Ecdysteroid Profile Research Articles

The ecdysteroidome of Drosophila: influence of diet and development.

Ecdysteroids are the hormones regulating development, physiology and fertility in arthropods, which synthesize them exclusively from dietary sterols. But how dietary sterol diversity influences the ecdysteroid profile, how animals ensure the production of desired hormones and whether there are functional differences between different ecdysteroids produced in vivo remains unknown. This is because currently there is no analytical technology for unbiased, comprehensive and quantitative assessment of the full complement of endogenous ecdysteroids. We developed a new LC-MS/MS method to screen the entire chemical space of ecdysteroid-related structures and to quantify known and newly discovered hormones and their catabolites. We quantified the ecdysteroidome in Drosophila melanogaster and investigated how the ecdysteroid profile varies with diet and development. We show that Drosophila can produce four different classes of ecdysteroids, which are obligatorily derived from four types of dietary sterol precursors. Drosophila makes makisterone A from plant sterols and epi-makisterone A from ergosterol, the major yeast sterol. However, they prefer to selectively utilize scarce ergosterol precursors to make a novel hormone 24,28-dehydromakisterone A and trace cholesterol to synthesize 20-hydroxyecdysone. Interestingly, epi-makisterone A supports only larval development, whereas all other ecdysteroids allow full adult development. We suggest that evolutionary pressure against producing epi-C-24 ecdysteroids might explain selective utilization of ergosterol precursors and the puzzling preference for cholesterol.

Changes in ecdysteroid levels and expression patterns of ecdysteroid-responsive factors and neuropeptide hormones during the embryogenesis of the blue crab, Callinectes sapidus

Embryogenesis requires the involvement and coordination of multiple networks of various genes, according to a timeline governing development. Crustacean embryogenesis usually includes the first molt, a process that is known to be positively controlled by ecdysteroids. We determined the amounts of ecdysteroids, as well as other related factors: the ecdysone receptor (CasEcR), the retinoid X receptor (CasRXR), the molt-inhibiting hormone (CasMIH), and crustacean hyperglycemic hormone (CasCHH) during the ovarian and embryonic developments of Callinectes sapidus. In summary, the ovaries at stages 1–4 have expression levels of maternal CasEcR and CasRXR 10–50 times higher than levels seen in embryos at the yolk stage. This large difference in the amount of the these factors in C. sapidus ovaries suggests that these maternal ecdysteroid-responsive factors may be utilized at the initiation of embryogenesis. During embryogenesis, the changes in total ecdysteroids and levels of CasEcR and CasRXR expression are similar to those observed in juvenile molts. The full-length cDNA sequence of the C. sapidus BTB domain protein (CasBTBDP) initially isolated from Y-organ cDNA, contains only Broad-Complex, Tramtrack, and Bric a brac (BTB) domains. The levels of CasBTBDP are kept constant throughout embryogenesis. The expression profiles of CasMIH and CasCHH are similar to the titers of ecdysteroids. However, the timing of their appearance is followed by increases in CasEcRs and CasRXRs, implying that the expressions of these neuropeptides may be influenced by ecdysteroids. Moreover, the ecdysteroid profile during embryogenesis may track directly with the timing of organogenesis of Y-organs and their activity. Our work reports, for first time, the observed expression and changes of ecdysteroid-responsive factors, along with CasCHH and CasMIH, during embryogenesis in the crustacean C. sapidus.

STUDY ON ECDYSTEROID LEVELS AND GENE EXPRESSION OF ENZYMES RELATED TO ECDYSTEROID BIOSYNTHESIS IN THE LARVAL TESTIS OF Spodoptera littoralis

We investigated here the ecdysteroid titers and the expression of six genes coding for known enzymes of the ecdysteroid biosynthesis in the testes of last instar larvae of the pest cotton leafworm, Spodoptera littoralis. We showed that the timing of the ecdysteroid profile was the same in testes and in hemolymph, with a small peak at day 2 and a large one at day 4 after ecdysis. Ecdysone and 20-hydroxyecdysone (20E) were detected in both tissues. 20E was the major ecdysteroid in testes and in hemolymph from day 4. Interestingly, the gene expression of the steroidogenetic enzymes, Neverland, and the five cytochrome P450 enzymes encoded by the Halloween genes was confirmed in the testes, and varied during the instar. However, from the data obtained so far, we cannot conclude that the measured ecdysteroids in the testes result from the activity of the genes under study. Indeed, it is suggested that the ecdysone produced centrally in the prothoracic glands, could have been transformed into 20E in the testes, where Sl-shade is well expressed.

Investigation of ecdysteroid content and composition of Silene repens indigenous in Mongolia and introduced into western Siberia

The ecdysteroid profile was investigated by HPLC, and it has been found that 2-deoxyecdysone, 2-deoxy-20-hydroxyecdysone, polypodine B, 20-hydroxyecdysone, and integristerone A are synthesized in the aerial part. The composition of biologically active substances (BAS) has been investigated, and the presence of triterpene glycosides, alkaloids and flavonoids has been confirmed; tannins have been detected for the first time. It has been shown that the ecological conditions, such as altitude above sea level and humidity, influence the ecdysteroid composition and BAS levels.

The phytoecdysteroid profiles of 7 species of Silene (Caryophyllaceae)

The phytoecdysteroid profiles of extracts of aerial parts of flowering plants of 7 ecdysteroid-containing species in the genus Silene (Caryophyllaceae; S. fridvaldszkyana Hampe, S. gigantea L., S. graminifolia Otth, S. mellifera Boiss. & Reuter, S. repens Patr., S. schmuckeri Wettst., and S. sendtneri Boiss.) have been examined and identified by HPLC and, in the case of two new compounds, by mass spectrometry and NMR. S. frivaldszkyana was found to contain predominantly 20-hydroxyecdysone (20E), with smaller amounts of 2-deoxyecdysone (2dE), 2-deoxy-20-hydroxyecdysone (2d20E), polypodine B (polB), integristerone A (IntA), 26-hydroxypolypodine B (26polB), and 20,26-dihydroxyecdysone (20,26E). Additionally, a new minor ecdysteroid, 26-hydroxyintegristerone A, has been identified from this species. S. gigantea contains 3 major ecdysteroids (2dE, 2d20E, and 20E) and much smaller amounts of intA and 2-deoxy-20-hydroxyecdysone 25-beta-D-glucoside, which is a new ecdysteroid. Ecdysteroids in the other 5 species have been identified by co-chromatography with reference compounds on RP- and NP-HPLC systems. There is considerable variability with regard to ecdysteroid profiles within the genus Silene. The chemotaxonomic value of ecdysteroid profiles within the genus Silene is discussed.

Hemolymph ecdysteroids during the last three molt cycles of the blue crab, Callinectes sapidus: quantitative and qualitative analyses and regulation

The profiles of circulating ecdysteroids during the three molt cycles prior to adulthood were monitored from the juvenile blue crab, Callinectes sapidus. Ecdysteroid patterns are remarkably similar in terms of peak concentrations ranging between 210-330 ng/ml hemolymph. Analysis of hemolymph at late premolt stage revealed six different types of ecdysteroids with ponasterone A (PoA) and 20-OH ecdysone (20-OH E) as the major forms. This ecdysteroid profile was consistent in all three molt cycles. Bilateral eyestalk ablation (EA) is a procedure that removes inhibitory neurohormones including crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) and often results in precocious molting in crustaceans. However, the inhibitory roles of these neuropeptides in vivo have not yet been tested in C. sapidus. We determined the regulatory roles of CHH and MIH in the circulating ecdysteroid from ablated animals through daily injection. A daily administration of purified native CHH and MIH at physiological concentration maintained intermolt levels of ecdysteroids in the EA animals. This suggests that Y organs (YO) require a brief exposure to CHH and MIH in order to maintain the low level of ecdysteroids. Compared to intact animals, the EA crabs did not exhibit the level of peak ecdysteroids, and the major ecdysteroid turned out to be 20-OH E, not PoA. These results further underscore the important actions of MIH and CHH in ecdysteroidogenesis, as they not only inhibit, but also control the composition of output of the YO activity.

Elevated Ecdysteroid Titer and Precocious Molt and Vitellogenesis Induced by Eyestalk Ablation in the Estuarine Crab, Metopograpsus Messor (Brachyura: Decapoda)

Abstract Premolt and vitellogenesis are mutually exclusive events in the wild population of the brachyuran crab, Metopograpsus messor; active vitellogenesis occurs only in intermolt females, at a relatively low ecdysteroid profile, judged from radioimmunoassay. Bilateral eyestalk ablation, however, has resulted in simultaneous precipitation of premolt and ovarian growth in the same crab, thus severing the normal antagonistic programming of these two high-energy demanding processes. This result argues for the necessity of eyestalk principles in maintaining the normal (antagonistic) programming of growth and reproduction in the species. Ovaries of the eyestalk ablated crabs precociously accumulate yolk and the oocytes could attain the size of a fully grown egg within 10 days post-ablation. The eyestalk ablated females have also shown dramatic rise in haemolymph ecdysteroid levels, even surpassing the levels (P < 0.05) of the normal premolt individuals, revealing that ovarian growth can occur under a high ec...

Cell Cultures of Ecdysteroid-Containing Ajuga reptans and Serratula coronata Plants

Cell suspension cultures of Ajuga reptans L. and Serratula coronata L. were derived from long-cultured calluses. Their growth patterns and morphophysiological characteristics indicated that these cells adapted well to grow in suspensions. During the growth cycle of cell suspension, the main ecdysteroid, among others present in plants, was 20-hydroxyecdyson (20E) for both cell lines. The content of 20E in cell suspension of A. reptans was 4–8 times higher than in the intact plant. After long culturing, the ecdysteroid profile in cell suspensions of S. coronata (20E, inokosterone, makisterone A, ecdyson, and a nonidentified metabolite) became similar to that of the intact plant. The ecdysteroids accumulated with a periodicity during subculturing cycle.

Applying fenoxycarb at the penultimate instar triggers an additional ecdysteroid surge and induces perfect extra larval molting in the silkworm

When the juvenile hormone analog fenoxycarb was topically applied to the silkworm Bombyx mori at the beginning of the 3rd or 4th (penultimate) instar, an extra larval molt was induced. The 5th instar period was shortened to about 5 days and the extra 6th instar ranged from 8 to more than 20 days, depending on the dose applied. Starvation before fenoxycarb treatment strongly enhanced the incidence of extra molting up to 100%. When 1 ng was applied in the 4th instar after a 2-day starvation, most larvae underwent an extra molt, metamorphosed to pupae, then to fertile adults. Combining starvation and fenoxycarb application thus induces a perfect extra molt efficiently. In perfect extra molting larvae, profiles of total ecdysteroid titer during the 4th and 5th instars were similar to that during the 4th instar in the control, and the ecdysteroid profile during the extra 6th instar was similar to that during the control 5th (last) instar. At ecdysteroid peaks, 20-hydroxyecdysone (20E) and ecdysone (E), generally regarded as the active molting hormone and its precursor, had similar titers in the 6th instar, whereas E was much less than 20E in the 4th and 5th instars in the extra molting larvae. E was also abundant only in the last larval instar in the control. These results suggest that both 20E and E contents are important for regulation of larval molt and metamorphosis in silkworms and that fenoxycarb triggers the extra molt by inducing an additional larval molt type of ecdysteroid surge before the last larval instar.

Phytoecdysteroid levels and distribution during development in Limnanthes alba Hartw. ex Benth. (Limnanthaceae).

Phytoecdysteroid (PE) production and accumulation in Limnanthes alba Hartw. ex Benth. is associated with flowering. PE content per plant remains fairly constant during the primary growth phase of the plant and only begins to increase significantly above amounts found in the seed once the development of the flower stalk has begun. Both content and concentration increase concomitantly from this point. Distributions in individual plants also associated the highest levels of PE accumulation with the reproductive tissues. This substantiates the association of PE with tissues of greatest fitness value and therefore the hypothesis that they contribute to defence. Analysis of extracts of L. alba tissues by reversed-phase HPLC coupled with ecdysteroid-specific RIA was used to monitor ecdysteroid profiles. RIA-positive peaks co-chromatographing with 20-hydroxyecdysone, ecdysone and ponasterone A were detected and several tissues also contain PE conjugates. Seedmeal of L. alba appears to be a convenient and promising source for the commercial isolation of the potent PE ponasterone A.

Identification and quantitative analysis of the phytoecdysteroids in Silene species (Caryophyllaceae) by high-performance liquid chromatography : Novel ecdysteroids from S. pseudotites

Many species in the genus Silene (Caryophyllaceae) have previously been shown to contain ecdysteroids and this genus is recognised as a good source of novel ecdysteroid analogues. We have used ecdysteroid-specific radioimmunoassays and the microplate-based Drosophila melanogaster B II cell bioassay for ecdysteroid agonist and antagonist activities to identify further phytoecdysteroid-containing species in this genus. The main ecdysteroid components from 10 Silene species ( S. antirrhina, S. chlorifolia, S. cretica, S. disticha, S. echinata, S. italica, S. portensis, S. pseudotites, S. radicosa, S. regia) were isolated and identified, mainly by normal-phase and reversed-phase high-performance liquid chromatography. The amount of each ecdysteroid was determined by comparing chromatogram peak areas with those for reference 20-hydroxyecdysone (20E) on reversed-phase HPLC. 20E is the most abundant ecdysteroid in each of the Silene extracts. Polypodine B, 2-deoxy-20-hydroxyecdysone and ecdysone are also common ecdysteroids in these Silene species, but the proportions of these ecdysteroids vary between the Silene species. HPLC proved to be a quick and effective way to screen Silene species, determine ecdysteroid profiles and, hence, identify extracts containing novel analogues. An extract of the aerial parts of S. pseudotites was found to contain several new ecdysteroids. These have been isolated and identified spectroscopically (by NMR and mass spectrometry) as 2-deoxyecdysone 22β- d-glucoside, 2-deoxy-20,26-dihydroxyecdysone and 2-deoxypolypodine B 3β- d-glucoside. Additionally, (5α-H)-2-deoxyintegristerone A (5α- 2H 91%, 5α- 1H 9%) was isolated as an artefact. This study contributes to the understanding of ecdysteroid distribution in Silene species and provides further information on the chemotaxonomic significance of ecdysteroids in Silene species.

Phytoecdysteroids in seeds of Lloydia serotina (Liliaceae)

Seeds of a number of species in the Liliaceae (sensu Brummitt, 1992) were examined for the presence of ecdysteroid agonist and antagonist activities. No species were antagonistic to 20-hydroxyecdysone action on the ecdysteroid-responsive Drosophila melanogaster B(II) cell line and only one extract, that of Lloydia serotina, was agonistic. This activity is attributable to the presence of phytoecdysteroids as detected by ecdysteroid-specific radioimmunoassay and the agonist version of the B(II) bioassay. HPLC in conjunction with radioimmunoassay and bioassay have been used to determine the ecdysteroid profile. The major ecdysteroids present are identified as 20-hydroxyecdysone and polypodine B (5beta,20-dihydroxyecdysone).

Phytoecdysteroids in the genus Asparagus (Asparagaceae)

Phytoecdysteroids, plant steroids which are analogues of invertebrate steroid hormones, probably contribute to the deterrence of phytophagous invertebrate predators. They also seem to possess antimicrobial activity and several pharmaceutical and medicinal benefits have been ascribed to them. Here, we present a survey of seeds of 16 species of the genus Asparagus (Asparagaceae), including the crop species A. officinalis, for ecdysteroid agonists (including phytoecdysteroids) and antagonists. Seven species were found to contain ecdysteroids with levels ranging from just detectable ( A. racemosus and A. sarmentosus) to relatively high ( A. laricinus). RP-HPLC/RIA/bioassay has been used to separate positive extracts of four species ( A. falcatus, A. laricinus, A. ramosissimus and A. scandens) and analyse the ecdysteroid profiles. The identities of the major ecdysteroids were confirmed by NP-HPLC. Seeds of A. officinalis do not contain detectable levels of ecdysteroids, but leaves, stems and roots contain low levels (detectable by RIA). This indicates that A. officinalis retains the genetic capacity to synthesise ecdysteroids and that future strategies could be developed for enhanced protection of asparagus spears through elevated ecdysteroid levels.

Phytoecdysteroid profiles in seeds of Sida spp. (Malvaceae).

Procedures are presented for the assessment of the phytoecdysteroid profiles in small plant samples (ca. 25 mg), using seeds of Sida spp. as an example. The procedures are suitable for the analysis of minute or valuable samples and provide copious information for chemotaxonomic purposes. Methanolic extracts of the plant material, after partitioning against hexane, were separated by reversed-phase gradient HPLC monitored by PAD, RIA and bioassay. Aliquots of the fractions were also treated with Helix pomatia hydrolases, followed by RIA and bioassay, in order to assess the presence of hydrolysable ecdysteroid conjugates. Further information could also be obtained by separation of samples using normal-phase gradient HPLC. Among 11 species of Sida examined, seed extracts of S. acuta (= S. carpinifolia) and S. rhombifolia were found to contain significant amounts of ecdysteroids, seed extracts of S. filicaulis contained only moderate levels, whilst the remaining species showed no detectable levels of ecdysteroids. The ecdysteroid profiles of the extracts of the three positive species were significantly different, demonstrating that phytoecdysteroids have chemotaxonomic value in this genus.

Phytoecdysteroids in seeds and plants of Rhagodia baccata (Labill.) Moq. (Chenopodiaceae).

Seeds of Rhagodia baccata afforded, in addition to 20-hydroxy-ecdysone and polypodine B, a novel phytoecdysteroid, (20R)-22-deoxy-20,21-dihydroxyecdysone, the structure of which was elucidated unequivocally by UV, LSIMS and NMR techniques. This compound possessed agonistic activity in the Drosophila melanogaster BII cell bioassay, with an ED50 value of 2.0 x 10(-7)M (ED50 value for 20-hydroxyecdysone = 7.5 x 10(-9)M). The distribution of ecdysteroids in plants of R. baccata has been determined. Highest levels are associated with the youngest aerial tissues and with the roots. Ecdysteroid profiles are qualitatively very similar throughout the plant, with 20-hydroxyecdysone and polypodine B predominating in all plant parts tested.

Phytoecdysteroids in the Genus Agapanthus (Alliaceae)

Bioassay/RIA-based analysis of seeds of 15 species, subspecies and varieties of Agapanthus revealed the presence of phytoecdysteroids in most of them. However, the concentrations varied markedly between samples. The accumulation of phytoecdysteroids was highest in a sample of A. campanulatus ssp. patens and a sample of A. inapertus ssp. hollandii (c. 1.5 mg ecdysone equivalents per g seed, using the DBL-1 antiserum). Moderate levels were present in a sample of A. inapertus ssp. inapertus. Phytoecdysteroid levels were almost undetectable in samples of A. coddii, A. caulescens ssp. angustifolius, A. minor var. alba and A. praecox ssp. orientalis. RP-HPLC separation coupled with monitoring by RIA and bioassay revealed that the samples contain significantly different ecdysteroid profiles. The major phytoecdysteroid in several of the extracts has been identified as 20-hydroxyecdysone on the basis of chromatographic evidence. It is concluded that ecdysteroid levels and profiles vary considerably in seeds of the genus Agapanthus, even between seed samples of the same species.

Changes in ecdysteroid levels during regeneration of a limb in Pilumnus hirtellus

Measurements of ecdysteroid levels were made in hemolymph, epidermis and regenerating limb of Pilumnus hirtellus crabs after autotomy of one pereiopod performed during stage C4 of the molting cycle. Comparison of the evolution of ecdysteroid profiles indicated that during the premolt period the epidermis and the regenerating limb behave like authentic target tissues, whereas during the transition period from intermolt to premolt, a hormone increase observed in the regenerate could be a result of autocrine activity. This increase is correlated with intense proliferative activity of the cells of the limb bud which enter their basal growth period.

Thyroxine-induced changes in ovarian protein and ecdysteroid levels in the silkworm, Bombyx mori L.: Effect on ovarian maturation and egg production

1. 1. Feeding of thyroxine to larvae of the bivoltine silkworm. Bombyx mori L. resulted in increased pupal hemolymph protein and ecdysteroid levels, increased yolk deposition and accelerated oocyte maturation. 2. 2. Due to early yolk deposition, mature oocytes were more numerous than vitellogenic oocytes in the ovarian tubules of thyroxine-treated animals scored on pupal day 8. 3. 3. Oocyte size was significantly higher following the thyroxine treatment. 4. 4. Pupal-adult eclosion was accelerated. 5. 5. Hemolymph and oocyte ecdysteroid profiles were quantitatively and qualitatively altered. 6. 6. The data suggest that the vertebrate hormone thyroxine effects changes in insect development and maturation through changes in ecdysteroid patterns.

Sterol and ecdysteroid profiles of Serratula tinctoria (L.): Plant and cell cultures producing steroids

Cell suspension cultures have been obtained from Serratula tinctoria, a plant producing ecdysteroids. Sterol profiles and ecdysteroid contents have been analysed and compared in plants and cell cultures. In particular, the composition of free and esterified sterols was investigated using appropriate analytical techniques. In plants, esterified sterols were more abundant (50–70% of the total sterol) than in cell cultures (13–36%). A selectivity for sterol esterification was noted: in plants, the triterpenes (as amyrins) were esterified, whereas it was the 4-desmethylsterols (sitosterol and cholesterol) in cell cultures. Ecdysteroids were present in higher quantities in plant (0.1–1.2% dry wt) than in cell cultures (0.01–0.03%). Analysis confirmed the presence of 20-hydroxyecdysone, 20-hydroxyecdysone-3-acetate and polypodine B as the main compounds. Roots were not only the richest organ in ecdysteroids, but also in cholesterol, lathosterol and 24-methylene-cholesterol. The results obtained suggest interesting relationships between free or esterified sterols and ecdysteroid contents.

Ovarian ecdysteroid levels and basal oöcyte development during maturation in the cockroach Blattella germanica (L.)

By using enzyme immunoassay techniques we have quatified the ovarian ecdysteroids during the 7-day cycle of oöcyte maturation in Blattella germanica. Immunoreactive ecdysteroid levels were low on days 0–2, increased to higher values on days 3–6, and peaked on day 7 (about 5 ng of 20-hydroxyecdysone equivalents per pair of ovaries). The predominant non-conjugated ovarian ecdysteroid detected by HPLC analysis was 20-hydroxyecdysone. During oöcyte maturation, the dynamics of patency, vitellogenin uptake, increase in follicle cells diameter (suggested to be due to polyploidy), and choriogenesis was studied. A comparison of the ovarian ecdysteroid profile with the succession of physiological processes occurring within the oöcyte maturation cycle suggests that these hormones could be directly or indirectly involved in the control of patency, polyploidy and choriogenesis in the follicle cells.