In vitro stimulation of the prothoracic gland of the insect as a test system (prothoracic gland test)

In vitro cultures of prothoracic glands of larvae of Periplaneta americana and of some Lepidoptera as biological tests are described. Incorporation of ³H-5-uridine in the RNA of the prothoracic glands represented the measure of the cellular activity of the glands.Activation factor I separated from extracts of corpora cardiaca of the cockroach Periplaneta americana by means of gel filtration techniques caused significant stimulation of RNA synthesis of the glands.     

The steroidogenic action of haemolymph stimulatory factor in Manduca sexta: Comparisons with prothoracicotropic hormone

A recently described protein, found in the haemolymph of Manduca sexta larvae, stimulates ecdysone synthesis by both larval and pupal prothoracic glands in vitro. The mode of action of this haemolymph stimulatory factor has been investigated, particularly as it compares to the action of the cerebral neuropeptide, prothoracicotropic hormone (PTTH). Unlike PTTH, the haemolymph factor does not stimulate ecdysone synthesis via an increase in the level of cAMP in the prothoracic glands. The haemolymph factor requires extracellular calcium for maximal stimulation of the prothoracic glands, but in contrast to PTTH, significant activity is retained in calcium-free medium. Exposure of the prothoracic glands to the haemolymph factor results in enhanced steroidogenesis within 1 min. This rapid stimulation contrasts with the 10–20 min lag period observed following PTTH exposure. However, the prolonged activation elicited by brief exposure to PTTH is not observed following exposure of the glands to the haemolymph stimulatory factor. Rather, the factor appears to be required as a sustained stimulus in order to exert its steroidogenic effects. The data indicate that the mode of action of the haemolymph factor is distinctly different from that reported previously for PTTH, and are consistent with the hypothesized role of the factor as a carrier of a sterol precursor utilized in ecdysone synthesis.     

Protein phosphorylation in the prothoracic glands as a cellular model for juvenile hormone-prothoracicotropic hormone interactions

Prothoracicotropic hormone (PTTH) is a brain peptide that initiates the molting process by acting directly at the cell membrane of the prothoracic glands to increase the intracellular levels of free Ca²⁺ and cyclic AMP (cAMP). This, in turn, leads to enhanced cAMP-dependent protein kinase activity resulting in the phosphorylation of a specific protein (M_r 34,000), and ultimately to a stimulation of ecdysone synthesis. When prothoracic glands are incubated in the presence of juvenile hormone (JH I) or (7S) hydroprene and then challenged with PTTH, the phosphorylation of the 34 kDa protein is decreased in a dose-dependent manner. The morphogenetically inactive methyl farnesoate is ineffective in preventing this downstream effect of PTTH. The JH effect does not appear to be stage specific, as early last larval, late last larval and pupal Manduca sexta prothoracic glands are similarly affected. The mechanism by which JH may prevent this PTTH-stimulated phosphorylation is discussed in terms of inhibition of phosphorylation via stimulation of an ATPase and stimulation of dephosphorylation by activation of a phosphoprotein phosphatase.     

Endocrine events during pre-diapause and non-diapause larval-pupal development of the tobacco hornworm, Manduca sexta

The haemolymph ecdysteroid titre and in vitro capacities of prothoracic glands and corpora allata to synthesize ecdysone and juvenile hormone, respectively, during the last-larval instar of diapause-destined (short-day) and non-diapause-destined (long-day) Manduca sexta were investigated. In general, the ecdysteroid titres for both populations of larvae were the same and exhibited the two peaks characteristic of the haemolymph titre during this developmental stage in Manduca. The only difference in the titre occurred between day 7 plus 12 h and day 7 plus 20 h, when the short-day larval titre did not decrease as quickly as the long-day titre. The in vitro synthesis of ecdysone by prothoracic glands of short- and long-day larvae during the pharate pupal phase of the instar were also essentially the same. Activity fluctuated at times which would support the idea that ecdysone synthesis by the glands is a major contributing factor to the changes in the haemolymph ecdysteroid titre. There was one subtle difference in prothoracic gland activity between the two populations, occurring on day 7 plus 2 h. By day 7 plus 10 h, however, rates of ecdysone synthesis by the short- and long-day glands were comparable. This elevated activity of the short-day glands occurred just prior to the period the haemolymph ecdysteroid titre remained elevated in these larvae. The capacities of corpora allata to synthesize juvenile hormone I and III in vitro were not markedly different in long- and short-day last-instar larvae. At the time of prothoracicotropic hormone release in the early pupa, activity of corpora allata from short- and long-day reared animals was low and also essentially the same. There were a few differences in the levels of synthesis at isolated times, but they were not consistent for both homologues. Overall, there are no compelling differences in the fluctuations of ecdysteroids and juvenile hormones between diapause-destined and non-diapause-destined Manduca larvae. Since these hormones do not appear to play any obviously significant role in the induction of pupal diapause in this insect, the photoperiodic induction of diapause in Manduca appears to be a predominantly brain-centred phenomenon not involving endocrine effectors.     

The effect of juvenile hormone on prothoracic gland function during the larval-pupal development of Manduca sexta: An in situ and in vitro analysis

The application of juvenile hormone I or ZR 512 to neck-ligated, day-5 fifth instar (V₅) larvae reduced the time to pupation in a dose-dependent manner when compared to neck-ligated controls treated with methyl epoxy stearate. Haemolymph ecdysteroid titres determined by radioimmunoassay (RIA) reflected the ability of juvenile hormone I and ZR 512 to stimulate larval-pupal development, i.e. the ecdysteroid titres were similar to those of normally developing larvae although the ecdysteroid peak elicited by ZR 512 lagged that in the normal titre by 1 day, while that elicited by juvenile hormone I lagged the ecdysteroid peak in normal larvae by 2 days. Neck-ligated V₅ larvae that were untreated ultimately pupated and the haemolymph ecdysteroid peak eliciting pupation in these animals was 7 μg/ml haemolymph, almost double that of normal animals and ZR 512- and juvenile hormone I-treated, ligated larvae. The data indicated that juvenile hormone I does stimulate the prothoracic glands but to determine whether this stimulation was direct or indirect, an in vitro approach was taken. Prothoracic glands from V₅, V₆ and V₇ larvae were incubated in vitro under conditions in which they could be stimulated by prothoracicotropic hormone, and were exposed to concentration of free juvenile hormones I, II, III or ZR 512 ranging from 10^?5M to 10^?10M. In no case were the prothoracic glands stimulated in a dose-dependent manner that would be indicative of hormone activation. Similar results were obtained when juvenile hormone bound to binding protein was incubated with the prothoracic glands. Studies with the acids of the three juvenile hormone homologues revealed them to be ineffective in activating prothoracic glands, although juvenile hormone III acid does appear to inhibit the synthesis of ecdysone by day-0 pupal prothoracic glands. The significance of the latter effect is unknown. It is concluded from these data that juvenile hormone can, indeed, activate late larval prothoracic glands in situ, but does so indirectly.     

Insulin stimulates ecdysteroidogenesis by prothoracic glands in the silkworm,Bombyx mori

It is generally accepted that the prothoracicotropic hormone (PTTH) is the stimulator of ecdysteroidogenesis by prothoracic glands in larval insects. In the present study, we investigated activation of ecdysteroidogenesis by bovine insulin in prothoracic glands of the silkworm, Bombyx mori. The results showed that the insulin stimulated ecdysteroidogenesis during a long-term incubation period and in a dose-dependent manner. In addition, insulin also stimulated both DNA synthesis and viability of prothoracic glands. Insulin-stimulated ecdysteroidogenesis was blocked by either LY294002 or wortmannin, indicating involvement of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Activation of ecdysteroidogenesis by insulin appeared to be developmentally regulated. Moreover, in vitro activation of ecdysteroidogenesis of prothoracic glands by insulin was also verified by in vivo experiments: injection of insulin into day 6 last instar larvae greatly increased both hemolymph ecdysteroid levels and ecdysteroidogenesis 24 h after the injection, indicating its possible in vivo function. Phosphorylation of Akt and the insulin receptor was stimulated by insulin, and stimulation of Akt phosphorylation appeared to be PI3K-dependent and developmentally regulated. Insulin did not stimulate extracellular signal-regulated kinase (ERK) signaling of the prothoracic glands. These results suggest that in silkworm prothoracic glands, in addition to the PTTH and an autocrine factor, ecdysteroidogenesis is also stimulated by insulin during development.     

Relationships between the parasitoid Hyposoter exiguae and Trichoplusia ni: Prevention of host pupation at the endocrine level

RNA synthesis in normal Trichoplusia ni fifth instars and hosts parasitized at ca. 12 hr post-ecdysis was followed by measuring ³H-uridine incorporation with an autoradiographic technique.Uptake of ³H-uridine was high in control prothoracic glands at 6 and 30 hr and their cytology indicated an active secretory phase which was most pronounced at 30 hr. At the same time, glands of parasitized larvae decreased incorporation and appeared less active than controls. At > 75 hr, control fat body cells incorporated almost no label but were filled with RNA-protein granules apparently sequestered from the haemolymph preparatory to pupation. With respect to incorporation and cytology, fat body of parasitized larvae was unchanged from earlier in the instar, which indicates that the changeover to pupal preparations had not taken place. Imaginal wing disks incorporated label and grew appreciably in control larvae but abruptly decreased uptake and showed no size increase in parasitized larvae. Incorporation of Malpighian tubule, midgut epithelium, and certain muscles at > 75 hr showed little change in parasitized larvae, but in controls activity was reduced and histolysis occasionally was evident in muscles.The parasitoid, Hyposoter exiguae, apparently prevented host larvae from pupating by preventing activation of host prothoracic glands in the fifth instar. Other tissues which are normally activated for metamorphosis by the prothoracic glands continued normal larval activities until the end of the association.     

Regulation of insect prothoracic glands: Existence of a haemolymph stimulatory factor in Manduca sexta

The primary regulator of ecdysone biosynthesis by insect prothoracic glands is the prothoracicotropic hormone. However, it now appears that other factors, secondary regulators, may modulate prothoracic gland activity. One such factor has been isolated from the haemolymph of Manduca larvae. This haemolymph factor stimulates in vitro ecdysone synthesis by larval and pupal prothoracic glands by approx. 5-fold. It has an apparent mol. wt of ～330 kD, is protease-sensitive and is heat labile, the latter clearly distinguishing it from the prothoracicotropic hormone. Further, its steroidogenic effects and those of prothoracicotropic hormone are additive. Treatment of larval or pupal prothoracic glands with both moieties simultaneously effects an approx. 10-fold increase in ecdysone synthesis. The haemolymph titre of the stimulatory factor is low at commitment of the last-larval instar, then increases by approx. 3-fold later in the instar during pharate-pupal development. This increase in the titre is sufficient to effect a significant increase in prothoracic gland activity that could be physiologically important. Thus, it appears that the fluctuating level of this haemolymph stimulatory factor may act in conjunction with prothoracicotropic hormone to regulate the haemolymph ecdysteroid titre by modulating the ecdysone biosynthetic activity of the prothoracic glands.     

Adenylate cyclase in prothoracic glands during the last larval instar of the silkworm, Bombyx mori

We have previously reported that the absence of prothoracicotropic hormone (PTTH) signal transduction during the early last larval instar of Bombyx mori plays a role in leading to very low ecdysteroid levels in the hemolymph, inactivation of the corpora allata, as well as larval-pupal transformation. In the present study, adenylate cyclase was characterized in crude preparations of prothoracic gland cell membranes in an effort to localize the cause of refractoriness to PTTH. It was found that cyclase activity of the prothoracic glands from the day 6 last instar showed activation responses to fluoride, a guanine nucleotide analogue, as well as calmodulin (CaM) in dose-dependent fashions. The additive effects of day 5 prothoracic gland adenylate cyclase stimulation by fluoride and CaM imply that there may exist Gs protein-dependent and CaM-dependent forms of adenylate cyclase. For day 1 last instar prothoracic glands, which showed no response to stimulation by PTTH in either cAMP generation or ecdysteroidogenesis, adenylate cyclase activity exhibited far less responsiveness to Ca(2+)/CaM than did that from day 5 glands. These findings suggest that day 1 prothoracic glands may possess some lesions in the receptor-Ca(2+) influx-adenylate cyclase signal transduction pathway and these impairments in PTTH signal transduction may be, at least in part, responsible for decreased ecdysteroidogenesis.     

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.     

Ring gland and prothoracic gland sensitivity to interspecific prothoracicotropic hormone extracts

Summary Using the techniques of intraspecific in vitro activation of prothoracic glands and ring glands by serial dilutions of prothoracicotropic hormone (PTTH) extracts from pupalManduca sexta (Lepidoptera) and larvalSarcophaga bullata (Diptera), a dose-response of activation was observed for both species. In both species maximum activation was at 0.5 brain equivalents while the number of brain equivalents necessary for half maximal stimulation (ED₅₀) was 0.20 forManduca and 0.15 forSarcophaga. When prothoracic glands or ring glands were challenged with interspecific PTTH extracts from a stage different from that of the gland donor, no dose-response of gland activation was observed. However, whenM. sexta larval prothoracic glands were challenged byS. bullata larval PTTH extract, activation was observed. The dose-response profile fell midway between the dose-response curves obtained for the intraspecific assays. Thus, PTTH extract from one insect has the ability to activate the prothoracic glands of an insect representing another order.     

Larval-pupal transformation of the prothoracic glands of Mamestra brassicae

The sensitivity of the prothoracic glands to juvenile hormone and prothoracicotropic hormone (PTTH) of penultimate (5th)-instar larvae of Mamestra brassicae was compared with that of the same-instar larvae destined for pupal ecdysis by allatectomy. The activity of the prothoracic glands was assessed using either moulting of isolated abdomens or ecdysone radioimmunoassay. Juvenile hormone application immediately after neck-ligation (which removes brain-corpora cardiaca-corpora allata complex) prevented prothoracic gland function in larvae at all stages. When larvae were allatectomized 12 hr after ecdysis, followed by neck-ligation at different times and given juvenile hormone immediately, the hormone inhibited the prothoracic glands of young larvae, but activated the prothoracic glands from day-5 or older larvae. Juvenile hormone I, juvenile hormone II and methoprene activated the prothoracic glands, but juvenile hormone III was relatively ineffective. Brain implantation instead of juvenile hormone application led to activation of the prothoracic glands at all stages.Allatectomy thus caused changes leading to metamorphosis including a transformation of the prothoracic glands from ‘larval’ to ‘pupal’ type. After this change these prothoracic glands were able to respond not only to PTTH but also to juvenile hormone just as in last-instar larvae.     

Indirect stimulation of the prothoracic glands of Manduca sexta by juvenile hormone: Evidence for a fat body stimulatory factor

Juvenile hormone or ZR512 applied topically to day-5, fifth-instar, neck-ligated Manduca sexta larvae results in the acceleration of pharate pupal development when compared to neck-ligated, untreated larvae. This occurs as a result of an increase in the haemolymph ecdysteroid titre. Juvenile hormone, therefore, appears to stimulate ecdysone synthesis by the prothoracic glands of these animals, but not directly as shown by in vitro analysis. When ecdysone synthesis by the prothoracic glands of these ZR512- or juvenile hormone-treated animals was analyzed in vitro, increased gland activity was demonstrated but this did not occur until at least 2 days after treatment. This time lag in response supports the concept of an indirect stimulation of the prothoracic glands. Incubation of fat body from these ZR512- or juvenile hormone-treated, neck-ligated, larvae in 19AB culture medium revealed that the resulting pre-conditioned medium was capable of stimulating prothoracic glands in vitro up to 9-fold in a dose-dependent manner. A developmental profile was generated of the amount of this stimulatory factor released into the medium by fat body of untreated larvae representing each day of the last instar, and revealed that maximal release occurred with fat body from day-9 animals. The alterations in the amount of factor release by the fat body during larval-pupal development roughly correlated with the juvenile hormone titre and suggested a possible role for this factor in the regulation of the ecdysteroid titre. In contrast to the prothoracicotropic hormone, the fat body stimulatory factor is heat labile and has an apparent mol. wt in the 30,000 Dalton range. These data, particularly the kinetics of prothoracic gland stimulation, suggest that the factor may be a protein transporting a substrate for ecdysone biosynthesis to the prothoracic glands.     

Growth and respiratory enzyme activity in the prothoracic glands of Galleria

In the penultimate-larval instar, the total volume of the prothoracic gland and the activities of some oxidative mitochondrial enzymes (cytochrome oxidase, NADH: cytochrome c oxidoreductase, succinate: cytochrome c oxidoreductase) undergo cyclic variations associated with larval growth. These specifically larval-larval growth cycles are absent in the prothoracic glands of normal last-instar larvae. Here the cycles can be induced artificially by implantation of brain or corpora cardiaca-allata complexes or, by exogenous application of juvenile hormone. The smallest size of the prothoracic gland in relation to the size of the body, as well as the minimal activity of all the three mitochondrial enzymes in the gland, have been found exactly at the moment of the pre-pupal peak of ecdysteroid in the body. The possibility that the prothoracic glands alone can synthetize ecdysteroid during the peak is questioned.     

Inhibition of RNA synthesis in salivary glands of Drosophilamelanogaster by 5′-methylthioadenosine

5′-Methylthioadenosine (MTA) inhibits the incorporation of [³H] uridine into RNA in salivary glands of $\text{Drosophila}\text{melanogaster}$. This effect is not due to an inhibition of [³H] uridine uptake into the glands. The inhibition of RNA synthesis by MTA is concentration dependent and maximum inhibition is observed after 45 minutes of incubation in the presence of 1 mM MTA. Experiments utilizing α-amanitin suggest that the synthesis of heterogeneous RNA is completely inhibited.     

Inhibition of isoproterenol-induced DNA and RNA synthesis in rat parotid and pancreas following removal of submandibular-sublingual glands

Summary [³H] thymidine incorporation into DNA of the parotid (PA) gland of adult and 20-day-old rats and into DNA of the pancreas (PANC) of 20-day-old rats was increased markedly following a 2-day regimen of isoproterenol (ISO) administration. However, when the submandibular-sublingual (SM-SL) glands had been removed just prior to initiation of the ISO injections, the [³H] thymidine incorporation into PA and PANC was inhibited, and cpm/mg protein of these organs was even lower than that of organs of untreated rats with SM-SL glands present. Removal of the PA glands just prior to initiation of the ISO regimen had no effect on the ISO-induced [³H] thymidine incorporation into DNA of PANC but partially inhibited that of the submandibular (SM) gland. It is suggested that the inhibitory effects on DNA and RNA synthesis that follow removal of SM-SL glands are attributable to the growth factors (epidermal growth factor and nerve growth factor) found in the rat SM gland. These factors appear to regulate normal DNA synthetic activity of exocrine glands as well as ₁-adrenoceptor mediated DNA synthesis. Cellular hypertrophy induced by the ISO was less markedly affected by absence of the SM glands, but a partial inhibition of [³H] uridine incorporation into RNA of PA of adult rats also occurred when SM-SL glands were removed prior to initiation of the ISO-regimen.     

Activation of the prothoracic gland by juvenile hormone and prothoracicotropic hormone in Mamestra brassicae

The effects of JHA (ZR-515) application or brain implantation on metamorphosis and adult development were examined in the last instar larvae and pupae of Mamestra brassicae. When JHA was applied to neck-ligated 4- or 5-day-old larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands taken from 5-day-old larvae, the insects pupated. Dauer pupae and diapausing pupae treated with JHA showed adult development. By contrast, pupation could not be induced by the application of JHA to 2- or 3-day-old neck-ligated larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands from 0-day-old larvae. Implantation of a brain into neck-ligated 3- or 5-day-old larvae (at the beginning of gut emptying and wandering) caused pupation of the host. A similar result was obtained when both a brain and the prothoracic glands from 0- or 5-day-old larvae were implanted into the isolated abdomens of 5-day-old larvae. These results indicate that activation of the prothoracic glands by application of JHA is temporally restricted to the last part of the last larval instar and to the pupal stage, while the activation by prothoracicotropic hormone (PTTH) can occur throughout the last larval instar and the pupal stage. In addition, the implantation of brains or application of JHA to neck-ligated 5-day-old larvae 25 days after ligation seldom induced pupation of the hosts, a result which suggests that larval prothoracic glands maintained under juvenile hormone (JH) or PTTH-free conditions for long periods of time may become insensitive to reactivation by both hormones.     

Effect of incubation and translation inhibitors on the transcriptional activity in salivary glands of Chironomus thummi

Short preincubations of excised salivary glands of Chironomus thummi in synthetic media modify both the activity of uridine uptake into the cells and its incorporation into RNA. The modification of uptake varies with the medium used. Incorporation into total RNA as well as into nucleolar preribosomal RNA is considerably decreased, while incorporation into non-nucleolar RNAs is little affected. When preincubated explanted glands are briefly treated with the protein synthesis inhibitors cycloheximide or anisomycin, the incorporation activity into preribosomal RNA is slightly recovered. This contrasts with the decrease of the labelling of preribosomal glandular RNA, when those drugs are applied in vivo to the larvae.     

The Use of Glucosamine and Actinomycin D in Radioactive Labeling of RNA in Cells in Culture

Pretreatment of confluent cultures of mouse L cells or of well-differentiated nervous system cells in primary cultures with 20–120 mM glucosamine resulted in a stimulation of the uptake of tritiated uridine, but not of adenosine. A marked stimulation of the incorporation of radioactive uridine into acid-precipitable macromolecules was also obtained, while adenosine incorporation was unchanged. Cultures of L cells in log phase of growth were similarly affected by glucosamine pretreatment. Uridine and cytidine uptakes were stimulated by 50%. Tritiated uridine incorporation was stimulated in a biphasic manner, with maximal stimulation (115%) after 15–60 min of labeling and at later times an inhibition of incorporation. The stimulation of cytidine incorporation paralleled the stimulation of its uptake. The data indicate that there is: a) a glucosamine-induced stimulation of pyrimidine nucleoside uptake, b) a marked stimulation of tritiated uridine incorporation into RNA due to depletion of the cellular pools of unlabeled uridine nucleotides during glucosamine pre-treatment, and c) a decrease in the rate of RNA synthesis after several hours of glucosamine treatment, probably related to diminished intracellular supplies of uridine nucleotides. In the presence of glucosamine, high concentrations of actinomycin D could be used to increase nuclear retention of pulse-labeled nascent RNA. Cordycepin treatment did not result in similar retention of RNA. These techniques will be useful in autoradiographic and biochemical studies of nuclear RNA synthesis.