Ecdysone stimulates and juvenile hormone inhibits follicle formation in a gall midge ovary in vitro

The larval ovaries of the paedogenetic gall midge Heteropeza pygmaea can be cultured in vitro throughout oögenesis. Addition of farnesol or C₁₆ juvenile hormone to the culture medium inhibits follicle formation in the ovaries; addition of ecdysterone, on the other hand, strongly accelerates the formation of follicles. It is suggested that ecdysone triggers or stimulates follicle formation in the ovaries also in vivo, and that juvenile hormone acts on oögenesis in a later stage of development.     

Adaptation of a radiochemical assay for juvenile hormone biosynthesis to study caste differentiation in a primitive termite

A radiochemical assay measuring juvenile hormone synthesis by corpora allata incubated in vitro was adapted for use with the termite Zootermopsis angusticollis. Corpora allata from 3–4-day old virgin female neotenic reproductives were used in these studies because this caste showed the highest rates of juvenile hormone synthesis (0.6 pmol h^?1 per pair corpora allata). Juvenile hormone-III synthesis was linear for up to 6 h over the range of concentrations of labelled l-methionine from 27–280 μM. Rates of juvenile hormone synthesis were stimulated up to 10-fold in a dose-dependent manner by the addition of farnesoic acid to the incubation medium. However, the relatively high concentration of 120 μM farnesoic acid reduced the rates of juvenile hormone synthesis. The radiochemical assay was used to determine rates of juvenile hormone synthesis in vitro by corpora allata from larvae with a queen and king vs orphaned larvae. The presence of reproductives resulted in a suppression of larval corpus allatum activity relative to orphaned controls.     

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.     

In vitro development of the strobilar stage of Mesocestoides corti

Thompson R. C. A., Jue Sue L. P. and Buckley S. J. 1982. In vitro development of the strobilar stage of Mesocestoides corti. International Journal for Parasitology12: 303–314. Sexually mature strobilated adults of Mesocestoides corti were grown consistently from undifferentiated tetrathyridia in vitro using a conventional diphasic culture system. Development (growth, strobilisation and maturation) was compared in vitro and in vivo. Although growth and strobilisation were comparable in vitro and in vivo, during the first 18 days, total length and numbers of proglottids decreased in vivo but continued to increase in vitro after day 18. Both male and female reproductive systems appeared to develop normally in vitro and self copulation was frequently observed in cultured worms. However, fully developed oncospheres were not produced in vitro.     

In vitro and in vivo activity of three sesquiterpenes against L(3) larvae of Anisakis type I

In order to investigate the possible use of terpenic derivatives to treat anisakiasis caused by L₃ larvae of Anisakis, we studied the in vitro and in vivo larvicidal activity of three sesquiterpenes (nerolidol, farnesol and elemol). In vitro experiments included the histological study of larval damage and in vivo studies the measurement of myeloperoxidase activity in rat gastrointestinal tract after administration of the sesquiterpenes. In the in vitro assays, the most active compound against the L₃ larvae was nerolidol, followed by farnesol; both caused the death of all nematodes, which showed cuticle changes and intestinal wall rupture. In the in vivo assays, only 20% of infected rats treated with nerolidol or farnesol showed gastric wall lesions in comparison to 86.6% of control animals. According to these results, nerolidol and farnesol are good candidates for further research as biocidal agents against L₃ larvae of Anisakis type I.     

Growth and silk formation of silkworm larvae influenced by phytoecdysones

The fourth and fifth instar larvae of the silkworm were reared on artificial diets containing ponasterone A, ecdysterone, and inokosterone. The growth of the larvae and their silk glands, fibroin-synthesizing activity, and silk formation have been investigated. With a diet containing ponasterone A, the fourth instar larvae grew slowly and only a few larvae could ecdyse, while the growth of the fifth instar larvae was disturbed and they died with a darkening of the skin. Ponasterone A also inhibited the growth of the silk glands during the fifth instar. In contrast, the other two phytoecdysones did not greatly influence larval growth. The fourth instar larvae grew rapidly and their ecdysis was advanced with a diet which contained 10 μg of inokosterone/1 g of dry diet. The diet which contained 5 μg of ecdysterone or 10 μg of inokosterone/1 g of dry diet accelerated maturation, while that containing 10 or 20 μg of ecdysterone, or 40 μg of inokosterone, delayed maturation of the fifth instar larvae.Only phytoecdysones caused a decrease in growth of the silk glands in the early half of the instar, and a large amount of phytoecdysones accelerated their growth during the last part of the fifth instar. The fibroin-synthesizing activity was levelled up by feeding ecdysterone and inokosterone, and inokosterone appreciably stimulated activity. Assay of in vitro fibroin synthesis showed that ponasterone A competed with ecdysterone in a stimulative action. Silk formation was much lower in larvae fed the diet containing 5 μg of ecdysterone or 10 μg of inokosterone/1 g of dry diet and was far greater in larvae fed the diet containing 40 μg of inokosterone than in the controls.     

THE CYTOLOGICAL EFFECT OF ECDYSTERONE ON THE MIDGUT CELLS OF THE FLESH-FLY SARCOPHAGA BULLATA

Larvae of the flesh-fly, Sarcophaga bullata, were injected with the synthetic moulting hormone ecdysterone or saline at the beginning of the third and final larval instar. One group was left untreated. The ecdysterone-injected larvae showed an increase in number of secondary lysosomes in the midgut epithelial cells similar to that observed at the onset of metamorphosis, an event which would normally occur about 48 hr later in these larvae.     

Hormonal Effects on the in vitro Larval Growth of the Swine Intestinal Roundworm,Ascaris suum

Summary

Several biogenic amines and insect juvenile hormone III were tested in a growth bioassay of the parasitic nematode, Ascaris suum. Compounds (1 to 1000μzmol) were placed in culture with third-stage larvae for 24 hr, larvae were then rinsed several times, and larval cultures were returned to incubators for 6 more days. By this time, larvae had developed to the fourth-stage. The larvae were fixed in hot formalin, and their lengths were measured. Epinephrine and norepinephrine oxidized and were nematocidal under these culture conditions. Histamine and serotonin had no effect on length of the larvae. Octopamine (10–50μmol) exposure resulted in a significant dose-dependent increase in length. When incubated with octopamine (10μzmol) for 7 days, larvae grew more slowly than controls without octopamine (P< 0.05). Juvenile hormone III stimulated a dose-dependent (0 to 10μmol) increase in length after a 24 hr exposure. No synergism was detected between juvenile hormone III and ecdysone when co-incubated with larvae. These results indicate that Ascaris larvae are growth-insensitive to low concentrations of biogenic amines of host origin. Conversely, biological transmitters of invertebrate origin are potent stimulators of larval growth.     

Relation between cell number and pupal development of wing disks in Bombyx mori

The effects of JH and ecdysone on pupal differentiation of the wing disk of Bombyx mori were studied in vivo and in vitro. JH prevents pupal differentiation during larval life by stopping a particular stage of the cell cycle. Immediately after allatectomy, a cell cycle sets in without ecdysone, but afterwards wing disks obtain the competence to differentiate to the pupal type in response to ecdysterone. Disks older than 2 days after allatectomy can develop to the pupal type with an abrupt increase of mitosis in a certain concentration of ecdysterone in vitro. Once the disks have gained such competence and begun pupal development, JH no longer exhibits the effect that prevents DNA synthesis, which is enhanced by ecdysterone. It is therefore suggested that there are two phases in DNA synthesis: one which is important for achieving competence and is inhibited by JH and relatively independent of ecdysone; and another which is important for morphogenetic development and depends on ecdysone but is not inhibited by JH.     

Aldehyde dehydrogenase 3 converts farnesal into farnesoic acid in the corpora allata of mosquitoes

The juvenile hormones (JHs) play a central role in insect reproduction, development and behavior. Interrupting JH biosynthesis has long been considered a promising strategy for the development of target-specific insecticides. Using a combination of RNAi, in vivo and in vitro studies we characterized the last unknown biosynthetic enzyme of the JH pathway, a fatty aldehyde dehydrogenase (AaALDH3) that oxidizes farnesal into farnesoic acid (FA) in the corpora allata (CA) of mosquitoes. The AaALDH3 is structurally and functionally a NAD⁺-dependent class 3 ALDH showing tissue- and developmental-stage-specific splice variants. Members of the ALDH3 family play critical roles in the development of cancer and Sjögren–Larsson syndrome in humans, but have not been studies in groups other than mammals. Using a newly developed assay utilizing fluorescent tags, we demonstrated that AaALDH3 activity, as well as the concentrations of farnesol, farnesal and FA were different in CA of sugar and blood-fed females. In CA of blood-fed females the low catalytic activity of AaALDH3 limited the flux of precursors and caused a remarkable increase in the pool of farnesal with a decrease in FA and JH synthesis. The accumulation of the potentially toxic farnesal stimulated the activity of a reductase that converted farnesal back into farnesol, resulting in farnesol leaking out of the CA. Our studies indicated AaALDH3 plays a key role in the regulation of JH synthesis in blood-fed females and mosquitoes seem to have developed a “trade-off” system to balance the key role of farnesal as a JH precursor with its potential toxicity.     

Effects of ecdysteroids on reproductive physiology of Nippostrongylus brasiliensis (nematoda) in vivo

1. The influence of ecdysteroids (ecdysone and ecdysterone) was investigated on the control of reproduction in the parasitic nematode Nippostrongylus brasiliensis in vivo.2. Infestive larvae (L₃) were immersed in solutions of ecdysteroids (2.2 μM) at 37°C for 4 hr before injection into the host. The effect on egg-laying was observed two stages later.3. The treatment increased egg-laying, but had no influence on the timing of the reproductive period. The greatest effect was observed with ecdysone, ecdysterone only inducing a small and non-significant stimulation under our experimental conditions.4. The physiological role of ecdysteroids in meiosis and gonadal development in nematodes is discussed.     

Acceleration of the growth of tetrathyridial populations of Mesocestoides Corti (Cestoda: Cyclophyllidea) by splenectomy

Acceleration of the growth of tetrathyridial populations of Mesocestoides corti (Cestoda: Cyclophyllidea) by splenectomy. International Journal for Parasitology4, 165–168. Experiments with LDF₁ hybrid mice showed that splenectomy 7 days prior to infection, increases the total biomass of tetrathyridial populations of Mesocestoides corti in hosts of both sexes.This increase is accompanied by a decrease in the size of individuals, and by an increase in the percentage of two-suckered and acephalic forms. Splenectomy thus accelerates both the growth of the biomass of populations and the asexual multiplication of the tetrathyridia.     

Effects of dietary sterols on development and reproduction of southwestern corn borer Diatraea grandiosella Dyar

Southwestern corn borer larvae, Diatraea grandiosella Dyar, were reared on artificial diets containing individual sterols (cholesterol, sitosterol, or stigmasterol) in concentrations ranging from 0.05 to 0.2%. Female larvae developed to pupae more rapidly as sitosterol and stigmasterol were increased in the diets. Increased cholesterol concentrations did not affect the larval period significantly, and development was not as rapid as with the phytosterols. Female larvae developed at significantly slower rates in all diets than did males, except at the highest concentrations of sitosterol and stigmasterol. Female pupae and adults were significantly heavier than the males, and pupal and adult weight increased as sterol concentrations increased. Number of eggs laid per fertilized female and egg hatchability were significantly increased as concentrations of the three sterols were increased in the larval diets. Sitosterol-reared females produced more eggs than did females reared on other sterols but egg hatchability was not significantly different among sterols.     

Ecdysterone responsive functions in the mutantl(1)su(f) ts67g ofDrosophila melanogaster

Summary Transferring the temperature sensitive mutantl(1)su(f) ^ts67g from 25° C to 30° C before or early in the third larval instar blocks the increase in the ecdysterone titer that normally occurs at the end of the larval period. Feeding exogenous ecdysterone to these hormone-deficient larvae results in the formation of pseudopupae. The mutant was used to study ecdysterone-inducible functions in late larval salivary glands by preparing three animal samples with different hormone titers: the titer was low in one sample because of an earlier temperature shift, high in a second sample because the larvae were subsequently transferred to ecdysterone-supplemented food, and also high in a third sample that was kept at 25°C, providing a control for normal development. The effect of the different hormone conditions was studied by³⁵S-methionine labeling of the salivary gland proteins during the larval to prepupal transition and the prepupal period. The results indicate that synthesis of several of the proteins normally appearing during the transition and prepupal period is induced by exogenous ecdysterone.     

Spontaneous sexual differentiation of Mesocestoides corti tetrathyridia in vitro

Barrett N. J., Smyth J. D. and Ong S. J. 1982. Spontaneous sexual differentiation of Mesocestoides corti tetrathyridia in vitro. International Journal for Parasitology12: 315–322. Tetrathyridia of Mesocestoides corti, from the body cavity of mice, maintained in the laboratory by intraperitoneal infection, were used for in vitro culture. In an initial experiment, after 50 days asexual multiplication in vitro one tetrathyridium spontaneously segmented and developed into a sexually mature adult. Further experiments were carried out in an attempt to determine the conditions favouring segmentation and sexual differentiation. A combination of 5 or 10 ml liquid medium S1OE.H (basically composed of CMRL 1066 and foetal calf serum with supplements) changed every 3 days, in a Leighton tube (19 × 105 mm), rotated at 38°C and gassed with 10 or 20% CO₂, containing between 100 and 200 tetrathyridia, has proved to be most suitable so far. Numerous adult worms with normal male and female genitalia have been obtained in this system. However, segmentation is sporadic, rather than consistent and only a few shelled eggs with hooked oncospheres have so far been obtained, suggesting that impregnation and fertilization in vitro is not fully comparable with that in vivo.     

Biosynthesis of ecdysterone from cholesterol in Taxus baccata

Biosynthesis of ecdysterone from [4-¹⁴C, 3-³H]cholesterol in Taxus baccata does not involve obligatory oxidation at C-3 during the formation of the A/B-cis ring junction.     

Hormonal regulation of epidermis-specific protein and messenger RNA synthesis in amphibian metamorphosis

Experiments using a monospecific antibody directed against one type of epidermis-specific keratin from adult skin of the amphibian Xenopus laevis have demonstrated that polysomes synthesizing this protein first appear within larval skin during natural metamorphosis. Further experiments demonstrated that the synthesis of keratin within larval skin could be induced precociously by the thyroid hormone, 3,3′,5-triiodo-l-thyronine, both in vivo and when the isolated larval skin is cultured in vitro. The earliest developmental age responsive to such hormone induction appeared to be Stage $\text{50}\text{52}$ of larval development. This is about 20–24 days before keratin would normally make its appearance within the skin during natural metamorphosis. Hormone treatment of tadpoles at this age will also cause a precocious increase in the amount of keratin messenger RNA present within larval skin. This has been demonstrated directly by the isolation of poly(A)-containing messenger RNA from hormone-treated larvae and its translation in a wheat germ cell-free system to give immunoprecipitable keratin. Peptide analysis of the in vitro translation product indicates that the hormone-induced mRNA probably codes for an initial protein product that is slightly larger than keratin itself.     

The influence of ecdysterone on gene amplification,DNA synthesis,and puff formation in the salivary gland chromosomes of Rhynchosciara hollaenderi

Many of the DNA and RNA puffing changes observed in Rhynchosciara during the prepupal period have been induced in younger larvae by injection of ecdysterone. However, the dose of hormone necessary for this induction is high, especially in the large cells of the proximal region of the gland. There are differences in the amplification and puffing response from that observed during normal development. Particular similarities and differences with possible explanations for the differences are discussed. Preceding and during the amplification which occurs at certain chromosomal regions, ecdysterone induces DNA synthesis along the entire chromosome. This induction of general DNA synthesis can occur independently of the amplification process. It appears to be similar in pattern to that occurring normally toward the end of larval life. — The normal prepupal behavior of Rhynchosciara was not induced by injection of ecdysterone into larvae of any age thus far examined.