fell throughout the remainder of the instar as the inherent ability of the prothoracic gland to maintain high levels of ecdysteroid synthesis in vitro in the absence of prothoracicotropic hormone increased. A two-phase in vitro assay for prothoracicotropic hormone was established using activition ratios. This assay showed saturable doseresponse kinetics for prothoracic gland ecdysone secretion and specificity to extracts prepared from brain or retrocerebral complexes. A comparable assay for prothoracicotropic hormone purification, based on net synthesis and requiring half the number of prothoracic glands was also established.Abbreviations A _r activation ratio - HPLC high performance liquid chromatography - HPSEC high performance size-exclusion chromatography - PG prothoracic gland - PTTH prothoracicotropic hormone - RIA radioimmunoassay     

Regulation and significance of ecdysteroid titre fluctuations in lepidopterous larvae and pupae

The last larval moult of Galleria mellonella is induced by an elevation of ecdysteroid titre to more than 200 ng/g. After ecdysis the titre remains very low until 70 hr of the last-instar when a slight elevation in ecdysteroid concentration initiates the onset of metamorphosis. An ecdysteroid peak (275 ng/g), which occurs between 108 and 144 hr, is associated with wandering and cocoon spinning. Pupal ecdysis follows about 20 hr after a large ecdysteroid peak (780 ng/g) with a maximum in slowly-mobile prepupae (160 hr of the last larval instar). The ecdysteroid decrease between the two peaks coincides with the period when the larvae exposed to unfavourable conditions enter diapause. The pupal-adult moult is initiated by a high ecdysteroid peak (1500–2500 ng/g) in early pupae and imaginal cuticle is secreted in response to a smaller peak (ca. 500 ng/g) in the middle of pupal instar.Until early pupae, the ecdysteroid content is regulated by the prothoracic glands. In decapitated larvae the glands become spontaneously active after 30–40 days and the body titre of ecdysteroids undergoes an increase; the glands revert to inactivity when the insects accomplish secretion of pupal cuticle. A similar ecdysteroid increase occurs within 10 days when the decapitated larvae receive implants of brains releasing the prothoracicotropic neurohormone (PTTH). In either case, the pupation-inducing increase of ecdysteroids is 3 times higher than the large ecdysteroid peak in the last-instar of intact larvae. This indicates that the function of prothoracic glands in intact larvae is restrained, probably by the juvenile hormone (JH). Exogenous JH suppresses the spontaneous activation of the prothoracic glands in decapitated larvae and reduces the ecdysteroid concentration in those larvae (both decapitated and intact), whose glands were activated by PTTH. Furthermore, JH influences the PTTH release from the brain in situ: depending on JH concentration and the age and size of treated larvae, the PTTH liberation is either accelerated or delayed.Neither in G. mellonella larvae, nor in the diapausing pupae of Hyalophora cecropia and Celerio euphorbiae, does JH directly activate the prothoracic glands. It is suggested that the induction of the moult by JH in decerebrate insects, which has been observed in some species, is either due to indirect stimulation of ecdysteroid production or to increased sensitivity of target tissues to ecdysteroids. In G. mellonella, a moult occurs at a 5–15 times lower than usual ecdysteroid concentration when the last-instar larvae are exposed to JH.     

Identification of a novel prothoracicostatic hormone and its receptor in the silkworm Bombyx mori

The insect brain regulates the activity of the prothoracic glands to secrete ecdysteroids, which affect growth, molting, and metamorphosis. Here we report the identification of a novel prothoracicostatic factor and its receptor in the silkworm Bombyx mori. The prothoracicostatic factor purified from pupal brains of B. mori is a decapeptide with the conserved structure of an insect myosuppressin and thus named Bommo-myosuppressin. Bommo-myosuppressin dose dependently suppressed the cAMP level and inhibited ecdysteroidogenesis in the larval prothoracic glands at much lower concentrations than the prothoracicostatic peptide, the other prothoracicostatic factor reported previously. In vitro analyses using a prothoracic gland incubation method revealed that Bommo-myosuppressin and prothoracicostatic peptide regulate the prothoracic gland activity via different receptors. In situ hybridization and immunohistochemistry revealed the existence of Bommo-myosuppressin in the brain neurosecretory cells projecting to neurohemal organs in which it is stored. We also identified and functionally characterized a specific receptor for Bommo-myosuppressin and showed its high expression in the prothoracic glands. All these results suggest that Bommo-myosuppressin functions as a prothoracicostatic hormone and plays an important role in controlling insect development.     

Stage dependent influences of polydnaviruses and the parasitoid larva on host ecdysteroids

In the solitary egg-larval parasitoid Chelonus inanitus (Braconidae) both polydnavirus and the parasitoid larva manipulate host development. Parasitization leads to a premature drop in juvenile hormone titre and a precocious onset of metamorphosis in the 5th larval instar. The C. inanitus bracovirus (CiBV) alone causes a reduction in host ecdysteroid titres at the pupal cell formation stage and prevents pupation. Here we report three new findings. (1) We show that parasitization causes a reduction in haemolymph ecdysteroid titre immediately after the moult to the 5th instar; similarly low values were seen in nonparasitized larvae after the moult to the 6th instar. These data along with parasitoid removal experiments indicate that the low ecdysteroid titre after the moult is a very early sign of the upcoming metamorphosis. (2) In vitro experiments with prothoracic glands and brain extracts showed that CiBV affects both prothoracic glands and prothoracicotropic hormone after the stage of pupal cell formation. (3) In the haemolymph of parasitized larvae the ecdysteroid titre increased in the late cell formation stage, i.e. immediately before egression of the parasitoid. In vitro experiments showed that late 2nd instar parasitoids release ecdysteroids and are thus very likely responsible for the rise in host ecdysteroids.     

Cellular events in the prothoracic glands and ecdysteroid titres during the last-larval instar of Spodoptera littoralis

Changes in prothoracic gland morphology were correlated to developmental events and ecdysteroid titres (20-hydroxyecdysone equivalents) during the last-larval instar in Spodoptera littoralis. After ecdysis to the last-larval instar the haemolymph ecdysteroid titre remained at about 45 ng/ml, when the prothoracic glands appeared quiescent. The first signs of distinct gland activity, indicated by increased cell size and radial channel formation, were observed at about 12 h prior to the cessation of feeding (36 h after the last-larval moult), accompanied by a gradual increase in ecdysteroid titre to 110 ng/ml haemolymph, at the onset of metamorphosis. During this phase ecdysteroid titres remained at a constant level (140–210 ng/ml haemolymph) and prothoracic gland cellular activity was absent for a short period. The construction of pupation cells occurred when haemolymph ecdysteroids titres increased to 700 ng/ml. A rapid increase in ecdysteroids began on the fourth night (1600 ng/ml haemolymph) reaching a maximal level (4000 ng/ml haemolymph) at the beginning of the fourth day. In freshly moulted pupae a relatively high ecdysteroid titre (1100 ng/ml haemolymph) was still observed, although during a decrease to almost negligible levels. The increase in ecdysteroid level during the third and the fourth nights of the last-larval instar was correlated with the period when almost all the prothoracic gland cells showed signs of high activity. Neck-ligation experiments indicated the necessity of head factors for normal metamorphosis up to the second to third day of the instar. The possibility that the prothoracic glands are under prothoracicotropic hormone regulation at these times is discussed.     

Switchover in the sensitivity of the prothoracic glands to juvenile hormone in the cotton leafworm, Spodoptera littoralis

Switchover in the sensitivity of the prothoracic glands to juvenile hormone analogue during the last-larval instar of Spodoptera littoralis occurs in the middle of the third scotophase i.e. at the end of phagoperiod when the body weight is maximal and the ecdysteroid is increasing in the haemolymph. Application of the analogue to larvae neck-ligated before the switchover completely inhibits or delays metamorphosis due to an inhibitory effect on the prothoracic gland cells and is not mediated by the nervous system. This inhibition by the analogue is dose-dependent, and when complete inhibition of metamorphosis occurs, the prothoracic glands cells degenerate. Treatment of neck-ligated larvae with the analogue after the switchover stimulates metamorphosis by accelerating the appearance of an ecdysteroids peak in the haemolymph. The stimulatory effect of the analogue to the prothoracic glands in neck-ligated larvae is not direct one, and some unknown factors seem to play a role therein.

The probale role of prothoracicotropic hormone as a synchronizing factor in the switchover in the sensitivity of the prothoracic glands to juvenile hormone is discussed.     

Regulation of ecdysteroidogenesis in prothoracic glands of the tobacco hornworm Manduca sexta

Ecdysteroidogenesis in Manduca sexta prothoracic glands is regulated by a set of bioregulatory molecules, including prothoracicotropic hormone (PTTH) and a protein factor present in larval hemolymph, and by the competence of the glands to synthesize ecdysteroids in response to those molecules. A larval molting bioassay was used to assess the in vivo activity of Manduca PTTHs. Crude PTTH, big PTTH, and small PTTH each elicited a larval molt in head-ligated larvae. However, big PTTH was approximately 10-fold more potent than crude PTTH, which was, in turn, several orders of magnitude more potent than small PTTH. When big and small PTTH were combined, the molting response was similar to that elicited with crude PTTH. The chemical nature of the hemolymph protein factor was also investigated. Injection of [3H]cholesterol into last-instar larvae and fractionation of the radiolabeled hemolymph by gel filtration chromatography revealed three peaks of radioactivity. One peak eluted in fractions containing the hemolymph protein factor, a result consistent with the notion that the factor transports a sterol substrate. The possibility that the factor is a 3(2)-ketoreductase was investigated by assessing the effect of the factor on the accumulation of RIA-detectable ecdysteroids in prothoracic-gland-conditioned medium. Three of five preparations of the factor significantly enhanced the amount of RIA-detectable ecdysteroids in conditioned medium, indicating that at least some preparations of the factor may contain ketoreductase activity. The above findings are discussed in the context of current hypotheses of how bioregulatory molecules interact with the prothoracic glands to regulate ecdysteroidogenesis in Manduca.     

Interendocrine regulation of the corpora allata and prothoracic glands of Manduca sexta

Regulation of the haemolymph titres of ecdysteroids and the juvenile hormones (JH) during larval-pupal development of the tobacco hornworm, Manduca sexta, involves the interendocrine control of the synthesis of each hormone by the other. Temporal relationships between the ecdysteroid titre peaks in the fourth and early fifth larval instar and the increases in corpora allata (CA) activity at these times suggests that ecdysteroids are evoking the increases. Incubation of brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes and isolated CA from these stages with 20-hydroxyecdysone (20-HE) revealed that 20-HE stimulates CA activity and that it does this indirectly via the Br-CC. The resulting increase in the JH titre after the commitment (first) peak in the fifth instar stimulates the fat body to secrete a factor which appears to be the same as a haemolymph stimulatory factor for the prothoracic glands. This moiety acts as a secondary effector that modulates the activity of the prothoracic glands and thus the ecdysteroid titre. These findings together have begun to elucidate the mechanisms by which the principal developmental hormones in the insect interact to regulate postembryonic development.     

Effect of allatectomy on ecdysteroid-dependent development of Rhodnius prolixus larvae

the regulation of haemolymph titres of ecdysteroids during larval development of the bloodsucking bug, Rhodnius prolixus was studied. Corpus allatum ablation in 4th-instar larvae 1 day after feeding was reflected in an increase of the intermoult period and in a high level of ecdysial arrest. These effects could be corrected by juvenile hormone and ecdysone therapies. Comparison of the ecdysteroid titres in haemolymph determined in control and allatectomized larvae, at different intervals after feeding, showed that allatectomy drastically depressed the ecdysteroid levels. Juvenile hormone treatment reestablished ecdysteroid titres in the haemolymph of allatectomized insects. Isolated prothoracic glands from allatectomized larvae had a very low production of ecdysteroid-RIA-activity when compared with prothoracic glands from control or allatectomized larvae which received in vivo juvenile hormone treatment. The complexity of the corpus allatum-prothoracic glands interaction in Rhodnius post-embryonic development is discussed.     

Activation of prothoracic glands by brains in vitro

The effects of brains from both diapausing and non-diapausing Mamestra brassicae pupae on the prothoracic glands from pupae of the same condition were studied by observations of the morphological changes and bioassay of the prothoracic glands in vitro.It was ascertained that the active brains intensified the hormonal activity of prothoracic glands from younger diapausing pupae more than those from older pupae. Further, these results coincided with the fact that the prothoracic glands from brainless pupae were more difficult to activate by active brains the longer the time after the glands had been extirpated.The brains from both younger and older diapausing M. brassicae pupae were able to activate co-cultured inactive prothoracic glands in vitro. These results suggest that even the brain from diapausing pupae of M. brassicae can synthesize and release the prothoracic gland activating hormone in vitro.     

Electron microscopical-immunocytochemical evidence of ecdysteroids in the prothoracic gland of Galleria mellonella

Summary Fixation of prothoracic glands of Galleria mellonella with a solution containing saponin permits immunocytochemical staining of the entire gland. By this means ecdysteroids were demonstrated electron microscopically to be present in the hyaloplasm and microtubules.Supported by Sächsische Akademie der Wissenschaften zu Leipzig     

Ultrastructure of the prothoracic gland of variously aged female pupae of Xyleborus ferrugineus and associated ecdysteroid titers

Summary The prothoracic glands of female pupae of Xyleborus ferrugineus at three ages (0-h, 48-h and 72-h-old) were examined for ultrastructural changes that correlate with high titers of ecdysteroids in the entire pupa.In all three ages, the prothoracic gland cells appear compact with a prominent nucleus. Lysosome-like structures with concentrically oriented internal membrane are observed in both 0-h and 72-h-old gland cells. Mitochondria are abundant in the glands of both ages, and rough endoplasmic reticulum is relatively sparse. Numerous microtubules are present in the prothoracic gland cells of 48-h-old female pupae that contain relatively high titers of ecdysteroids. Rough endoplasmic reticulum also is especially abundant at this age of the pupa. Ecdysone titers, estimated by the radioimmunoassay method (RIA), were 161.04±22.61 pg/mg, 704.25±69.02 pg/mg and 298.72±26.80pg/mg body weight in 0-h, 48-h and 72-h-old female pupae, respectively.This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison; and in part by research grants No. RR-00779, Division of Research Resources, and No. AG-01271, Institute on Aging, of the National Institutes of Health; and No. PFR 77-08279 from the National Science Foundation to D.M.N.We thank especially Jon Gundersen and Steven Schnurrer for extensive and invaluable technical assistance during this research