Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. VI. Inhibition by juvenile hormones.

In the salivary gland chromosomes of late-third instar larvae and in late (8- to 12-hr) prepupae of Drosophila melanogaster, there are ecdysone-induced sequences of puffing patterns which can be reproduced in vitro. These two sequences are separated by a period when the glands are thought to be exposed to a low titer of β-ecdysone and during which they acquire the competence to respond to ecdysone at the late prepupal puff sites. Attempts to modify either the late larval or the late prepupal responses to ecdysone in vitro by the simultaneous addition of juvenile hormone (JH) with ecdysone, to larval or prepupal glands, respectively, are unsuccessful. If, however, JH (ca. 10^?6M) is added to larval glands cultured 6 hr in ecdysone and then 3 hr in JH alone, the subsequent induction of prepupal ecdysone puffs is inhibited. Thus the role of JH appears to lie in modifying the acquisition of competence to respond to ecdysone rather than in a direct antagonism between the two hormones.     

Cytogenetic analysis of the X chromosome region 2B3-4 — 2B11 of Drosophila melanogaster

Mutation t467, belonging to the swi complementation group, and causing death in late prepupa, is located in the interval from 2B6 to the left part of 2B7-8. In this region puffing is absent in salivary gland chromosomes. In t467/t467 homozygotes intermoult early and early-late larval 20-OH ecdysone puffs do not differ from the controls. Mid-prepupal puffs are normal too with a few exceptions. However, all late larval and prepupal puffs are reduced or absent in the mutant. Both, hormone incubation of t467 glands in vitro and hormone injection have shown: i) 20-OH ecdysone in vitro does not restore the normal larval puffing pattern. ii) Withdrawal of the hormone from glands at PS6 causes premature appearance of late larval puffs, which, however, do not reach control sizes. It is concluded that the swi gene product is necessary for induction of late puffs. Thus in the 2B3-4—2B7-8 region three genes, affecting 20-OH ecdysone induction processes, have become known.     

Die puff-Musterfolgen der Speicheldrüsenchromosomen aus wanderlarven und vorpuppen von Drosophila melanogaster in vitro

Late larval salivary glands of D. melanogaster of an exactly defined developmental stage (VP 0, i.e. prepupae ot later than 15 min after formation of the puparium) are cultured under sterile conditions in three standard media for insect tissue culture and in Ringer solution. In chromosomes II and III, variations in puff number and size are the same in vivo and in vitro, and almost all changes in puffing pattern are very similar to those appearing in normal development. They are the same in the four media. No additional puff is ever induced due to the medium. By contrast, salivary gland chromosomes from larvae of the late third instar before pupation do show different alterations in vitro than in vivo. This points to a threshold in the course of the puffing pattern between puff stage 8/9 and 10/11. The appearance of a substance causing prepupal changes in puffing is strictly correlated with the formation of the pupanium and the beginning of the intermoult phase in the prepupa. Comparing the results of the experiments it can be stated that the new control system is not based solely on the absence of ecdysone, but also on the existence of another inducer. Immediately after puparium formation the control by ecdysone is still active, together with the control by the supposed inducer. Later, control by ecdysone respectively by the puffs of the ecdysone cycle is substituted by the new control system, up to the next moult. As far as the chemical nature of the puffing inducer in the intermoult phase is concerned, further investigations are necessary.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

A technique for the short term organ culture of larval salivary glands of D. melanogaster is described. Cultured Puff Stage 1 glands respond to 20-OH ecdysone by initiating the cycle of puffing activity characteristic of late larval development and puparium formation. This puffing cycle involves the sequential activation of at least 125 puffs. Their response to ecdysone allows these puffs to be divided into 3 main classes: a) PS1 puffs that regress (e.g. 25AC); b) puffs activated very rapidly (within 5 min) (e.g. 23E, 74EF, 75B) and c) puffs activated only after longer periods (>4 h) (e.g. 62E, 78D, 22C, 63E and 82F). The detailed behaviour of representatives of each class is described. These data support Clever's distinction of ‘early’ and ‘late’ ecdysone responsive sites.     

The control of prepupal puffing patterns in vitro; Implications for prepupal ecdysone titres in Drosophilia melanogaster

In late third instar larvae and prepupae of Drosophila melanogaster there is a complex change in puffing patterns in the salivary gland chromosomes. There are two peaks of activity in this period. The first, in larvae, is known to be under the control of the moulting hormone ecdysone. The second, in prepupae, is now shown by the in vitro culture of prepupal glands to be under the specific control of β-ecdysone in a manner similar to the first. A new class of puffs, active between these two peaks, whose induction is inhibited by ecdysone in vitro, is described. The behaviour of these puffs, exemplified by 75CD and 63E, suggests a period of very low ecdysone titre in vivo. The developmental significance of the role of ecdysone during prepupal development is discussed.     

Developmental regulation of granule size and numbers in larval salivary glands of drosophila by steroid hormone ecdysone

The size and number of secretory granules in late larval salivary glands of Drosophila melanogaster have been related to interecdysial and early metamorphic development represented by well-known puffs in polytene chromosomes. Interecdysial period (puff stage 1 (PS1)) is characterized by presence of numerous small granules (11,000 per cell). The transition from PSI to early metamorphic phase (PS2 and upwards), induced by rapid elevation in endogenous steroid hormone ecdysone, is accompanied by continuous growth of granule diameter with concomitant reduction in their number per cell. In the PS4, just prior to secretion, approximately 3000 mature granules occur per cell. The mature state is associated with the change from hyperbolic to Gaussian distribution of granule number over their size range. Similar changes in secretory granule parameters were observed in interecdysial salivary glands explanted from 3rd instar larvae and cultured in vitro in medium containing 5x10(-6) m ecdysone.     

Activité,in vivo, d''inhibiteurs de la biosynthèse de l''ecdysone chezLocusta migratoria

Summary Among about 50 compounds synthesized to inhibit enzymes involved in the biosynthesis pathway of ecdysone we selected seven molecules which showed a strong effect on ecdysone production byLocusta migratoria prothoracic glands incubatedin vitro. These molecules mostly possess a specific activity on ecdysone biosynthesis which is irreversible. The compounds were administered in one or several injections of aqueous or oily solutions at different times in the course of the two last larval instars. Three inhibitors led in a 10% ratio to a prolongation (sometimes more than 3 times the standard length) of the instar, pointing out a decrease in the ecdysone biosynthesis. Two other inhibitors induced some morphogenetic modifications in the adults, as size reduction or wing alterations, and metamorphosis difficulties. Thein vivo low activity compared with the strong onein vitro could be due to difficulties for the compounds to reach the prothoracic glands without degradation. The variation of inhibitory activity which appearsin vivo between the seven compounds studied is not linked either with the chemical structures of the molecules (which are very near one another) or with theirin vitro activity.

     

Cytogenetic analysis of the X-chromosome region 2B1-2-2B9-10 of Drosophila melanogaster

In salivary glands of yellow control stock the puffing pattern in the ecdysone-added artificial C46P medium was on the whole similar to that observed during larval development in vivo. However, underdevelopment of a series of late puffs and a delay in the regression of early puffs were observed. In addition a set of medium puffs not visible in vivo appeared. Late puffs differed from those developing in Grace medium.When salivary glands of homozygotes for the lethal dor ^lt187, a mutation that causes death in the third instar with no signs of ecdysone induction were incubated with ecdysterone, the development of puffs was restored, i.e., the puffing pattern of mutant cells in vitro practically did not differ from that in cells of the control stock. This implies that the dor ^lt187 lethal allele belongs to the class of ecdysone-deficient mutations.     

The time during which beta-ecdysone is required for the differentiation in vitro and in situ of wing imaginal discs of Drosophila melanogaster.

The time during which β-ecdysone is required for the apolysis and imaginal differentiation of wing discs of Drosophila both in vitro and in situ has been examined, and it is concluded that β-ecdysone is required as a sustained stimulus rather than as a trigger for differentiation. These results are compared with the requirement for β-ecdysone for the puffing of salivary gland polytene chromosomes during the prepupal stage (Richards, G. P., 1976, Develop. Biol.48, 191–195). It is suggested that imaginal discs and larval salivary glands require different exposures to β-ecdysone to fulfill their developmental commitments and that the drop in β-ecdysone titer during the early prepupal stage, which is necessary for the subsequent puffing of the polytene chromosomes, plays little or no part in imaginal disc differentiation.     

Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. I. Dependence upon ecdysone concentration

The response of the three major classes of puff in salivary gland chromosomes of larval Drosophila melanogaster to varying β-ecdysone concentrations has been studied in in vitro cultured glands. Two (25AC and 68C) of the intermolt puffs regress at a rate dependent upon the hormone concentration. Three rapidly reacting puffs (23E, 74EF and 75B) respond in a graded way to β-ecdysone concentrations over a range of at least 600 ×. In contrast, five late-reacting puffs (62E, 78D, 22C, 63E, and 82F) do not respond below 5 × 10^?8M and at 2.5 × 10^?7M react maximally. The 50% response of the early puff sites 74EF and 75B and of the late puff sites occurs at 1 × 10^?7M. Two points are discussed in detail: whether ecdysone is necessary as a sustained stimulus or only as a trigger for the sequential puffing response and an evaluation of the absolute ecdysone concentration necessary for induction.     

The hormonal control of salivary gland secretion in Drosophila melanogaster: Studies in vitro

The larval salivary gland of Drosophila melanogaster synthesises a complex secretion, known as ‘glue’. which is secreted at puparium formation and then cements the puparium to its substrate. This secretion is made during the third larval instar and is stored in the gland cells as large granules. A few hours before puparium formation it is secreted into the gland's lumen by exocytosis. This process is induced by ecdysone and can be studied in vitro. Secretion is initiated about 3.5 hr after exposure of glands to ecdysone and is complete by 8 hr. The effects of varying the ecdysone concentration, of inhibitors of RNA or protein synthesis, and of withdrawing the hormone at various times after initial exposure on the process of secretion have been studied. We conclude that some event(s) occurring during the first 3 hr exposure to ecdysone is necessary to initiate secretion of the glue into the gland lumen. The possible relationship between this event(s) and the ecdysone induced changes in gene activity (puffs) which occur in the salivary glands at the same time is discussed.     

Ecdysone-regulated chromosome puffing in the salivary glands of the Mediterranean fruit fly, Ceratitis capitata.

The effect of ecdysone on the puffing activity of the polytene chromosomes of Ceratitis capitata has been studied in organ cultures of late-larval salivary glands. Culture of glands from 120-h-old larvae (puff stage 1) in the presence of ecdysone resulted in the initiation of the late-larval puffing cycle that is normally observed in 145-h-old larvae (puff stage 4). During a 7-h period in the presence of ecdysone, the puffing patterns of most loci resembled the in vivo patterns observed in the period between puff stages 4 and 10, indicating that the first puffing cycle can be initiated by the hormone and proceed almost to completion, in vitro. Culture of salivary glands in the presence of ecdysone and a protein-synthesis inhibitor, as well as ecdysone withdrawal and readdition experiments, indicated that most of the ecdysone-regulated puffs could be categorized into three classes: (i) the puffs that were suppressed immediately by ecdysone, even in the absence of protein synthesis; (ii) the puffs that were induced directly by ecdysone; and (iii) the puffs that were induced indirectly by ecdysone, that is, they were induced after a lag period of a few hours and required protein synthesis for their induction.     

Comparative characteristics of the puffing pattern and the endocrine system in an oregon laboratory stock and in l(2)gl Drosophila melanogaster mutants differing in the time of their death

This study shows that homozygotes for different alleles of the lethal mutant, l(2)gl, differing in the time of death also vary in the state of their endocrine system and the puffing patterns of their salivary gland chromosomes. Homozygotes which die at the larval stage have underdeveloped prothoracic glands and normal corpora allata (CA); in those dying at the prepupal stage both the prothoracic glands and the CA are equally underdeveloped. — All the early third instar larval puffs (96–110 h., PS 1–2) develop in homozygotes; however, the reduction of some early larval puffs, normally occurring before pupariation or at puparium formation, is delayed. Some puffs are more developed than normal. — The differences in puffing patterns chiefly concerned puffs which normally appear 4–5 h before puparium formation and at puparium formation. In homozygotes lethal as larvae some of the puffs normally active at this time did not develop. However, along with some of the late larval puffs, there appeared many puffs characteristic of prepupae. — In homozygotes lethal as prepupae only the time and sequence of puff appearance was altered. Many late larval puffs were active in prepupae rather than in larvae, whereas some of the puffs, normally appearing in prepupae, were active in the larval stage.Accordingly, we propose to distinguish two groups of puff loci. 1) Hormone dependent puffs: These do not develop in larval lethals and are active only after puparium formation in pupariated lethals. 2) Autonomous puffs: Their appearance depends more on the time of development, than on hormonal background. It is suggested that the induction of hormone dependent puffs and of puparium formation is possible at low ecdysone levels, provided that the juvenile hormone level is also low.     

Developmental changes in fat body and midgut chromosomes of Drosophila auraria

Changes in puffing activity of fat body (FB) and midgut (MG) chromosomes of Drosophila auraria during late larval and white prepupal development as well as after in vitro culture with or without ecdysterone were studied and compared with those of the salivary gland (SG). The Balbiani Rings characteristic of the SG chromosomes of D. auraria, are not formed in FB and MG. Most of the inverted tandem chromosomal duplications that have been found to be common to all three tissues showed differentiation of puffing activity of the bands considered to be homologous. The major early ecdysone puffs 73A and 73B (considered to be homologues of D. melanogaster puffs 74EF and 75B, respectively), together with other early ecdysone puffs were present in all three tissues. Clear intermoult and postintermoult puffs were not evident in FB and MG chromosomes. However, a small set of late ecdysone puffs could be scored in FB, while no late ecdysone puffs were abserved in MG. Other tissue-specific puffs were identified, but a very small number of them were limited to MG.by W. Beermann     

Ecdysteroid titers and changes in chromosomal activity in the salivary glands of Rhynchosciara americana

Titers of ecdysone and 20-OH ecdysone were measured separately in both hemolymph and salivary glands of metamorphosing Rhynchosciara larvae. Gland titers were consistently higher than hemolymph titers. Although 20-OH ecdysone was the most prominent form of the hormone, measurable quantities of ecdysone were also observed throughout development in both tissues. Changes in salivary gland replication and puffing activity could be correlated with changes in gland 20-OH ecdysone titers. This was true for both developmentally changing RNA puffs and DNA puffs, which occur during the prepupal period. The DNA puffs are tied to the final DNA replication cycle, and both this cycle and the period of amplification can be correlated with increases in gland 20-OH ecdysone content. Various aspects and possible interpretations of the above correlations are discussed.This work is dedicated to the memory of Prof. Hans D. Berendes     

The apparent requirement of two hormones, alpha- and beta-ecdysone, for molting induction in insects

Puff formations at loci I-18-C and IV-2-B of the salivary gland chromosomes are early indications of a beginning molting process in Chironomus tentans larvae. The effectiveness of the two ecdysone analogs, α- and β-ecdysone, in inducing these puffs was compared. Incubation of salivary glands in vitro with β-ecdysone causes only puff IV-2-B to appear; incubation with α-ecdysone stimulates initially puffing at only I-18-C. After an injection of α-ecdysone, puffing at I-18-C begins within less than 15 min, whereas puffing at IV-2-B is delayed for more than 30 min. Following an injection of β-ecdysone, puffing at IV-2-B begins within less than 15 min, whereas puffing at I-18-C is delayed. Injected ³H-α-ecdysone is converted to β-ecdysone and a polar compound. Injected ³H-β-ecdysone is converted to a compound less polar than α-ecdysone and a polar metabolite which stimulates puffing at I-18-C, like α-ecdysone. It is suggested that the two ecdysones have different targets in the cell, that they can be rapidly converted to compounds with the activity of the other analog, and that the induction of a complete molt requires the action of both hormones.     

Infectivity of Leucocytozoon caulleryi sporozoites developed in vitro and in vivo

Morii T., Matsui T., Iijima T. and Fiotnaoa F. 1984. Infectivity of Leucocytozoon caulleryi sporozoites developed in vitro and in vivo. International Journal for Parasitology14: 135–139. Infectivity of Leucocytozoon caulleryi sporozoites isolated from various sites in Culicoides arakawae and from the midguts and the salivary glands which had been cultured in vitro after the infective blood meals was studied. Sporozoites isolated from the midguts, the abdominal and thoracic hemocoel and the salivary glands of biting midges on the 2nd day after feeding did not show infectivity to any of the chickens inoculated. Sporozoites obtained from the salivary glands on the 3rd day after feeding caused infection in all the inoculated chickens. The results indicated that sporozoites which had been just released from oocysts or had just reached the salivary glands cannot induce infection in chickens. Sporozoites were produced in the midguts which had been cultured in vitro in Medium 199 or Grace's medium after the infective blood meals, but they showed lower infectivity than those isolated from the salivary glands which had been cultured by the same methods as the midgut cultivation. The development of infectivity of L. caulleryi sporozoites seems to be site-dependent rather than time-dependent. High infectivity of sporozoites develops during their residence in the salivary glands of biting midges.     

DNA-puffing patterns in the salivary glands of Trichosia pubescens (Diptera-Sciaridae)

The puffing pattern in polytene chromosomes from salivary glands of fourth-instar larvae of Trichosia pubescens was studied. It was found that the puffing pattern is rather constant during most of larval life but changes continuously in a precise sequential order during the period preceding pupation. During this period, characterized by drastic changes in the puffing pattern, amplification of specific genes and expansion of the DNA-puffs occur. The pattern of protein synthesis in the salivary gland has also been studied by means of SDS-polyacrylamide gel electrophoresis and fluorography. It changes drastically and continuously during the phases preceding pupation. These changes in protein synthesis could be correlated with the changes in the puffing pattern, essentially with the activity of the DNA-puffs.