Occurrence of 28- and 27-Carbon ecdysteroids and sterols in developing worker pupae of the leaf-cutting ant acromyrmex octospinosus (reich) (hymenoptera,formicidae: Attini)

The major ecdysteroids in large worker pupae of the leaf-cutting ant Acromyrmex octospinosus were characterized at the peak ecdysteroid concentration by using high-performance liquid chromatography, enzyme immunoassay, and mass spectrometry. In decreasing amounts, they were determined to be makisterone A, an unidentified C₂₈ ecdysteroid bearing a molecular weight of 494, 20-hydroxyecdysone (ratio of 1 to 6 as compared to makisterone A), and putative but negligible ecdysone. The presence of both C₂₈ and C₂₇ ecdysteroids is discussed in relation to the content of 4-desmethylsterols determined by gas chromatography and mass spectrometry to be ergosta-5,7,24 (28)-trien-3β-ol, ergosterol, ergosta-5,7-dien-3β-ol and ergosta-7,24(28)-dien-3β-ol for the main sterols, and with a small amount of cholesterol.     

The neutral sterols of Megalotomus quinquespinosus (say) (hemiptera: Alydidae) and identification of makisterone a as the major free ecdysteroid

Last-stage nymphs of the broad-headed bug, Megalotomus quinquespinosus contain the C₂₈ ecdysteroid makisterone A as their major ecdysteroid. No ecdysone or 20-hydroxyecdysone was detected in whole body extracts analyzed by high performance liquid chromatography and radioimmune assay. Analyses of the neutral sterols of this phytophagous hemipteran revealed that the sterol composition of the nymphs was highly reflective of their dietary sterols. The most abundant nymphal sterols were sitosterol (46.6%), Δ⁷-stigmastenol (13.8%) and spinasterol (13.4%). Cholesterol accounted for only 0.2% of the total sterols and indicates that this species is incapable of converting 24-alkyl sterols to cholesterol.     

Makisterone C: A 29-carbon ecdysteroid from developing embryos of the cotton stainer bug,Dysdercus fasciatus

An ecdysteroid RIA was used to determine the ecdysteroid titer in developing embryos of Dysdercus fasciatus and revealed that peak titer occurred approximately 120 h post-oviposition. Analysis of neutral sterols at this time indicated sitosterol to be the predominant neutral sterol with lesser amounts of campesterol. Embryonic sterols were highly reflective of the sterols found in the cotton seed diet upon which previous generations of the bugs had fed. Analysis of the embryonic extract for ecdysteroids indicated the presence of both makisterone A and the 29-carbon ecdysteroid makisterone C. Isolation of these compounds was accomplished by reversed-phase and silica HPLC in conjunction with RIA, and the identification of both compounds was confirmed by mass spectrometry. No ecdysone or 20-hydroxyecdysone was detected in the embryonic sample.     

Biosynthesis of makisterone A and 20-hydroxyecdysone from labeled sterols by the honey bee,Apis mellifera

In an effort to determine the sterol precursor(s) of the 28-carbon ecdysteroid, makisterone A, honey bee pupae (13 days post-oviposition) were injected with radiolabeled sterols and subsequently examined for labeled ecdysteroids. High performance liquid chromatography of the pupal extracts revealed that [³H]campesterol was converted to a compound that behaved chromatographically identical to authentic makisterone A, and [¹⁴C]cholesterol was incorporated into a compound chromatographically like 20-hydroxyecdysone. No incorporation of either 24-[³H]methylenecholesterol or [¹⁴C]sitosterol into an ecdysteroid was observed. The neutral sterols of uninjected honey bee pupae contained 49.8% 24-methylenecholesterol on a relative percent basis and, with three other C²⁸ and C²⁹ sterols, accounted for over 99% of the total sterols present.     

Relationship of the neutral sterols and ecdysteroids of the parasitic mite,Varroa jacobsoni to those of the honey bee,Apis mellifera

The neutral sterols of the parasitic mite Varroa jacobsoni were compared with Apis mellifera carnica drone pupae. Analysis by GLC-mass spectrometry indicated mite sterols were reflective of the sterol composition of the drones; 24-methylenecholesterol was the major sterol in both species, with lesser amounts of sitosterol and isofucosterol. Cholesterol accounted for less than 1% of the total sterols. Ecdysteroid analyses indicated drones contained primarily makisterone A. In addition to makisterone A, mites contained ecdysone and 20-hydroxyecdysone, which accounted for over 66% of the ecdysteroid detected. These results indicate that while V. jacobsoni are apparently unable to convert dietary sterols to cholesterol, they are able to produce significant amount of C(27) ecdysteroids in a low cholesterol environment.     

Absence of phytosterol dealkylation and identification of the major ecdysteroid as makisterone A in Dysdercus fasciatus (Heteroptera,Pyrrhocoridae)

The absence of phytosterol dealkylation in the cotton stainer bug, Dysdercus fasciatus, has been established and the major ecdysteroid in the fifth-stage larvae identified. The demonstration that the free and esterified sterols in D fasciatus consisted of 95–96% sitosterol and 4–5% campesterol, a similar composition to the cottonseed diet, together with the lack of conversion of [¹⁴C]sitosterol into cholesterol, establishes that phytosterol dealkylation does not occur in this insect species. The ecdysteroid titer determined by radioimmunoassay in the fifth instar of D fasciatus shows a distinct peak at day 6, the instar lasting for 7 days. Makisterone A was purified by HPLC from insects at a time of high ecdysteroid titer and identified as a major component by both fast atom bombardment and electron impact mass spectrometry. Gas-liquid chromatography/mass spectrometry (selected ion monitoring) confirmed the occurrence of makisterone A and revealed the presence of two unidentified compounds. One of these occurs in a similar amount to makisterone A and may be 26-hydroxymakisterone A, whereas only a minute amount of the other compound, which may be 20-deoxymakisterone A, was present; further identification of the latter compounds is necessary. C₂₇ ecdysteroids (eg, ecdysone and 20-hydroxyecdysone) and C₂₉ ecdysteroids (eg, podecdysone A) were undetectable. The specificity of the enzymes of ecdysteroid biosynthesis is discussed.     

Neutral sterols of representatives of two groups of hemiptera and their correlation to ecdysteroid content

The sterols of four species of Pentatomomorpha—Oncopeltus fasciatus (Dallas), Nezara viridula (L.), Dysdercus cingulatus (F.), and Podisus maculiventris (Say)—and threé species of Cimicomorpha—Rhodnius prolixus Stal, Arilus cristatus (L), and Cimex lectularius (L.)—were isolated and examined in order to compare neutral sterol utilization and content with the known ecdysteroids of these species. In the phytophagous Pentatomomorpha, O. fasciatus, N. viridula, and D. cingulatus, the low content of cholesterol and the high content of C₂₈ and C₂₉ phytosterols (< 1% and > 99% of the tissue sterols, respectively) indicated that these species are unable to dealkylate the C-24 position of sterols. These insects appear to have adapted to the challenge of both insufficient dietary cholesterol and inability to dealkylate phytosterols by evolving the ability to produce a C₂₈ ecdysteroid (makisterone A). The secondarily predacious P. maculiventris has adequate cholesterol available for C₂₇ ecdysteroid production, but appears to have retained the ecdysteroid biosynthetic pathways of its phytophagous ancestors because it produces a C₂₈ ecdysteroid. Cholesterol was the major sterol of each of the three species of Cimicomorpha, and these insects are only able to produce C₂₇ ecdysteroids.     

Makisterone A: The major ecdysteroid from the pupa of the honey bee,Apis mellifera

Makisterone $\text{A}$, a 28-carbon moulting hormone, has been identified as the major free pupal ecdysteroid in the honey bee, Apis mellifera. The pupal ecdysteroid was isolated and identified by normal and reversed-phase high performance liquid chromatography in conjunction with a radioimmune assay. The compound was further characterized physico-chemically by both mass spectrometry and nuclear magnetic resonance spectroscopy. No C₂₇ ecdysteroids (i.e. 20-hydroxyecdysone or ecdysone) were detected at this stage of development. This is the first isolation and identification of a 28-carbon ecdysteroid in an insect species from the order Hymenoptera. Utilization of dietary sterols by honey bees is also discussed.     

A new ecdysteroid, 2-deoxy-5beta,20-dihydroxyecdysone from the fruits of Diploclisia glaucescens

Chemical investigation of ethyl acetate extract of the fruits of Diploclisia glaucescens of the family Menispermaceae furnished a new ecdysteroid 2-deoxy-5beta,20-dihydroxyecdysone, together with 20-hydroxyecdysone, 3-deoxy-1beta,20-dihydroxyecdysone, 2-deoxy-20-hydroxyecdysone, 24-ethyl-20-hydroxyecdysone (makisterone C). Latter two ecdysteroids are reported first time from the family Menispermaceae.     

Ecdysone metabolism in Pieris brassicae during the feeding last larval instar

Ecdysone metabolism in Pieris brassicae during the feeding last larval stage was investigated by using ³H-labeled ecdysteroid injections followed by high-performance liquid chromatographic (HPLC

1 Abbreviations: 3DE = 3-dehydroecdysone; 3D20E = 3-dehydro-20-hydroxyecdysone; 2026E = 20,26-dihydroxyecdysone; E = ecdysone; Eoic = ecdysonoic acid; 2026E′ = 3-epi-20,26-dihydroxyecdysone; E′ = 3-epiecdysone; E′oic = 3-epiecdysonoic acid; E′8P = 3-epiecdysone 3-phosphate; 20E′ = 3-epi-20-hydroxyecdysone; 20E′3P = 3-epi-20-hydroxyecdysone 3-phosphate; FT = Fourier transform; HPLC = high-performance liquid chromatography; 20E = 20-hydroxyecdysone; 20Eoic = 20-hydroxyecdysonoic acid; NMR = nuclear magnetic resonance; NP-HPLC = normal phase HPLC; RP-HPLC = reverse phase HPLC; TFA = trifluoroacetic acid; Tris = tris(hydroxymethyl)-aminomethane.

) analysis of metabolites. Metabolites were generally identified by comigration with available references in different HPLC systems. Analysis of compounds for which no reference was available required a large-scale preparation and purification for their identification by ¹H nuclear magnetic resonance spectrometry. The metabolic reactions affect the ecdysone molecule at C-3, C-20, and C-26, leading to molecules which are modified at one, two, or three of these positions. At C-20, hydroxylation leads to 20-hydroxyecdysteroids. At C-26, hydroxylation leads to 26-hydroxyecdysteroids which can be further converted into 26-oic derivatives (ecdysonoic acids) by oxidation. At C-3, there are several possibilities: there may be oxidation into 3-dehydroecdysteroids, or epimerization possibly followed by phosphate conjugation. Thus, injected 20-hydroxyecdysone was converted principally into 20-hydroxyecdysonoic acid, 3-dehydro-20-hydroxyecdysone, and 3-epi-20-hydroxyecdysone 3-phosphate. Labelled ecdysone mainly gave the same metabolites doubled by a homologous series lacking the 20-hydroxyl group.     

Larval moulting hormone of trichophoran Hemiptera-Heteroptera: Makisterone A,not 20-hydroxyecdysone

The haemolymph ecdysteroids were examined in fifth-stage larvae of Nezara viridula, Podisus maculiventris and Dysdercus cingulatus (Hemiptera-Heteroptera) using high-pressure liquid chromatography to separate the ecdysteroids and a radioimmunoassay to detect the fractionated ecdysteroids. The length of the fifth stage ranged from 5 to 8 days, and a peak in ecdysteroid titre (1700–2650 ng/ml) occurred 2–3 days prior to ecdysis to the adult. An ecdysteroid matching the retention time of makisterone A (24-methyl-20-hydroxyecdysone) was clearly present in haemolymph taken at the time of peak titre in all 3 of these true bugs, whereas little, if any, ecdysone or 20-hydroxyecdysone was detected. These data, along with previously reported data for the milkweed bug Oncopeltus fasciatus, are persuasive evidence that makisterone A is the larval moulting hormone of a group of closely related Heteroptera called the Trichophora (Lygaeoida, Pentatomoidea, Pyrrhocoroidea and Coreoidea).     

Further investigations on the action of ecdysteroids on the salivary glands of the female tickAmblyomma americanum

Two phytecdysteroids (abutasterone, makisterone A) and five synthetic ecdysteroid analogues, all at 1 g/ml, were tested on salivary glands from the female tick,Amblyomma americanum L. (Acari:Ixodiae), held in organ culture for four days. All of these substances caused a significant reduction in fluid secretory competence of salivary glands in vitro. This constitutes further evidence that the structural requirements for causing salivary-gland degeneration in ticks are similar to those generally required for ecdysteroid activity in other arthropods. Although vertebrate steroids are known to augment fluid secretory competence by salivary glands in organ culture, -estradiol was not able to attenuate the degenerating effect of 20-hydroxyecdysone, supporting the suggestion that ecdysteroids and vertebrate steroids have distinct sites/mechanisms of action on this tissue.     

Sterol utilization and ecdysteroid content in the house fly,Musca domestica (L.)

Larvae of the house fly, Musca domestica were reared aseptically on diets which contained either cholesterol, campesterol or sitosterol as the dietary sterol at a concentration of 0.1% dry wt. Analysis of puraria (24 h post-pupariation) reared on campesterol or sitosterol diets revealed they contained from 2.7 to 4.6% cholesterol, indicating an ability to accumulate this sterol even where it is present in only minute quantities. Purparia on all diets produced the 27-carbon molting hormones, ecdysone and 20-hydroxyecdysone. When the concentration of campesterol was increased to 0.2% dry wt, puparia also contained the 28-carbon ecdysteroid, makisterone A, although it accounted for only 20.7% of the total ecdysteroid produced.     

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).     

Chitin synthesis in Spodoptera frugiperda wing imaginal discs. III: Role of the peripodial membrane

We determined the contribution of the peripodial membrane to chitin synthesis in cultured wing imaginal discs of Spodoptera frugiperda. This was accomplished by examining chitin synthesis in vitro in intact imaginal discs, in the peripodial membrane, and in imaginal discs in which the peripodial membrane had been injured. Chitin synthesis in peripodial membrane-deprived imaginal discs, peripodial membrane injured imaginal discs, and peripodial membrane fragments was assessed by measuring incorporation of [¹⁴C]GlcNAc after treatment with 20-hydroxyecdysone in tissue culture. Removing or injuring the peripodial membrane resulted in a marked decrease in ecdysteroid-dependent chitin synthesis in these wing discs compared with intact wing discs. In addition, a break in the ecdysteroid treatment of 4 h reduced chitin synthesis in the wing discs substantially. These biochemical experiments were supplemented with ultrastructural and immunocytochemical approaches. A wheat germ agglutinin colloidal gold complex was used to visualize the presence of chitin synthesized by wing discs including the peripodial membrane. These experiments confirmed the importance of an intact peripodial membrane for optimal production of cuticle by the wing pouch. Our results demonstrate that for opti-ma1 production of chitin in tissue culture, wing discs must be treated with 20-hydroxyecdysone for an uninterrupted period of 48 h, and the peripodial membrane of these imaginal discs must be present and uninjured. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Sterol composition of steryl chlorin esters (SCEs) formed through grazing of algae by freshwater crustaceans: relevance to the composition of sedimentary SCEs

In a laboratory experiment, we studied the composition of sterols in steryl chlorin esters (SCEs) egested in fecal pellets of freshwater crustaceans (Daphnia magna and Asellus hilgendorfi) fed on a single green algae (Chlorella, Scendesmus, or Stigeoclonium) or on phytoplankton collected from a shallow pond abundant in diatoms. Both unaltered sterols present in dietary phytoplankton and sterols formed by metabolism in crustaceans were incorporated in the SCEs. C₂₇ sterols except for cholesterol (C₂₇⁵) and C₂₈ sterols, major sterols in diatoms, were scarce in the SCEs compared with those in the dietary algae, whereas cholesterol, which could be formed by crustacean metabolism, was relatively abundant in the SCEs. Therefore, the contribution of diatoms to the total phytoplankton population would be underestimated if diatom-specific C₂₇ and C₂₈ sterols in sedimentary SCEs were used in estimations as biomarkers of diatoms.     

Temporal patterns of protein synthesis inManduca epidermis during the change to pupal commitment in vitro: their modulation by 20-hydroxyecdysone and juvenile hormone

Summary The epidermis of final instar tobacco hornworm larvae,Manduca sexta, becomes committed to pupal differentiation in response to ecdysteroid in the absence of juvenile hormone (JH). Many changes in protein synthetic patterns have been noted during this time (Kiely and Riddiford 1985). To determine which of these changes are caused by ecdysteroid and which are important for the change of commitment, we have incubated larvally-committed epidermis for 24 h with 1 g/ml 20-hydroxyecdysone (20HE) and 3 g/ml epoxygeranylsesamole (EGS) (a JH mimic), with 3 g/ml EGS alone, or in hormone-free medium. Synthesis of larval-specific proteins such as insecticyanin and larval cuticular proteins was reduced to trace amounts or was undetectable after culture with 20HE for 24 h. The larval cuticular proteins that are greatly increasedin vivo on day 3 were not synthesized after exposure to 20HEin vitro. Ecdysteroid increased the synthesis of many of the proteins first seenin vivo on day 3 or during the wandering stage. The synthesis of about half of these latter proteins was inhibited by JH, indicating that they were likely part of the change of commitment. Other proteins that appear at this stagein vivo showed increased synthesis also in hormone-free medium and therefore were independent of the change of commitment.     

STEROLS OF 14 SPECIES OF MARINE DIATOMS (BACILLARIOPHYTA)1

The sterol compositions of 14 species of marine diatoms were determined by gas chromatography and gas chromatography-mass spectrometry. A variety of sterol profiles were found. The sterols 24-methylcholesta-5,22E-dien-3β-ol, cholest-5-en-3β-ol, and 24-methylcholesta-5,24(28)-dien-3β-ol, previously described as the most common sterols found in diatoms, were major sterols in only a few of the species. In light of this and other recent data, it is clear that these three sterols are not typical constituents of many diatom species. Most of the centric species examined had 24-methylcholesta-5,24(28)-dien-3β-ol and 24-methylcholest-5-en-3β-ol as two of their major sterols. The exception was Rhizosolenia setigera, which possessed cholesta-5,24-dien-3β-ol as its single major sterol. In contrast to the centric species, the pennate diatoms examined did not have any particular sterols common to most species. Minor levels ofΔ⁷-sterols, rarely found in large amounts in diatoms, were found in four species. C₂₉sterols were found in many species; seven contained 24-ethylcholest-5-en-3β-ol and three contained 24-ethylcholesta-5,22E-dien-3β-ol, reinforcing previous suggestions that C₂₉ sterols are not restricted to higher plants and macroalgae. 24-Ethylcholesta-5,22E-dien-3β-ol may prove to be useful for taxonomy of the genus Amphora and the order Thalassiophysales. A major sterol of Fragilaria pinnata was the uncommon algal sterol 23,24-dimethylcholesta-5,22E-dien-3β-ol. Cholesta-5,24-dien-3β-ol was the only sterol found in the culture of Nitzschia closterium. This differed from previous reports of 24-methylcholesta-5,22E-dien-3β-ol as the single major sterol in N. closterium. Two C₂₈ sterols possessing an unusual side chain were found in Thalassi-onema nitzschioides, a C_28:2 sterol (16%) and a C_28:1 sterol in lower abundance (2.5%), which may be 23-methylcholesta-5,22E-dien-3β-ol and 23-methyl-5α-cholest-22E-en-3β-ol, respectively. The species Cylindrotheca fusiformis, T. nitzschioides, and Skeletonema sp. may be useful as direct sources of cholesterol in mariculture feeds due to their moderate to high content of this sterol.     

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.     

Additional minor ecdysteroid components of Leuzea carthamoides

Seventeen additional minor ecdysteroid compounds were isolated and identified from the roots of Leuzea carthamoides (Wild.) DC. Eight of them are new phytoecdysteroids: carthamoleusterone (13) is a new side-chain cyclo-ether with five-membered ring; 14-epi-ponasterone A 22-glucoside (12) is a rare and unusual natural 14 beta-OH epimer; 15-hydroxyponasterone A (11) is also new and rare with its C-15 substituted position, as well as 22-deoxy-28-hydroxymakisterone C (18) possessing secondary hydroxyl in position C-28 and 26-hydroxymakisterone C (20) with hydroxy groups in positions 25 and 26. New are also 1 beta-hydroxymakisterone C (21) and 20,22-acetonides of inokosterone (8) and integristerone A (10). Series of already known ecdysteroids: ecdysone (1), 20-hydroxyecdysone 2- and 3-acetates (3 and 4), turkesterone (6), inokosterone (7), 24-epi-makisterone A (14), and amarasterone A (22) are reported here as new constituents of L. carthamoides. Seven earlier reported Leuzea ecdysteroids: 20-hydroxyecdysone (2), ajugasterone C (5), integristerone A (9), 24(28)-dehydromakisterone A (15), 24(28)-dehydroamarasterone B (16), (24Z)-29-hydroxy-24(28)-dehydromakisterone C (17) and makisterone C (19) are also included because they are now better characterized.