The identification and titres of conjugated and free ecdysteroids in developing ovaries and newly-laid eggs of Schistocerca gregaria

Ecdysteroid levels throughout ovarian development and in newly-laid eggs of S. gregaria have been determined. A simple method for the separation of free and conjugated ecdysteroids is described. Both free and polar conjugated ecdysteroids are present at the end of oögenesis and in newly-laid eggs, but the polar conjugated ecdysteroids always predominate; 95% of the total ecdysteroid in newly-laid eggs is in the conjugated form. Ecdysone, 2-deoxyecdysone and 20-hydroxyecdysone have been fully characterized from both the ‘free’ and ‘conjugated’ fractions. The presence of traces of 26-hydroxyecdysone in the ‘conjugate’ fraction was indicated by HPLC analyses. The levels of ecdysteroid released from the conjugates of newly-laid eggs were 35 μg/egg pod (44 μg/g wet weight) for ecdysone, 16 μg/egg pod (19.4 μg/g) for 2-deoxyecdysone and 5 μg/egg pod (6.1 μg/g) for 20-hydroxyecdysone. The level of free ecdysone found in newly-laid eggs was 2 μg/egg pod (2.6 μg/g).     

Catalytical oxidation of ecdysteroids to 3-dehydro products and their biological activities

Oxidation of ecdysone and ecdysterone with platinum as catalyst gives rise to several polar and 4–5 apolar substances, among them 3-dehydroecdysone and 3-dehydroecdysterone. Highest yields in the 3-dehydro products were reached after 4 to 8 hr of oxidation. 3-dehydroecdysone and 3-dehydroecdysterone are less active in inducing ecdysone specific puffs in Drosophila hydei salivary gland giant chromosomes but still contain a remarkable biological activity when compared with the original ecdysteroids. The activity of these two compounds is about one tenth that of ecdysone in the Calliphora assay.     

Controlled ecdysteroid accumulation in eggs of the silkworm,Bombyx mori,by an imidazole compound (KK-42), and embryogenesis in these eggs

An imidazole compound (KK-42), a potent inhibitor of ecdysone synthesis, was applied to the female pharate adult of the silkworm, Bombyx mori, to control ecdysteroid accumulation in developing ovaries and mature eggs. KK-42 applied on day 2 or later completely suppressed an increase in ecdysteroid content in developing ovaries. The inhibitory action of KK-42 was restricted to vitellogenic follicles, i.e., those in which active ecdysteroid synthesis is occurring. Ecdysteroid content in the mature eggs of moths remained at the level accumulated in ovaries before KK-42 application. Thus, KK-42 was shown to be a novel agent to suppress the ecdysteroid accumulation in eggs. Eggs containing different amounts of ecdysteroids showed different levels of embryonic development. About 80% of the eggs which contained less than 10 ng free ecdysteroids/g eggs were not fertilized. More than 80% of the eggs containing less than 40 ng/g eggs of free ecdysteroids initiated embryogenesis but failed to hatch. Larvae hatched from almost all eggs which accumulated free ecdysteroids of more than 150 ng/g. Thus, maternal ecdysteroids appear to be required at different titers for fertilization, embryogenesis, and hatching of the silkworm larvae. © 1994 Wiley-Liss, Inc.     

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.     

Ecdysteroids during oögenesis in the ovoviviparous cockroach Nauphoeta cinerea

By using thin-layer chromatography and high-pressure liquid chromatography combined with radioimmunoassay as well as gas chromatography-mass spectrometry we have identified and quantified ecdysteroids in ovaries and haemolymph of adult female Nauphoeta cinerea. Our analyses demonstrate the presence of ecdysone and 20-hydroxyecdysone, the latter being clearly predominant in all stages investigated. Titre determinations of free ecdysteroids in ovaries show that the 20-hydroxyecdysone concentration is highest (approximately 400 ng/g) at the beginning of chorion formation, suggesting an involvement in this process. Towards ovulation, the titre of free ecdysteroid drops and is low in the newly ovulated egg case. Measurement of immunoreactive highly polar products demonstrates that their concentration remains on a low level throughout the oöcyte maturation period; hydrolysis experiments with Helix pomatia enzymes reveal that, compared to the free ecdysteroids in the ovary, only small quantities of ecdysteroids are present as Helix hydrolysable conjugates. If one compares the quantities of free ecdysteroids in the ovary with those in the haemolymph it becomes apparent that the concentration in the haemolymph is about 10 times lower than that in the ovary.In vitro incubation of follicle cells from oöcytes at stages around chorion formation reveals that these cells are able to produce ecdysone and 20-hydroxyecdysone, and incubation with [³H]-ecdysone demonstrates that ecdysone is efficiently converted to 20-hydroxyecdysone in a stage-dependent manner. These observations strongly suggest that the follicle cells are the site of ecdysteroid biosynthesis and of C-20-ecdysone hydroxylation.A comparison of these findings with observations made of other insects such as locusts and mosquitoes demonstrates significant differences in quality, composition, titre fluctuation and distribution of ecdysteroids in adult females from different species and suggests that these ecdysteroids might fulfil multiple and various biological functions.     

A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various ecdysteroids and the amounts of these ecdysteroids fluctuate during embryonic development. In order to know the function of egg ecdysteroids in embryonic development of B. mori, we examined the biological activities of various egg ecdysteroids by in vitro ligand-binding assay and bioassay using B. mori eggs. First, using the ecdysteroid receptor of B. mori (BmEcR-B1/BmUSP heterodimer) prepared by yeast and Escherichia coli expression systems, the interaction between the ecdysteroid receptor and various egg ecdysteroids of B. mori was analyzed. The relative binding affinities of egg ecdysteroids to the BmEcR-B1/BmUSP heterodimer decreased in the order of 20-hydroxyecdysone > 2-deoxy-20-hydroxyecdysone > 22-deoxy-20-hydroxyecdysone > ecdysone > 2-deoxyecdysone > ecdysone 22-phosphate. Next, several egg ecdysteroids of B. mori were injected into the prospective diapause eggs, which show a very low level of free ecdysteroids at the onset of embryonic diapause (gastrula stage). Approximately 7% of them (P < 0.002, chi(2)-test) developed beyond the gastrula stage without entering diapause by the injection of 20-hydroxyecdysone (25 ng/egg). In contrast, the injection of other ecdysteroids was not effective in inducing embryonic development. These results suggest that 20-hydroxyecdysone, via the ecdysteroid receptor, is responsible for the developmental difference between diapause and non-diapause in B. mori embryos. Furthermore, it was suggested that continuous supply of 20-hydroxyecdysone may be required to induce embryonic development.     

Comparative studies on ecdysone metabolism between mature larvae and pharate pupae in the fleshfly,Sarcophaga peregrina

Metabolites of radioactive ecdysone or 20-hydroxyecdysone in larvae and pharate pupae of Sarcophaga peregrina were separated and identified by using thin-layer chromatography, high-performance liquid chromatography, and chemical methods. At the larval stage ecdysone was metabolized to biologically less active ecdysteroids predominantly through 20-hydroxyecydsone, at the pharate pupal stage, to other ecdysteroids which were tentatively identified as 26-hydroxyecdysone, 3-epi-26-hydroxyecdysone, and 3-epi-20,26-dihydroxyecdysone. Ecdysteroid acids were found in the polar metabolites during pharate pupal-pupal transformation, but scarcely detected in the larval metabolites. These acids were presumed to be ecdysonoic acid, 20-hydroxyecdysonoic acid, and their epimers. The conjugates of ecdysteroid that released the free ecdysteroids by enzymatic hydrolysis were produced more in larvae than in pupae, whereas the very polar ecdysteroids that were not affected by the enzyme were found more in pupae. Therefore, there are different metabolic pathways of ecdysone between these two successive developmental stages, and the alteration of the metabolic pathway may serve as one of the important factors in a regulatory mechanism of molting hormone activity which is responsible for normal development of this insect.     

Ovarian ecdysteroids and their secretion in late-pharate adults of Galleria mellonella

Approximately two-thirds of the total amount of ecdysteroids in late—pharate adults of the wax moth, Galleria mellonella, were found in the ovaries and one-third in the ovariectomized body. Chemical analysis of these ecdysteroids by thin-layer and high-pressure liquid chromatography, coupled with an ecdysteroid radioimmunoassay, revealed the presence of 2-deoxyecdysone, ecdysone and 20-hydroxyecdysone, as well as high and low polarity unknowns. The predominant identifiable ecdysteroid in both the ovaries and ovariectomized body was 2-deoxyecdysone, followed by lesser amounts of ecdysone and 20-hydroxyecdysone, respectively. Incubation of late-pharate adult ovaries in culture medium revealed that they synthesize and secrete ecdysteroids in vitro. The in vitro distribution of ecdysteroids between ovaries and incubation medium was similar to that observed between ovaries and ovariectomized bodies in situ and the predominant identifiable moiety both retained and released by the ovaries in vitro was 2-deoxyecdysone, followed by lesser amounts of ecdysone and 20-hydroxyecdysone. Collectively, these results support the idea that the ecdysteroids synthesized by the ovaries of late-pharate adult Galleria are both stored and secreted and that the quantity of a specifically secreted ecdysteroid is precisely controlled. This apparent regulation of the distribution of ovarian ecdysteroids raises the possibility that the stored and secreted forms have distinct functions in the reproductive physiology of this insect.     

Induction of egg diapause by implantation of corpora cardiaca and corpora allata in Bombyx mori

Diapause egg production was examined in non-diapause egg producers by implantation of various cephalic organs into pharate adults 4 days after larval-pupal ecdysis. The implantation of five pairs of corpora cardiaca or corpora allata induced a great amount of egg diapause. Implantation of these organs was effective in inducing egg diapause even when the suboesophageal ganglion of the recipients had been removed, although the effect of the corpora allata decreased moderately. The injection of juvenile hormone into 4-day-old pharate adults did not greatly increase production of diapause eggs.     

Ecdysteroid profiles for hemolymph and testes from larvae,pupae, and pharate adults of the European corn borer,Ostrinia nubilalis hubner

Total ecdysteroid levels as well as concentrations of several individual ecdysteroids were determined for hemolymph and testes of fifth instars, pupae, and pharate adults of the European corn borer, Ostrinia nubilalis (Hubner). For total levels, the patterns of fluctuation in hemolymph and testes were similar, but the concentrations in testes were lower than those in hemolymph. In both hemolymph and testes there were two ecdysteroid peaks: the first just prior to the formation of the pharate pupa, the second just prior to the formation of the pharate adult. An examination of ecdysteroid profiles revealed some important differences. Ecdysone was either absent or present at extremely low levels in larval testes, whereas in hemolymph there was a premolt ecdysone peak. In pupal testes, ecdysone was present, but levels of 26-hydroxyecdysone were much lower than those in hemolymph. Thus, in regard to ecdysteroids, testes have the ability to control their own internal milieu.     

Autoradiographic identification of ecdysteroid-binding cells in the nervous system of the moth Manduca sexta

The steroid hormone 20-hydroxyecdysone regulates many aspects of nervous system development in the moth Manduca sexta, including stage-specific neuronal morphology and stage-specific neuronal death. We have used steroid hormone autoradiography to study the distribution of cells that concentrate ecdysteroids in the ventral nervous system of this insect. The ligand was [3H]-ponasterone A, a bioactive phytoecdysone. Tissue was examined from three stages of development: the end of larval life (first day of wandering), the end of metamorphosis (pharate adult), and 4-day-old adults. In the abdominal ganglia of wandering larvae and pharate adults, a subset of neurons including both motoneurons and interneurons exhibited a nuclear concentration of radiolabeled hormone. The pattern of binding was reproducible but stage-specific, with a greater proportion of neurons showing binding in the larvae than in pharate adults. No labeled neurons were found in abdominal ganglia from mature (4-day-old) adults. In the case of the pharate adult ganglia, the ecdysteroid receptor content of specific, identified motoneurons was determined. These results are discussed in light of the responses of these neurons to physiological changes in levels of circulating ecdysteroids.     

Ecdysteroids in the developing eggs of the desert locust, Schistocerca gregaria

Ecdysteroid levels in the developing eggs of Schistocerca gregaria were determined, at daily intervals, using gas chromatography and electron capture detection of ecdysteroid derivatives. Ecdysone and 20-hydroxyecdysone were present both as the free ecdysteroid and as polar conjugates. Total ecdysteroids reached a maximum of 40 ng/egg with ecdysone contributing the greater part.     

Embryonic ecdysteroids in a mole crab,Emerita asiatica (Milne-Edwards)

Using radioimmunoassay (RIA) and high performance liquid chromatography (HPLC), the presence of a complex mixture of free and conjugated ecdysteroids is reported in the embryonated eggs of a mole crab,Emerita asiatica. From an initial low value of 6.5 ng/g egg wet weight in stage I, the total ecdysteroids increased in concentration to 15.2 ng/g egg wet weight in stage III. This was followed by a sharp fall in stage IV, but again increased to 15.0 ng/g egg wet weight in stage VI. After a further decline in stage VII, the total ecdysteroids registered the highest value of 36.2 ng/g egg wet weight in stage VIII. This value, however, declined to a low level in the prehatching stage (IX). The concentration of the free ecdysteroids always predominated over the conjugated ones. The HPLC analysis of free ecdysteroids demonstrated the presence of 20-hydroxyecdysone and ecdysone in the ratio of 2.5. Purified lipovitellin II also contained free and conjugated ecdysteroids. The functional significance of the embryonic ecdysteroids as well as their nature of synthesis and storage within the eggs is discussed in the light of the information available on insect embryogenesis.     

Induction of egg diapause in Bombyx mori by some cephalo-thoracic organs of the cockroach, Periplaneta americana

Induction of egg diapause in the silkworm, Bombyx mori by some cephalo-thoracic organs of the cockroach, Periplaneta americana was examined. All tissues tested such as brain, corpora cardiaca, corpora allata, suboesophageal and thoracic ganglia and nerve cords between thoracic ganglia were able to produce diapause eggs in non-diapause egg producers both by transplantation and injection of their crude homogenates. The homogenate of thoracic ganglia was effective even in pharate adults with the suboesophageal ganglion removed or in isolated abdomens of pharate adults.From these results, it was surmised that some endocrine organs, as well as the central nervous system in the cephalo-thorax of Periplaneta americana, contained the active principle responsible for egg diapause in Bombyx mori.     

Ecdysteroid biosynthesis in workers of the European honeybee Apis mellifera L

We previously reported preferential expression of genes for ecdysteroid signaling in the mushroom bodies of honeybee workers, suggesting a role of ecdysteroid signaling in regulating honeybee behaviors. The organs that produce ecdysteroids in worker honeybees, however, remain unknown. We show here that the expression of neverland and Non-molting glossy/shroud, which are involved in early steps of ecdysteroid synthesis, was enhanced in the ovary, while the expression of CYP306A1 and CYP302A1, which are involved in later steps of ecdysone synthesis, was enhanced in the brain, and the expression of CYP314A1, which is involved in converting ecdysone into active 20-hydroxyecdysone (20E), was enhanced in the brain, fat body, and ovary. In in vitro organ culture, a significant amount of ecdysteroids was detected in the culture medium of the brain, fat body, and hypopharyngeal glands. The ecdysteroids detected in the culture medium of the fat body were identified as ecdysone and 20E. These findings suggest that, in worker honeybees, cholesterol is converted into intermediate ecdysteroids in the ovary, whereas ecdysone is synthesized and secreted mainly by the brain and converted into 20E in the brain and fat body.     

An active principle from the corpora cardiaca,corpora allata and suboesophageal ganglion of the locust,inducing egg diapause in Bombyx mori

The activity of the substance(s) which are contained in the cephalic endocrine organs of the locust which induce egg diapause in Bombyx mori was examined by implantation and injection of saline extracts of these organs. Extracts from the median and lateral neurosecretory parts of the locust brain were not effective in inducing egg diapause. Extracts of the corpora cardiaca, corpora allata, and suboesophageal ganglion of the locust induced diapause eggs in Bombyx pharate adults from which the suboesophageal ganglion had been removed. The first two extracts could induce egg diapause even in isolated abdomens of pharate adults of Bombyx. In the locust corpora cardiaca, the activity was present only in the glandular lobe and not in the nervous region. This activity decreased when the nervi corporis cardiaci I and II and of nervi corporis allati I were cut. Allatectomy also brought about a decrease in the activity in the glandular lobe which could not be restored by the injection of juvenile hormone. The activity in the corpora allata was enhanced slightly by the disconnection though not significantly.From these results, it is assumed that the corpora cardiaca, corpora allata and suboesophageal ganglion of the locust contain and active principle(s) capable of inducing egg diapause in Bombyx mori. The nervous connections between the brain, corpora cardiaca, and corpora allata are essential for the accumulation of the active substance(s) in the glandular lobes of the corpora cardiaca.     

In vivo uptake and metabolism of [3H]ecdysone in the vitellogenic follicles of Sarcophaga bullata (Diptera) and its localisation by autoradiography

Within 4 h after injection of [³H]ecdysone, almost all tritiated material has disappeared from the haemolymph, indicating that the uptake by the tissues is very fast. After only 15 min, 19% of the label was found in the ecdysterone fraction and 4% in the highly polar products (HPP) fraction. The uptake of [³H]ecdysone by the ovary (mid-vitellogenic) is almost complete within 1 h after injection. The pattern of [³H]ecdysteroids in the ovaries follows a well ordered sequence: firstly, [³H]ecdysone is the major component of the [³ H]ecdysteroids but it disappears within 2 h, next a peak value of [³H]ecdysterone was found at 1 h, whereafter this also disappeared, and from 2 h on, there was a considerable increase in HPP. The HPP consisted of 3 fractions (A, B and C). Glusulase treatment revealed that apparently only fraction B consisted of glucuronide and/or sulphate-conjugates of ecdysteroids. Autoradiographic experiments confirmed that the uptake of [³H]ecdysone was a very rapid process. In ovaries fixed 1 h after injection, the silver grains were abundant in the ooplasm but were also found in the follicle cell cytoplasm and in trophocytes. In follicles examined 16 h after injection, only a few silver grains were observed in the trophocytes and follicle cells. However, the cytoplasm of the oocyte was labelled. The border cells also accumulated label.

The major results indicate that all cell types of the follicle seem to be able to absorb ecdysone from the haemolymph and that there seems to be a rather selective uptake of ecdysone. In the ooplasm, ecdysone is converted to highly polar conjugates.     

Ecdysteroids during embryonation of eggs of Ascaris suum

1. The optimal temperature for in vitro development of fertilized eggs of Ascaris suum was 24 degrees C. 2. Samples (2 X 10(7) eggs) were obtained from in vitro embryonating cultures every 3 days for 4 weeks; lipids were extracted, partially purified, fractionated with HPLC and analyzed for ecdysteroids by radioimmunoassay. 3. Free ecdysone and 20-hydroxyecdysone (20-HE) were at low levels (less than 20 pg) in freshly excised eggs and rose to maximal values on day 6 of embryonation. 4. Conjugated ecdysone and conjugated 20-HE rose to maximal values on day 9. 5. Both free and conjugated ecdysteroids were undetectable from days 15 to 27 of cultivation. 6. These profiles indicate that ecdysteroids might have a selective role in nematode embryonation and/or tanning of the egg shell.     

Organic,inorganic, and caloric composition of eggs,pentaculae, and adults of the brooding sea cucumber Cucumaria curata cowles (Echinodermata: Holothuroidea)

Levels (percentage composition) of water, ash, carbohydrate, lipid, protein, and calories were determined for eggs, pentaculae, and adults of the sea cucumber Cucumaria curata Cowles. Component contents (μg/individual) were calculated for eggs and pentaculae. During the 28 days of development to hatching, the large yolky eggs gain water and ash, the total dry weight increasing from 169 to 190 μg/egg during embryogenesis. There were no statistically significant changes in lipid, protein, and caloric contents during embryogenesis, but carbohydrate decreased by 0.82 μg/egg.The decrease in carbohydrate is sufficient to account for estimated embryonic energy requirements. Based on the utilization of carbohydrate, embryos of C. curata show a nutritional pattern similar to that of the planktonic embryos of sea urchins and different from that of embryos developing from terrestrial eggs, freshwater eggs, and planktonic and demersal marine eggs.Although broods varied widely in egg number and mean egg dry weight, C. curata gives eggs which contain a constant proportion of organic components.Levels of ash, water, and protein in the adults exceeded those in the pentacula, and lipid comprises a much smaller proportion of the adult body than it did of the pentacula.     

Regulation of ecdysone hydroxylation in Locusta migratoria: Rôle of the moulting hormone level

After repetitive injections of moderate doses of ecdysone, ecdysterone or phenobarbital to young Vth (last) instar larvae of Locusta migratoria, the conversion rate of ecdysone to ecdysterone in vivo is significantly higher than in control insects. Similarly, 5 hr after injection of a low dose of ecdysone or ecdysterone, a strong ‘induction’ of ecdysone 20-monooxygenase activity occurs. This ‘inductive’ effect is blocked by cycloheximide.Simultaneous injections of ecdysone and ecdysterone show that hydroxylation of ecdysone is inhibited by the product of the reaction, ecdysterone. Removal of the prothoracic glands and X-ray treatment of the hemocytopoietic tissue do not affect ecdysone hydroxylation. The mechanism of induction and inhibition of ecdysone 20-monooxygenase shown in this study is probably responsible for the important variations of this key enzyme which have been reported from several insect species.