New insights into the regulation mechanism of Pacific white shrimp (Litopenaeus vannamei) hepatopancreas under 4-nonylphenol exposure using transcriptome analysis.

4-Nonylphenol (4-NP) is one of the common endocrine-disrupting chemicals (EDCs) in estuaries and coastal zones, which can exert detrimental effects on the physiological function of aquatic organisms. However, the molecular response triggered by 4-NP remains largely unknown in Pacific white shrimp (Litopenaeus vannamei). In this study, transcriptomic analysis was performed to investigate the underlying mechanisms of 4-NP toxicity in the hepatopancreas of L. vannamei. Nine RNA-Seq libraries were generated from L. vannamei at 0 h, 24 h, and 48 h following exposure to 4-NP. Compared with 0 h vs 24 h, 962 up- and 463 down-regulated differentially expressed genes (DEGs) were identified, indicating that many genes in L. vannamei were induced to resist adverse circumstances by 4-NP exposure. In contrast, 902 up- and 1027 down-regulated DEGs were revealed in the comparison of 0 h vs 48 h, demonstrating that prolonged exposure to the stress from 4-NP resulted in more inhibited genes. To validate the accuracy of the transcriptome data, eight DEGs were selected for quantitative real-time polymerase chain reaction (qRT-PCR), which were consistent with the RNA-Seq results. Through KEGG pathway enrichment analysis, three specific pathways related to hormonal effects and endocrine function of L. vannamei were enriched significantly, including tyrosine metabolism, insect hormone biosynthesis, and melanogenesis. After 4-NP stress, genes involved in tyrosine metabolism (Tyr) and melanogenesis pathway (AC, CBP, Wnt, Frizzled, Tcf, and Ras) were induced to promote melanin pigment to help shrimp resist adverse environments. In the insect hormone biosynthesis, ALDH, CYP15A1, CYP15A1/C1, and JHE genes were activated to synthesize juvenile hormone (JH), while Spook, Phm, Sad, and CYP18A1 were induced to generate molting hormone. There is an enhanced interaction between the molting hormone and JH, with JH playing a dominant role and maintaining its "classic status quo action". Our study demonstrated that 4-NP exposure led to impairments of biological functions in L. vannamei hepatopancreas. The genes and pathways identified provide novel insights into the molecular mechanisms underlying 4-NP toxicity effects in prawns and enrich the information on the toxicity mechanism of crustaceans in response to EDCs exposure.

Ecdysteroid-containing food supplements from Cyanotis arachnoidea on the European market: evidence for spinach product counterfeiting.

Phytoecdysteroids like 20-hydroxyecdysone ("ecdysterone") can exert a mild, non-hormonal anabolic/adaptogenic activity in mammals, and as such, are frequently used in food supplements. Spinach is well-known for its relatively low ecdysteroid content. Cyanotis arachnoidea, a plant native in China, is among the richest sources of phytoecdysteroids, and extracts of this plant are marketed in tons per year amounts via the internet at highly competitive prices. Here we report the investigation of a series of food supplements produced in Germany and claimed to contain spinach extracts. Twelve ecdysteroids including two new compounds were isolated and utilized as marker compounds. A comparative analysis of the products with Cyanotis and spinach extracts provides evidence that they were manufactured from Cyanotis extracts instead of spinach as stated. Based on the chromatographic fingerprints, 20-hydroxyecdysone 2- and 3-acetate are suggested as diagnostic markers for related quality control. This case appears to represent an unusual type of dietary supplement counterfeiting: undeclared extracts from alternative plants would supposedly 'guarantee' product efficacy.

High performance liquid chromatography used for quality control of Achyranthis Radix.

To establish a standard of quality control and to identify reliable Achyranthis Radix, three phytoecdysones including ecdysterone (1), 25R-inokosterone (2) and 25S-inokosterone (3) were determined by quantitative HPLC/UV analysis. Three phytoecdysones were separated with an YMC J'sphere ODS C(18) column (250 mm × 4.6 mm, 4 µm) by isocratic elution using 0.1% formic acid in water and acetonitrile (85:15, v/v%) as the mobile phase. The flow rate was 1.0 mL/min and the UV detector wavelength was set at 245 nm. The standards were quantified by HPLC/UV from Achyranthes bidentata Blume and Achyranthes japonica Nakai, as well as Cyathula capitata Moq. and Cyathula officinalis Kuan, which are of a different genus but are comparative herbs. The method was successfully used in the analysis of Achyranthis Radix of different geographical origin or genera with relatively simple conditions and procedures, and the assay results were satisfactory for linearity, recovery, precision, accuracy, stability and robustness. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of eighteen A. bidentata Blume samples and ten A. japonica Nakai samples. The results indicate that the established HPLC/UV method is suitable for quantitation and pattern recognition analyses for quality evaluation of Achyranthis Radix.

Simultaneous determination of main phytoecdysones and triterpenoids in radix achyranthis bidentatae by high-performance liquid chromatography with diode array-evaporative light scattering detectors and mass spectrometry.

A liquid chromatographic method was developed for simultaneous determination of two main types of bioactive compounds: four phytoecdysones and eight triterpenoids in radix achyranthis bidentatae (RAB), i.e., polypodine B (1), ecdysterone (2), 25-R inokosterone (3), 25-S inokosterone (4), ginsenoside Ro (5), chikusetsusaponin IVa (6), zingibroside R1 (7), chikusetsusaponin IVa ethyl ester (8), 28-deglucosyl-chikusetsusaponin IVa (9), PJS-1 (10), 28-deglucosyl-chikusetsusaponin IVa butyl ester (11), and oleanolic acid (12). Optimum separations were obtained with a Zorbax C18 column, using a gradient elution with 0.08% aqueous formic acid (containing 5% isopropyl alcohol) and acetonitrile as mobile phase. Phytoecdysones were detected by diode array detector (DAD) at 242 nm, whereas triterpenoids were monitored by evaporative light scattering detector (ELSD) connected in series with DAD, temperature for the drift tube was 110 degrees C and the nitrogen flow rate was 3.2 L min(-1). The identity of the analytes was confirmed using retention times, ultraviolet absorbance and mass spectral data in comparison with reference compounds. The method was validated for acceptable precision (intra- and inter-day variation < or = 4.87%), accuracy (recovery > or = 88.9%) and sensitivity (LOD < or = 0.43 microg mL(-1) (DAD) and 26.0 microg mL(-1) (ELSD), LOQ < or = 0.97 microg mL(-1) (DAD) and 46.5 microg mL(-1) (ELSD), respectively). This rapid and reliable method was applied for the analysis of four cultivated and ten commercial samples. The results demonstrated that the method is suitable for routine analysis and quality control of RAB.

Simultaneous qualitation and quantification of four phytoecdysones in Radix Achyranthis Bidentatae by high-performance liquid chromatography with diode array detection.

A high-performance liquid chromatographic method with diode array detection was developed and validated to simultaneously identify and quantify four phytoecdysones,i.e., polypodine B (1), ecdysterone (2), 25-R inokosterone (3) and 25-S inokosterone (4), in Radix Achyranthis Bidentatae. The analysis was performed using a C(18) column with 6% tetrahydrofuran aqueous and acetonitrile isocratic elution, and the detection was carried out by DAD at 242 nm. The identities of the analytes were determined by comparing the retention time and UV spectrum with those of reference compounds. The validation of the method included linearity, sensitivity, recovery and stability. All calibration curves of the four phytoecdysones showed good linear regression (r(2) >or= 0.9993). The limit of detection (S/N = 3) and limit of quantification (S/N = 10) were less than 7.5 and 12.3 ng, respectively. The method provided desirable repeatability with overall intra- and inter-day variations of less than 4.67%. The obtained recoveries varied between 95.1 and 104.4% while the relative standard deviations were below 4.85% (n = 3). The analysis results indicated that the contents of the investigated phytoecdysones in Radix Achyranthis Bidentatae from different locations were highly variant, and the genuine crude drug indigenous to Henan province did not possess the highest concentration of phytoecdysones.

Detection of saponins in extracts from the rhizomes of Paris species and prepared Chinese medicines by high performance liquid chromatography-electrospray ionization mass spectrometry.

A highly sensitive and specific method, based on high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS), was developed for simultaneous detection of eleven steroidal saponins and one phytoecdysone in extracts from the rhizomes of thirteen PARIS species and four prepared Chinese medicines (PCMs). The HPLC experiments were performed by means of a reversed-phase C18 column (4.6 mm x 150 mm, 5 microm) and mobile phase system consisting of 0.1 % aqueous formic acid and acetonitrile under gradient elution conditions. The most intensive electrospray ionization signals were found in the negative ion spectra due to HCOO- adducts. The limits of detection (LODs) for the saponins were lower than 40 ng/mL in selected ion monitoring (SIM) mode. The identification of the saponins in the extracts of PARIS species and PCMs was confirmed using their retention times and mass spectral comparisons to standard compounds. The validated method was successfully applied for simultaneous detection of twelve standard compounds in the analytes so that it provided a new means for quality evaluation of Rhizoma Paridis and Chinese multiherb remedies that contain Rhizoma Paridis. The results showed that most species of PARIS contain steroidal saponins, which provided evidence for expanding the botanical origin of Rhizoma Paridis.

Extraction and monitoring of phytoecdysteroids through HPLC.

The size of the phytoecdysteroids family is rapidly growing. Recent data shows over 250 ecdysteroid analogs have been identified so far in plants. It is theorized that there are over 1000 possible structures, which might occur in nature, but it is a fact that ecdysteroids usually occur in plants as a complex cocktail of structurally different compounds. Among these compounds, the major component is usually the common ecdysteroid-like 20-hydroxyecdysone. Ecdysteroids are polar steroids, almost sugar-like in their solubility properties. Extraction and purification of ecdysteroids (polyhydroxy steroids) is complicated by their polar nature and poor crystallizing properties. These properties make them difficult to separate from other polar plant constituents. Besides, this plant extract is very often processed by multistep procedures to isolate the major and minor ecdysteroids from the new or existing sources. A simplified scheme consisting of a few extraction steps for the purification of ecdysteroid from plants is in great demand. A quantitative approach through high-performance liquid chromatography has been initiated for developing an easy method for the extraction of ecdysteroids from Ipomoea hederacea (kaladana) seeds.

Identification and determination of ecdysone and phenylpropanoid glucoside and flavonoids in Lamium maculatum by capillary zone electrophoresis.

The simultaneous determination of 20-hydroxy ecdysone (1), 3,7-dimethoxy-quercetin (2), acteoside (3) and rutin (4) in the mixture of leaf and stem, and the flower of Lamium maculatum has been investigated by capillary zone electrophoresis for the first time. With an electrolyte containing 30 mmol/L borate, at pH 9.47 and 20 kV applied voltage, the four active compounds were completely separated within 5 min with satisfactory results. The effects of concentration of borate and electrolyte pH on electrophoretic behavior and separation were studied. Regression equations revealed linear relationships (correlation coefficients 0.9998-0.9999) between the peak area of each analyte and the concentration. The levels of analytes in the different parts of Lamium maculatum were easily determined with recoveries ranging from 98.3 to 105.0%.

Woc (without children) gene control of ecdysone biosynthesis in Drosophila melanogaster.

The first step in ecdysteroidogenesis, i.e. the 7,8-dehydrogenation of dietary cholesterol (C) to 7-dehydrocholesterol (7dC), is blocked in Drosophila melanogaster homozygous woc (without children) third instar larval ring glands (source of ecdysone). Unlike ring glands from wild-type D. melanogaster larvae, glands from woc mutants cannot convert radiolabelled C or 25-hydroxycholesterol (25C) to 7dC or 7-dehydro-25-hydroxycholesterol (7d25C) in vitro, nor to ecdysone (E). Yet, when these same glands are incubated with synthetic tracer 7d25C, the rate of metabolism of this polar Delta(5,7)-sterol into E is identical to that observed with glands from comparably staged wild-type larvae. The absence of this enzymatic activity in vivo is probably the direct cause of the observed low whole-body ecdysteroid titers in late third instar homozygous mutant larvae, the low ecdysteroid secretory activity in vitro of brain-ring gland complexes from these animals, and the failure of the larvae to pupariate (undergo metamorphosis). Oral administration of 7dC, but not C, results in a dramatic increase in ecdysteroid production both in vivo and in vitro by the woc mutant brain-ring gland complexes and affects a partial rescue to the beginning of pupal-adult development, but no further, despite elevated whole-body ecdysteroid titers. Data previously reported (Wismar et al., 2000) indicate that the woc gene encodes a zinc-finger protein that apparently modulates the activity of the 7,8-dehydrogenase.

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.

Ecdysteroids in stress responsive and nonresponsive Drosophila virilis lines under stress conditions.

After exposure to thermal stress or a control temperature, the relative abundance of ecdysone (E) and 20-hydroxyecdysone (20E) was measured in a wild-type line of Drosophila virilis (101) that is stress responsive and in a mutant line (147) that is not stress responsive. In line 101, the 20E content was higher and E content lower in females than in males. The abundance of E and 20E in females of line 147 was significantly higher than that in females of line 101. Females of line 101 were found to respond to 60 min of heat stress (38 degrees C) by an increase in the abundance of both E and 20E, while in males of this line the amount of 20E increased and that of E declined. A role of the ecdysteroids in the control of reproduction of D. virilis under stress is discussed.

Ecdysteroid titers during postembryonic development of Dermanyssus gallinae (Acari: Dermanyssidae).

Ecdysteroid titers in methanolic extracts of protonymphal and deutonymphal stages of the chicken mite, Dermanyssus gallinae (De Geer), were measured using radioimmunoassay. Highest titers of ecdysteroids were present 24 h after feeding. Further studies using high-performance liquid chromatography radioimmunoassay showed that ecdysteroid immunopositive material had the same retention time as 20-hydroxyecdysone and ecdysone. Ecdysteroid conjugates appear to be present in polar and apolar fractions.

Changes in morphogenetic hormone titers in isolated workers of the termite Reticulitermes flavipes (Kollar).

The daily titers of juvenile hormone and ecdysteroids were determined for workers of the Eastern subterranean termite Reticulitermes flavipes (Kollar) (Isoptera, Rhinotermitidae) following isolation of the workers from soldiers and reproductives. Experiments have demonstrated that isolation leads to biochemical and physiological changes that result in a presolider molt. Juvenile hormone (JH) and molting hormone (MH) titers were determined in hemolymph samples using radioimmunoassay. The identity of the ecdysteroids as predominantly 20-hydroxyecdysone (90%) and ecdysone (10%) was determined by HPLC using pooled extracts of hemolymph.

Analysis of ecdysteroids in the trematodes, Schistosoma mansoni and Fasciola hepatica.

Adult Schistosoma mansoni from experimentally infected mice and Fasciola hepatica recovered from ovine livers post mortem were analyzed for free and conjugated ecdysteroids by radioimmunoassay, high-performance liquid chromatography monitoring fractions by radioimmunoassay, and by capillary gas chromatography/mass spectrometry (selected ion monitoring). Both species contained ecdysone and 20-hydroxyecdysone as free ecdysteroids and as polar conjugates. F. hepatica also contained a polar conjugate of 2-deoxyecdysone. Evidence of apolar ecdysteroid conjugates was only obtained for F. hepatica. The free ecdysteroid-containing fraction of S. mansoni contained small amounts of unidentified immunoreactive material.

Patterns of E74A RNA and protein expression at the onset of metamorphosis in Drosophila.

Metamorphosis in Drosophila is triggered by a pulse of the steroid hormone ecdysone at the end of larval development. Ecdysone initiates a genetic hierarchy that can be visualized as a series of puffs in the larval salivary gland polytene chromosomes. The E74 gene is responsible for the early ecdysone-inducible puff at position 74EF and encodes two related DNA-binding proteins which appear to play a regulatory role in the hierarchy. Here we describe the spatial and temporal patterns of E74A RNA and protein expression at the onset of metamorphosis. We use in situ hybridization, antibody stains, and western and northern blot analyses to follow E74A expression from its initial appearance as nascent transcripts on the polytene chromosomes, to spliced mRNA, to post-translationally modified nuclear E74A protein. E74A is expressed in a wide variety of late-third instar tissues, suggesting that it plays a broad pleiotropic role in response to the hormone. In early prepupae, when the overall levels of E74A mRNA are decreasing, relatively high levels of E74A RNA persist in the gut, peripodial membranes of the imaginal discs, and proliferation centers of the brain. The spatial distribution of nuclear E74A protein correlates with the RNA distribution with the single exception that no E74A protein can be detected in the proliferation centers of the brain. There is also a temporal discrepancy between E74A mRNA and protein accumulation. The peak of E74A protein induced by the late larval ecdysone pulse follows the peak of E74A mRNA by approximately 2 h. This delay is not seen in 10 h prepupae, when the next pulse of ecdysone induces the simultaneous expression of E74A mRNA and protein. We discuss possible mechanisms for post-transcriptional regulation of E74A expression and suggest that the unusually long and complex 5' leader in the E74A mRNA may regulate its translation.

Involvement of translation and transcription in insect steroidogenesis.

The involvement of protein and RNA synthesis in insect steroidogenesis was investigated using the prothoracic glands of the tobacco hornworm Manduca sexta. Ecdysone secretion stimulated by prothoracicotropic hormone (PTTH) and by cAMP analogs such as dibutyryl cAMP (dbcAMP), was suppressed by the translation inhibitors cycloheximide and puromycin, and by the transcription inhibitor actinomycin D. Inhibition of protein synthesis did not prevent the activation of glandular kinases, as indicated by continued protein phosphorylation in the presence of cycloheximide. Incorporation of radiolabeled amino acids and uridine increased within 60 min of glandular activation, suggesting that ecdysteroid secretion was accompanied by enhanced protein and RNA synthesis. One-dimensional gel electrophoresis revealed an increase in the translation of glandular proteins within 20 min of activation. The results suggest that the translation of protein from short-lived mRNA is necessary for optimal synthesis of ecdysteroids, and that the requisite proteins act beyond the activation of cAMP-dependent protein kinase.

A competitive enzyme-linked immunosorbent assay: applications in the assay of peptides, steroids, and cyclic nucleotides.

Indirect competitive enzyme-linked immunosorbent assays (ELISAs) that can be used to quantify several types of small, bioactive molecules, including peptides, steroids, and cyclic nucleotides, are described. The assays require no special expertise to perform, and the sensitivities are very high, equally or exceeding what is commonly achieved in radioimmunoassay (RIA). The molecule to be assayed or a synthetic derivative is coupled to a protein carrier (= conjugate). The conjugate is adsorbed to the wells of a microtiter plate where it is bound by antibody in inverse proportion to free hapten in a sample or standard. Bound antibody is then quantified with enzyme-labeled anti-immunoglobulin and appropriate substrate. The assay of peptides is illustrated for the sulfated cholecystokinin octapeptide, in which an ED50 of 20 fmol (2 x 10(-10) M in 100 microliters assay volume) is attained. The ED50's and slopes of the dose-response curves in the steroid and cyclic nucleotide ELISAs are compared with those parameters obtained earlier by RIA using the same antisera. This comparison indicates that a steroid, ecdysone, can be quantified with no apparent participation of the bridging group of the conjugate in the competitive assay. Furthermore, the ED50's in the ecdysone assays (ecdysone 2 beta, 3 beta, 14 alpha, 22R, 25-pentahydroxy-5 beta-cholest-7-en-6-one, 7.7 fmol; 20-hydroxyecdysone, 16 fmol) are 19- to 38-fold lower for ELISA than for RIA. In the cyclic nucleotide assay, the bridge of a cAMP conjugate (homologous with the bridge of the immunogen) decreases the slope of the dose-response curve. This effect is minimized by the use of short incubations with anti-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemolymph ecdysteroids and molt cycle in males and females of Siriella armata M-Edw. (Crustacea: Mysidacea): possible control by the MI-ME X-organ of the eyestalk.

Hemolymph ecdysteroids were quantified by radioimmunoassay (RIA) at successive stages of the molt cycle in the mysid Siriella armata. Profiles showed a single peak during premolt, at stage D1 for males, and D2 for reproducing females who displayed ecdysteroid levels 10 times higher than males. Titers were also measured for individuals which had been molt inhibited by early electrocauterization of the eyestalk MI-ME X-organ. In the case of total inhibition of molt preparation, the ecdysteroid peak was suppressed. It was displaced toward the end of the cycle when only ecdysis was inhibited. Ecdysone and 20-hydroxyecdysone were characterized in the hemolymph of both sexes using high-pressure liquid chromatography followed by RIA. High-polarity products, abundant in the female hemolymph, were resolved into 20-hydroxyecdysone and ecdysone by enzymatic hydrolysis and thin-layer chromatography. The quantitative and qualitative variations of ecdysteroid in the different situations (male or female, normal or inhibited cycles) are presented in relation to apolysis, epidermic activity, ecdysis, and secondary vitellogenesis in females, emphasizing the importance not only of ecdysteroids, but also of the MI-ME X-organ in monitoring molt and blood preparation in mysids.

Analysis of ecdysteroids in different developmental stages of Hymenolepis diminuta.

Prepatent and patent adult Hymenolepis diminuta from the intestines of rats, H. diminuta eggs recovered from the faeces of rats harbouring patent infections, and infective cysticercoids from the beetle intermediate host were analysed for free and conjugated ecdysteroids. Adult worms and eggs contained both free ecdysteroids and hydrolysable polar conjugated ecdysteroids, with comparatively large amounts of immunoreactive material also being detected following hydrolysis of the possible apolar conjugated ecdysteroid fraction. Free ecdysteroids were not detected in the cysticercoid sample. The concentration of free ecdysteroids in H. diminuta eggs was higher than that detected in the tissues of the adult worms. Ecdysone and 20-hydroxyecdysone were the major identified compounds of the free ecdysteroid fraction, whereas in the hydrolysed polar conjugated ecdysteroid fraction these two compounds were accompanied by 20,26-dihydroxyecdysone. The free ecdysteroid fraction also contained comparatively large amounts of unidentified immunoreactive material.