The molting hormones from the embryonated egg of the tobacco hornworm, Manduca sexta (L.)

26-Hydroxyecdysone is the predominant molting hormone in 24- to 44-hour-old embryonated tobacco hornworm eggs, accounting for approximately 80% of the ecdysones present at this stage of development. This molting hormone was previously shown to be the major ecdysone present in 48- to 64-hour-old embryonated eggs of this insect. During both of these periods of embryonic development in the hornworm 20-hydroxyecdysone is a minor component, in contrast to its presence as the major ecdysone in the hornworm during certain stages of post-embryonic development.     

The ecdysteroids from the tobacco hornworm during pupal-adult development five days after peak titer of molting hormone activity.

Six naturally occurring C27 ecdysteroids were isolated and identified from the tobacco hornworm during pupal-adult development five days after peak titer of molting hormone activity. In order of decreasing quantities the hormones were: 20,26-dihydroxyecdysone, 3-epi-20-hydroxyecdysone, 20hydroxyecdysone, 3-epi-20,26-dihydroxyecdysone, 3-epi-ecdysone, and ecdysone. 20-Hydroxyecdysone, in an earlier study, was the major molting hormone present at peak titer during pupal-adult development. The major ecdysteroid present during embryonic development in this insect, 26-hydroxyecdysone, was not detected. The copresence of all six of these ecdysteroids from a single developmental stage of an insect provides information on the metabolic interrelationships that exist among these steroids and on their possible function(s) in insects. The 3alpha-ecdysteroids were far less active than the 3 beta-epimers in the house fly assay. The significance of epimerization is discussed.     

Metabolism of 26-[14C]hydroxyecdysone 26-phosphate in the tobacco hornworm,Manduca sexta L., to a new ecdysteroid conjugate: 26-[14C]hydroxyecdysone 22-glucoside

Following injection into female Manduca sexta pupae, [¹⁴C]cholesterol is converted to a radiolabeled C₂₁ nonecdysteroid conjugate as well as ecdysteroid conjugates, which in ovaries and newly-laid eggs consist mainly of labeled 26-hydroxyecdysone 26-phosphate. During embryogenesis, as the level of 26-hydroxyecdysone 26-phosphate decreases there is a concurrent increase in the amount of a new, labeled ecdysteroid conjugate. This conjugate, which is the major ecdysteroid conjugate (9.4 μg/g) in 0- to 1-hour-old larvae was identified as 26-hydroxyecdysone 22-glucoside by nuclear magnetic resonance and chemical ionization mass spectrometry. This is the first ecdysteroid glucoside to be identified from an insect. The disappearance of 26-hydroxyecdysone 26-phosphate in 0- to 1-hour-old larvae indicates that the 26-hydroxyecdysone 22-glucoside is derived from 26-hydroxyecdysone 26-phosphate. 3-Epi-26-hydroxyecdysone was the major free ecdysteroid isolated from these larvae and 3-epi-20,26-dihydroxyecdysone was the next most abundant ecdysteroid isolated. Interestingly, the 0- to 1-hour-old larvae contained the highest levels of 3α-ecdysteroids per gram of insect tissue (8.7 μg/g) to be isolated from an insect, yet there was a complete absence of the corresponding free 3β-epimers. The ecdysteroid conjugate profiles of ovaries and 0- to 1-hour-old larvae are discussed. Methodology is presented that permits the efficient separation of free and conjugated ecdysteroids and nonecdysteroid conjugates (C₂₁-steroid conjugates).     

Profile of free and conjugated ecdysteroids and ecdysteroid acids during embryonic development of Manduca sexta (L.) following maternal incorporation of [14C]cholesterol

The levels of both free and conjugated ecdysteroids, maternally labeled from [¹⁴C]cholesterol, of six different age groups of Manduca sexta eggs were quantitatively determined. Eggs 0–1-h old contain about 2.5 and 35 μ/g of the 2- and 26-phosphates of 26-hydroxyecdysone, respectively, and 1 μg/g of 26-hydroxyecdysone. During embryogenesis of 26-hydroxyedcdysone 26-phosphate is hydrolyzed to 26-hydroxyecdysone, which reaches a peak titer in 1–18-h-old eggs; the level of 26-hydroxyecdysone 2-phosphate remains rather constant. Additionally, other metabolic modifications such as hydroxylation, conjugation, epimerization, and oxidation are occurring; and as the level of the 26-hydroxyecdysone 26-phosphate decreases there is a progression of other ecdysteroids. C-20 hydroxylation first appears in 24–40-h-old eggs and reaches peak activity in 48–64-h-old eggs, where 20-hydroxyecdysone and 20, 26-dihydroxyecdysone are both present at peak titer but the latter is the major free ecdysteroid. Ecdysone is observed at measurable levels only in the three age groups of eggs between 1 and 64 h-old. C-3 epimerase activity also appears at 24–40 h and continually increases throughout embryogenesis to the point that 3-epi-26-hydroxyecdysone and 3-epi-20, 26-dihydroxyecdysone are the major free ecdysteroids in 96-h-old eggs. A new ecdysteroid conjugate, 26-hydroxyecdysone 22-glucoside, first appears at 24–40h and becomes the major conjugate in 72–80-h-old eggs; it represents an apparent end-product as its peak titer is reached and maintained throughout the final embryonic stages. 20-Hydroxyecdysonoic acid occurs in 48–64-h-old eggs, and along with 3-epi-20-hydroxyecdysonoic and ecdysonoic acids in 72–88-h-old eggs. 20-Hydroxyecdysonoic acid peaks during the latter time interval, and as its titer subsequently falls, there is a concurrent increase in the level of 3-epi-20-hydroxyecdysonoic which was identified as the second major component of the ecdysteroid conjugate fraction of 0–1-h-old larvae. Our results indicate that there is little or no biosynthesis of ecdysteroids during embryogenesis; that the materal ecdysteroid conjugate 26-hydroxyecdysone 26-phosphate serves as source for 26-hydroxyecdysone and the numerous metabolites; that 26-hydroxyecdysone and 20,26-dihydroxyecdysone may be the active hormones during embryonic development; and that glucosylation, epimerization, and formation of acids cosntitute inactivation processes. A scheme of the proposed pathways involved in the metabolism of 26–hydroxyecdysone 26-phosphate in the developing eggs of m. sexta is presented.     

Qualitative and quantitative analyses of juvenile hormone and ecdysteroids from the egg to the pupal molt in Trichoplusia ni

A method was developed to determine in the same extract juvenile hormone and various types of ecdysteroids in precisely staged eggs and larvae of Trichoplusia ni. Ecdysteroids were tentatively identified on the basis of their retention time in ion suppression reversed-phase HPLC and their cross-reactivity with two relatively non-specific, complimentary antibodies, whereas juvenile hormone was identified using reversed-phase HPLC combined with Galleria bioassay. Freshly laid eggs contained low levels of immunoreactive ecdysteroids. Mid-polar ecdysteroids increased in the phase of segmentation (14-18 h) and 1st larval cuticle formation (36-44 h), when 20-hydroxyecdysone and 20,26-dihydroxyecdysone were found to be predominant. Only traces of ecdysone and 26-hydroxyecdysone were seen. Toward hatching ecdysteroids decreased and represented mainly compounds more polar than 20,26-dihydroxyecdysone. In larval development ecdysteroids were low at the beginning of the feeding phases, increased toward cessation of feeding, and reached highest levels 12-15 h before ecdysis. In feeding stages ecdysone and 20-hydroxyecdysone were predominant, whereas in molting stages they were seen together with 20,26-dihydroxyecdysone and 20-hydroxyecdysonoic acid. The juvenile hormone titer was very low in freshly laid eggs and was high (approximately 25 ng/g) in embryos at the stage of 1st larval cuticle formation and eye pigmentation. In eggs we tentatively identified juvenile hormones I and II, whereas in larval stages juvenile hormone II appeared to be the predominant or exclusive juvenile hormone. Its titer fluctuated rapidly and was high in early 1st-instar larvae and again before the molts into the 3rd, 4th, and 5th instar. Highest titers were reached concomitant with the peak in 20-hydroxyecdysone 12-15 h before ecdysis.     

Endocrine interrelationship between the parasitoid Chelonus sp. and its host Trichoplusia ni

The egg-larval parasitoid Chelonus sp. induces the precocious onset of metamorphosis in the 4th (penultimate) stadium of its host Trichoplusia ni, emerges from the prepupa, and then feeds on it. Qualitative and quantitative changes in ecdysteroids and juvenile hormone were measured. Hemolymph of 3rd-to 4th-instar host larvae and the parasitoids they contained, as well as nonparasitized and parasitized eggs, were analyzed. In the host hemolymph a broad peak of ecdysteroids during molting into the 4th stadium and a continuous increase from day 2 (onset of precocious wandering) until day 4 (emergence of parasitoid) were observed; 20-hydroxyecdysone and 20,26-dihydroxyecdysone were predominant. The juvenile hormone titer fluctuated in the 3rd and early 4th stadium and fell to undetectable levels shortly before the precocious onset of wandering. The parasitoid's ecdysteroids started to increase on the molt to the 2nd instar (= early 4th instar of the host) and thereafter fluctuated on a high level, 20-hydroxyecdysone, 20,26-dihydroxy-ecdysone, and ecdysone being predominant. The juvenile hormone titer was high in late 1st-instar parasitoids, decreased to low levels at ecdysis into the 2nd instar, and increased again to high levels in the 2nd-instar larvae at the time when their shape changed from flat to cylindrical. After ecdysis to the 3rd instar the juvenile hormone titer fell. A comparison revealed that both ecdysteroids and juvenile hormone fluctuate independently in parasitoid and host at most stages, suggesting that the parasitoid produces its own hormones. The first data on ecdysteroids and juvenile hormones in the egg stage of a parasitoid/host system are reported. At the stage of eye pigmentation parasitized eggs contained more immunoreactive midpolar ecdysteroids than non-parasitized ones. 20-Hydroxyecdysone and 20,26-dihydroxyecdysone were the predominant ecdysteroids in both nonparasitized and parasitized eggs, but the latter contained several additional ecdysteroids which were not seen in nonparasitized eggs. The titer of juvenile hormone was similar in both. Shortly before hatching the ecdysteroids were low in parasitized and nonparasitized eggs, but the content of juvenile hormone was much higher in the former. At this stage the majority of parasitoids have already eclosed and teratocytes are released. The results of HPLC analysis indicated the presence of juvenile hormone III together with juvenile hormones I and II in parasitized eggs, but only juvenile hormones I and II in nonparasitized eggs.     

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.     

Metabolism of [14C]cholesterol in Manduca sexta pupae: Isolation and identification of sterol sulfates,free ecdysteroids,and ecdysteroid acids

[¹⁴C]Cholesterol was injected into fifth-instar larvae of Manduca sexta, and the metabolites were isolated and identified from 8-day-old male and female pupae. A major portion of the metabolized cholesterol was esterified either with a sulfate group or with fatty acids. The predominant ecdysteroid metabolites were 20-hydroxyecdysone, 20,26-dihydroxyecdysone, 20-hydroxyecdysonoic acid, and 3-epi-20-hydroxyecdysonoic acid. Smaller amounts of ecdysteroids were identified as conjugates of 26-hydroxyecdysone, 3-epi-20-hydroxyecdysone, 20,26-dihydroxyecdysone, and its 3α-epimer. The metabolic profiles were similar for both male and female pupae. The two ecdysteroid acids were identified by nuclear magnetic resonance spectroscopy and chemical ionization mass spectrometry and by mass spectral analyses of their methyl esters. Detection of 3-epi-20-hydroxyecdysonoic acid as a major metabolite is significant, as its occurrence has been scarcely reported. 3-Epiecdysteroid acid formation is discussed as a possible ecdysteroid-inactivating pathway that may be operating specifically in lepidopterous insects or in particular developmental stages such as eggs or pupae.     

Metabolism of ecdysteroid by testes of the tobacco budworm,Heliothis virescens

Testes from late last stage larvae of the tobacco budworm, Heliothis virescens, were incubated with [³H]ecdysone and [³H]cholesterol. [³H]Ecdysone was converted to six other major ecdysteroids, identified by cochromatography in reverse-phase high-pressure liquid chromatography (RPHPLC); four of them were verified by normal-phase HPLC. A highly polar fraction, moderately polar ecdysteroids (20,26-dihydroxyecdysone, 3-epi-20-hydroxyecdysone, and 20-hydroxyecdysone) and low-polarity ecdysteroids, including 2-deoxyecdysone, were detected after incubation with [³H]ecdysone. Compounds that reacted positively to antibodies to progesterone and testosterone were detected in the low-polarity fractions. Testes were incubated in fractions corresponding to each of the major ecdysteroid peaks derived from [³H]ecdysone metabolism. Although most of the radioactive ecdysteroid fractions were further metabolized to high- and low-polarity endpoints, 88% of the [³H]20-hydroxyecdysone peak apparently remained unmetabolized. 20-Hydroxyecdysone may be the primary ecdysteroid product of testes of H. virescens. [³H]Cholesterol was not metabolized to any appreciable extent.     

Ecdysteroids in developing ovaries and eggs of the tobacco hornworm

Ecdysteroids of ovaries and newly-laid eggs (0- to 1-hour-old) of the tobacco hornworm are present mainly as conjugates (greater than 95%). Newly-laid eggs contain ecdysteroid conjugates equivalent to 21 micrograms of 26-hydroxyecdysone and 0.73 micrograms of ecdysone per gram of eggs. These levels are similar in ovaries of 93-hour-old adult females. In 1- to 18-hour-old eggs more than 63% of the ecdysteroids exist in the free form and the proportion is similar in 48- to 64-hour-old eggs. The ratio of 26-hydroxyecdysone to ecdysone in the conjugated form remains constant during oocyte maturation and embryogenesis. Though 26-hydroxyecdysone is without molting hormone activity in the house fly assay, the exceptionally high concentration of 26-hydroxyecdysone conjugate(s) in ovaries and newly-laid eggs, together with the fact that it is being released during embryogenesis, indicate some physiological role for 26-hydroxyecdysone.     

Metabolism of [3H]-ecdysone in Schistocerca gregaria; formation of ecdysteroid acids together with free and phosphorylated ecdysteroid acetates

The metabolism of [³H]-ecdysone has been investigated at times of low and high endogenous ecdysteroid tit re, in early and late fifth-instar Schistocerca gregaria larvae, respectively. Ecdysone-3-acetate, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone were identified as metabolites in both the free form and as polar conjugates. Comparison of the intact polar conjugates of the ecdysteroid acetates on two HPLC systems with the corresponding authentic compounds indicated that they were 3-acetylecdysone-2-phosphate and 3-acetyl-20-hydroxyecdysone-2-phosphate. Other major polar metabolites were identified as ecdysonoic acid and 20-hydroxyecdysonoic acid. Ecdysone metabolism in fifth-instar S. gregaria is apparently an age-dependent process. Early in the instar, excretion of both free and conjugated ecdysteroids, as well as ecdysteroid 26-acids, occurs. At this stage the level of ecdysteroid acetates in the conjugated (phosphate) form is high, in contrast to the free ecdysteroids, where ecdysone predominates. When the endogenous hormone titre is high, the formation of ecdysteroid acetates is less, the major excreted matabolites at that stage being conjugated 20-hydroxyecdysone together with ecdysteroid-26-acids, but little free ecdysteroids. Acetylation of ecdysone occurs primarily in the gastric caecae. Ecdysone-3-acetate (mainly as polar conjugate) is also a major product of ingested ecdysone in early fifth-instar Locusta migratoria.     

Identification of the molting hormone of the sweet potato (Bemisia tabaci) and greenhouse (Trialeurodes vaporariorum) whitefly

In order to identify the whitefly molting hormone, whole body extracts of mature 4th instar and newly formed pharate adult Bemisia tabaci (Biotype B) and Trialeurodes vaporariorum were prepared and subjected to reverse phase high performance liquid chromatography (RPHPLC). Ecdysteroid content of fractions was determined by enzymeimmunoassay (EIA). The only detectable ecdysteroids that were present in significant amounts in whitefly extracts were ecdysone and 20-hydroxyecdysone. The concentrations of 20-hydroxyecdysone in B. tabaci and T. vaporariorum extracts, respectively, were 40 and 15 times greater than the concentrations of ecdysone. The identity of the two ecdysteroids was confirmed by normal phase high performance liquid chromatography (NPHPLC). When ecdysteroid content of RPHPLC fractions was assayed by radioimmunoassay (RIA), small amounts of polar ecdysteroids were also detected indicating that these ecdysteroids have a very low affinity for the antiserum used in the EIA. Ecdysteroid at 10.4 mM administered by feeding stimulated 2nd instar whitefly nymphs to molt. Based on our results, it appears that 20-hydroxyecdysone is the whitefly molting hormone.     

Conversion of ecdysone and 20-hydroxyecdysone into 3-dehydroecdysteroids is a major pathway in third instar Drosophila melanogaster larvae

Ecdysone and 20-hydroxyecdysone metabolism was investigated in third instar Drosophila larvae both in vivo by injecting radiolabelled ecdysteroids and in vitro by incubating various tissues with labelled ecdysteroids.Ecdysone metabolism proceeds through different pathways: (1) C-20 hydroxylation; (2) C-26 hydroxylation and C-26 oxidation leading to the formation of 26-hydroxyecdysteroids (26-hydroxyecdysone and 20,26-dihydroxyecdysone) and acidic compounds (ecdysonoic acid and 20-hydroxyecdysonoic acid); C-3 oxidation and C-3 epimerization then conjugation leading to the formation of 3-dehydrocompounds (3-dehydroecdysone and 3-dehydro-20-hydroxyecdysone), 3-epimers (3-epiecdysone and 3-epi-20-hydroxyecdysone) and conjugates (only one conjugate was tentatively characterized as 3-epi-20-hydroxyecdysone-3-phosphate). 3-Dehydrocompounds are the major metabolites formed in third instar Drosophila larvae and C-3 oxidation occurs in various tissues. Experiments using tritiated cholesterol provided evidence that 3-dehydroecdysone and 3-dehydro-20-hydroxyecdysone are true endogenous ecdysteroids in Drosophila larvae.     

Ecdysteroids in Carausius eggs during embryonic development

Peaks of ecdysteroids were observed during the different phases of embryonic development of intact Carausius eggs or eggs precociously deprived of their exochorion and cultivated under paraffin oil. Several groups of ecdysteroids were separated and analyzed by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) combined with radioimmunoassay. Ecdysteroids were similar in the two categories of eggs, including high-polarity products (essentially conjugates hydrolyzable by Helix pomatia digestive juice, or alkaline phosphatase), possible ecdysonoic acids (unhydrolyzable polar substances), free hormones, and nonpolar ecdysteroids. Four ecdysteroids were identified by co-elution during HPLC with reference compounds of 20,26-dihydroxyecdysone, 20-hydroxyecdysone, ecdysone, and 2-deoxy-20-hydroxyecdysone. Concentrations of these substances (free and conjugated forms) were studied during the different stages of embryonic development: 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone were the major free ecdysteroids. They showed parallel variations with large peaks at stages VI₈ and VII₆ whereas ecdysone titers were consistently low. Injected labelled ecdysone was converted efficiently into 20-hydroxyecdysone, and both compounds underwent 26-hydroxylation and/or conjugation to polar or apolar metabolites.     

Ecdysteroid binding sites in gastrolith forming tissue and stomach during the molting cycle of crayfish Procambarus clarkii.

The distribution of ecdysteroid binding sites in the stomach and gastrolith disc tissue of crayfish (Procambarus clarkii) was examined in relation to the molting stage by thaw-mount autoradiography. The radiolabeled hormone analogue ponasterone A (25-deoxy-20-hydroxyecdysone) was used. Ecdysteroid binding sites were demonstrated only in certain molting stages, the small gastrolith period and the aftermolt stage. In gastrolith epithelium, ponasterone A binding sites first appeared in the cytoplasm, and then in the nuclei and cytoplasm. In the stomach epithelium, many nuclear binding sites were detectable during the period of gastrolith secretion. These periodical changes in specific ponasterone A binding when correlated with the molting stages clearly show that ecdysteroids may function as an initiator for gastrolith formation and reabsorption. The findings also suggest that ecdysteroids control calcium transport in the stomach epithelium. The time-related and functional differences of cytoplasmic and nuclear concentration of ecdysteroid receptors indicate the presence of cytoplasmic and nuclear receptors associated with specific actions.     

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.     

Developmental Changes of Ecdysteroids in the Eggs of the Silkworm, Bombyx mori

Ecdysteroids were studied in relation to embryonic development and diapause of the silkworm, Bombyx mori . The majority of the ecdysteroids was found to be in the conjugated form, and minor part, in the free form. In the developing eggs, 2-deoxyecdysone, 2-deoxy-20-hydroxy-ecdysone and 3-epi-ecdysone were found to have the free ecdysteroid form as well as the conjugated ecdysteroid form. Ecdysone and 20-hydroxyecdysone almost exclusively had the conjugated form. The concentration of ecdysteroids in the embryo was higher than that in yolkplasm in the early embryonic stages. During the embryonic diapause, the concentration of free ecdysteroids decreased to a low level while the conjugated form maintained the original level.     

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).     

The effect of glucose on the activity of phosphofructokinase in the mucosa of rat small intestine.

Maturing eggs of the desert locust, Schistocerca gregaria, contain a variety of ecdysteroid (insect moulting hormone) conjugates and metabolites, four of which have been previously isolated from polar extracts and identified as ecdysonoic acid, 20-hydroxyecdysonoic acid, 3-acetylecdysone 2-phosphate and ecdysone 2-phosphate. In the present study we have isolated eight additional ecdysteroids from similar late-stage eggs by high-performance liquid chromatography. The 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone, all of which were first identified as ecdysteroid components of newly-laid eggs of S. gregaria, were identified by co-chromatography with authentic compounds and by physicochemical techniques. The remaining compounds were identified as 3-acetyl-20-hydroxyecdysone 2-phosphate, 3-epi-2-deoxyecdysone 3-phosphate, 3-acetylecdysone 22-phosphate and 2-acetylecdysone 22-phosphate by fast atom bombardment mass spectrometry, p.m.r. spectroscopy and analysis of the steroid moieties after enzymic hydrolysis. The latter two compounds, after isolation, are susceptible to nonenzymic acetyl migration and deacetylation to give mixtures of ecdysone 22-phosphate and its 2- and 3-acetate derivatives. The possible role and significance of these ecdysteroid conjugates with respect to the control of hormone titres in insect eggs is discussed.