Production Of 20-hydroxyecdysone Research Articles

20-Hydroxyecdysone Primes Innate Immune Responses That Limit Bacterial and Malarial Parasite Survival in Anopheles gambiae.

Blood feeding is an integral behavior of mosquitoes to acquire nutritional resources needed for reproduction. This requirement also enables mosquitoes to serve as efficient vectors to acquire and potentially transmit a multitude of mosquito-borne diseases, most notably malaria. Recent studies suggest that mosquito immunity is stimulated following a blood meal, independent of infection status. Since blood feeding promotes production of the hormone 20-hydroxyecdysone (20E), we hypothesized that 20E plays an important role in priming the immune response for pathogen challenge. Here, we examine the immunological effects of priming Anopheles gambiae with 20E prior to pathogen infection, demonstrating a significant reduction in bacteria and Plasmodium berghei survival in the mosquito host. Transcriptome sequencing (RNA-seq) analysis following 20E treatment identifies several known 20E-regulated genes, as well as several immune genes with previously reported function in antipathogen defense. Together, these data demonstrate that 20E influences cellular immune function and antipathogen immunity following mosquito blood feeding, arguing the importance of hormones in the regulation of mosquito innate immune function.IMPORTANCE Blood feeding is required to provide nutrients for mosquito egg production and serves as a mechanism to acquire and transmit pathogens. Shortly after a blood meal is taken, there is a peak in the production of 20-hydroxyecdysone (20E), a mosquito hormone that initiates physiological changes, including yolk protein production and mating refractoriness. Here, we examine additional roles of 20E in the regulation of mosquito immunity, demonstrating that priming the immune system with 20E increases mosquito resistance to pathogens. We identify differentially expressed genes in response to 20E treatment, including several involved in innate immune function as well as lipid metabolism and transport. Together, these data argue that 20E stimulates mosquito cellular immune function and innate immunity shortly after blood feeding.

De novo assembly and transcriptome of Pfaffia glomerata uncovers the role of photoautotrophy and the P450 family genes in 20-hydroxyecdysone production.

Pfaffia glomerata is a medically important species because it produces the phytoecdysteroid 20-hydroxyecdysone (20-E). However, there has been no ready-to-use transcriptome data available in the literature for this plant. Here, we present de novo transcriptome sequencing of RNA from P. glomerata in order to investigate the 20-E production as well as to understand the biochemical pathway of secondary metabolites in this non-model species. We then analyze the effect of photoautotrophy on the production of 20-E genes phylogenetically identified followed by expression analysis. For this, total messenger RNA (mRNA) from leaves, stems, roots, and flowers was used to construct indexed mRNA libraries. Based on the similarity searches against plant non-redundant protein database, gene ontology, and eukaryotic orthologous groups, 164,439 transcripts were annotated. In addition, the effect of photoautotrophy in two genes putatively involved in the 20-E synthesis pathway was analyzed. The Phantom gene (CYP76C), a precursor of the route, showed increased expression in P. glomerata plants cultured under photoautotrophic conditions. This was accompanied by increased production of this metabolite indicating a putative involvement in 20-E synthesis. This work reveals that several genes in the P. glomerata transcriptome are related to secondary metabolism and stresses, that genes of the P450 family participate in the 20-E biosynthesis route, and that plants cultured under photoautotrophic conditions promote an upregulated Phantom gene and enhance the productivity of 20-E. The data will be used for future investigations of the 20-E synthesis pathway in P. glomerata while offering a better understanding of the metabolism of the species.

Induction of vitellogenesis by glass bottom boat in the female banana shrimp, Fenneropenaeus merguiensis de Man

In shrimp aquaculture, eyestalk ablation is the only technique that is widely used to accelerate ovarian development. Alternative methods for producing improved ovarian development in broodstock are currently being investigated. Several factors involved in the regulation of ovarian development in shrimp have been investigated. Among these factors, growth factors in the transforming growth factor beta (TGF-β) superfamily have been implicated as playing potential roles in the regulation of gonad development. In this work, a member of the TGF-β superfamily known as glass bottom boat (GBB), an ortholog of bone morphogenetic protein (BMP), was investigated to uncover its role in ovarian development in the banana shrimp Fenneropenaeus merguiensis. Full-length cDNA of FmGBB was obtained from transcriptome data. Phylogenetic analysis indicated that the sequence of FmGBB from banana shrimp was similar to those of other arthropods and vertebrate BMP 5/6/7, but was different from those of decapentaplegic proteins and vertebrate BMP 2/4. The FmGBB transcript was found to be widely expressed in shrimp tissues, and its expression in the ovary was dramatically increased in early and late vitellogenic stages during ovarian development and decreased in the mature stage, suggesting its role in vitellogenesis. To study the effects of FmGBB, a soluble recombinant mature FmGBB peptide (His-TF-rgbb) containing both monomers and homodimers was successfully expressed in Escherichia coli. The His-TF-rgbb peptide triggered oocyte proliferation in both cultured ovarian explants and in previtellogenic shrimp upon injection. Interestingly, the injection of His-TF-rgbb into previtellogenic female shrimp stimulated an increase in Vg expression in their ovaries while suppressing production of 20-hydroxyecdysone. Our results suggest the potential role of FmGBB in oocyte proliferation and vitellogenesis; this novel finding can be utilized to stimulate ovarian development in cultured shrimp.

Evaluation of Spinacia oleracea (L.) for phytodesalination and augmented production of bioactive metabolite, 20-hydroxyecdysone

ABSTRACTIn this study, adaptive features of Spinacia oleracea to different levels of salinity, its use in desalination and production of 20-Hydroxyecdysone were studied. Plants showed survival up to EC 12 dS/m with reduced growth as compared with control. Net photosynthesis rate, transpiration, stomatal conductance, and water use efficiency of salt treated plants declines with increasing salinity stress. Higher antioxidant enzyme activities and compatible solutes accumulation were observed in salt treated plants as function of osmotic adjustment. Significant Na+ sequestration and Na/K ratio were noted with increase in salt stress in comparison to the control. Since the plant accumulates a bioactive, secondary metabolite 20-Hydroxyecdysone (20E), we observed significant 20E content in plants grown at EC 4–12 dS/m in comparison to control. Furthermore, a preliminary field experiment, showed significant reduction in the soil electrical conductivity by 1.8 ds/m after 90 days of plant growth with Na+ sequestration in plant biomass. Subsequent to this growth period, the phytodesalinized soil supported the significant growth of a glycophyte (rice). Our results suggest that S. oleracea can adapt to saline conditions with antioxidant defense and osmotic adjustment. The plant can be used as a potential candidate for desalination and also for enhanced production of 20-Hydroxyecdysone.

Effect of elicitation and precursor feeding on accumulation of 20-hydroxyecdysone in Achyranthes aspera Linn. cell suspension cultures.

20-Hydroxyecdysone is one of the most common ecdysteroids in plants with potential therapeutic applications. In this study, cell suspension cultures of Achyranthes aspera were raised in shake flasks to investigate the production of 20-hydroxyecdysone. The quantification and characterization of 20-hydroxyecdysone in the cultures were done by High performance liquid chromatography (HPLC) and Liquid Chromatography-quadrupole time-of- flight mass spectrometry (LC-Q-TOF) analyses. For raising the suspension, calli initiated from in vitro grown leaf explants were cultured in liquid Murashige and Skoog (MS) medium augmented with combinations of 2, 4-dichlorophenoxyacetic acid (1mgL-1) and α-naphthaleneacetic acid (1mgL-1). Maximum growth index of the cell suspension was 9.9, which was achieved during 20th day of culture (final phase of exponential growth). At this stage, the biomass accumulated was 1.09±0.09g dry weight (DW) and the 20-hydroxyecdysone concentration was 0.24mgg-1 DW. Eliciting the cultures with 0.6mM Methyl jasmonate for 6days; enhanced the production of 20-hydroxyecdysone production to 0.35mgg-1 DW. By augmenting the cultures with the precursors namely cholesterol (10mgL-1) and 7-dehydrocholesterol (10mgL-1), production of 20-hydroxyecdysone was boosted to 0.31mgg-1 DW and 0.28mgg-1 DW respectively.

Growth control through regulation of insulin signalling by nutrition-activated steroid hormone in Drosophila.

Growth and maturation are coordinated processes in all animals. Integration of internal cues, such as signalling pathways, with external cues, such as nutritional status, is paramount for an orderly progression of development and growth. In Drosophila, this involves insulin and steroid signalling, but the underlying mechanisms and their coordination are incompletely understood. We show that bioactive 20-hydroxyecdysone production by the enzyme Shade in the fat body is a nutrient-dependent process. We demonstrate that under fed conditions, Shade plays a role in growth control. We identify the trachea and the insulin-producing cells in the brain as direct targets through which 20-hydroxyecdysone regulates insulin signalling. The identification of trachea-dependent regulation of insulin signalling exposes an important variable that may have been overlooked in other studies focusing on insulin signalling in Drosophila Our findings provide a potentially conserved, novel mechanism by which nutrition can modulate steroid hormone bioactivation, reveal an important caveat of a commonly used transgenic tool to study insulin-producing cell function, and yield further insights into how steroid and insulin signalling are coordinated during development to regulate growth and developmental timing.

Hairy-root cultures of Achyranthes aspera Linn. as a novel route for the production of 20-hydroxyecdysone

An efficient transformation system for hairy-root induction in Achyranthes aspera L. was established and investigated as a novel method for the production of 20-hydroxyecdysone. Four Agrobacterium rhizogenes strains (MTCC 2364, NCIM 5140, A4, and ATCC 15834) were examined for their frequency of transformation in the presence of various concentrations of acetosyringone. Strain ATCC 15834 showed significantly higher transformation efficiency and was capable of inducing hairy roots from different explants (young leaf, hypocotyls, cotyledon, and stem segments) of A. aspera in the presence of 100 µM acetosyringone. The hairy root transformation of the lines (AS001 and AS002) was confirmed by PCR amplification of rol B locus using primers specific for rol B gene. Hairy roots grown in the Murashige and Skoog (MS) liquid medium augmented with 30 g/L sucrose exhibited the highest biomass accumulation and this medium was found to be superior to Gamborg’s B5 medium and Whites root culture medium. HPLC and LC-Q-TOF analyses of the hairy-root extract of A. aspera revealed the ability of hairy-root lines to synthesise the phytoecdysteroid 20-hydroxyecdysone.

Impact of Gamma Irradiation Induced Variation in Cell Growth and Phytoecdysteroid Production in Sesuvium portulacastrum

Abiotic stress in the form of gamma irradiation has been shown a potent inducer of oxidative stress in plant cell cultures which produce higher amounts of commercially important secondary metabolites. In the present study, the impact of low doses of gamma irradiation on growth and accumulation of 20-hydroxyecdysone of Sesuvium portulacastrum was investigated. Shoot cultures were established on Murashige and Skoog medium supplemented with indole-3-acetic acid (0.5 mg L−1) and N 6-benzylaminopurine (2.0 mg L−1). Mutations were induced in tissue culture by treating multiple shoots at low doses of gamma irradiation in the range from 5 to 40 Gy. In vitro cell growth and 20-hydroxyecdysone were assessed during M1T1, M1T2, M1T3, and M1T4 generations. A gamma radiation dose of 20 Gy was calculated as 50% of the lethal value (LD50). The survival rates of multiple shoot cultures exposed to high doses were gradually reduced in the course of increased order of generations. High-gamma irradiation doses were harmful to growth and 20-hydroxyecdysone production. The accumulation of 20-hydroxyecdysone of 0.139 mg/plant dry weight was significantly two-fold higher than non-irradiated shoot cultures. The stressed shoots increased 13-fold 20-hydroxyecdysone at 20 Gy during the M1T4 generation compared to the yield of the M1T1 generation. The ex vitro plants produced 0.321 mg/plant dry weight of 20-hydroxyecdysone which was remarkably greater than the untreated control. The present study postulated that gamma radiation induced metabolic changes and easy-to-achieve putative mutant comprised with the high amount of 20-hydroxyecdysone.

In vitro propagation of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen] as affected by carbon sources

This study aimed to establish a protocol for in vitro propagation of two accessions (Ac) of Pfaffia glomerata (Ac 4 and Ac 13) and to evaluate the effect of different carbon sources on the production of 20-hydroxyecdysone (20E) in leaves and roots. For the assessment of axillary shoot proliferation in vitro, nodal segments were inoculated onto Murashige and Skoog (MS) medium supplemented with 2.22 μM 6-benzyladenine and 2.68 μM α-naphthaleneacetic acid and carbon sources (glucose or sucrose) at varying concentrations (0.1, 0.2, or 0.3 M). To assess the in vitro production of 20E, nodal segments were inoculated into Magenta® containers containing MS medium with different carbon sources (glucose, sucrose, or glucose + sucrose at 0.1 or 0.2 M) and placed in plastic bags with bacterial filters. Both experiments were composed of five repetitions for each treatment and analyzed after 30 d of culture. Multiple shoot formations were genotype-dependent when segments were cultivated on a medium supplemented with glucose or sucrose at 0.1 M, yielding 35 and 43 shoots per explant for Ac 4 and 4.4 and 2.8 shoots per explant for Ac 13, respectively. For the 20E content, significant effects were also observed among accessions and carbon sources. Ac 13 had the highest average 20E levels for both roots and leaves. Under the experimental conditions, Ac 4 had more favorable characteristics for large-scale multiplication than Ac 13, and glucose at 0.2 M was the best carbon source for the cultivation of Pfaffia, both for producing multiple shoots and for in vitro 20E production. This is the first report using a combination of auxin and cytokinin to enable effective Pfaffia in vitro axillary shoot proliferation from nodal explants.

Cloning and characterization of an elicitor-responsive gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase involved in 20-hydroxyecdysone production in cell cultures of Cyanotis arachnoidea

Cyanotis arachnoidea contains a rich source of bioactive phytoecdysteroids (i.e. analogues of insect steroid hormones). 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) supplies mevalonate for the synthesis of many secondary metabolites including 20-hydroxyecdysone (20E), one of metabolism-enhancing phytoecdysteroids. In this study, in order to develop a sustainable source of 20E, cell suspension cultures were established from shoot cultures of C. arachnoidea, and a full length cDNA encoding HMGR (designated as CaHMGR) was cloned and characterized. The cDNA contained 2037 nucleotides with a complete open reading frame (ORF) of 1800 nucleotides, which was predicted to encode a peptide of 599 amino acids. Expression analysis by real-time PCR revealed that CaHMGR mRNA was abundant in C. arachnoidea stems, roots and leaves. When cultivated in Murashige & Skoog medium supplemented with 0.2 mg L−1 1-naphthlcetic acid (NAA) and 3.0 mg L−1 6-benzyladenine (6-BA), C. arachnoidea cells in suspension culture grew rapidly, yielding 20E (124.14 μg L−1) after 12 days. The content of 20E in cell cultures elicited by 0.2 mM methyl jasmonate (MeJA), 100 mg L−1 yeast elicitor (YE) or 25 μM AgNO3 was increased 8-, 2-, and 6-fold over the control, respectively. Quantitative real-time PCR analysis showed that CaHMGR was expressed at a higher level under the treatment of MeJA or Ag+ elicitor. Our results suggested that 20E accumulation may be the result of the expression up-regulation of CaHMGR involved in the biosynthesis under the treatment of various elicitors.

Enhance production of 20-hydroxyecdysone in cell suspension cultures of Vitex glabrata R.Br. by elicitor feeding

The effects of chitosan and methyl jasmonate on growth and 20-hydroxyecdysone production in cell suspension cultures of Vitex glabrata, a medicinal plant in Thailand, were investigated. Elicitation with chitosan at 50 mg/L resulted in 17.16 g/L biomass and 377.09 mg/100 g dry weight (DW) 20-hydroxyecdysone, which were 1.62 and 8.33 times higher than the control cultures, respectively. Likewise, addition of methyl jasmonate at 100 µM also enhanced growth and production of 20-hydroxyecdysone. The highest growth and 20-hydroxyecdysone production reached 14.44 g/L and 621.76 mg/100 g DW, which were 1.35 and 14.54 times higher in comparison to the control cultures, respectively. This is the first report to indicate that elicitation with chitosan and methyl jasmonate enhanced the production of 20-hydroxyecdysone in cell suspension cultures of V. glabrata. Key words: Vitex glabrata, 20-hydroxyecdysone, cell suspension culture, chitosan, methyl jasmonate.

Induced accumulation of 20-hydroxyecdysone in cell suspension cultures of Vitex glabrata R.Br.

This study describes the effects of culture medium, culture temperature, sucrose concentration and cholesterol feeding on cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata , an important medicinal plant in Thailand. Cell growth and 20-hydroxyecdysone production were not significantly different when cells were cultivated on B5 or half-strength MS medium. However, cultivation of V. glabrata cell cultures at 25°C yielded 1.06- and 1.09-fold higher values of cell growth and 20-hydroxyecdysone content, respectively than those at 30°C. Sucrose at 30 and 40 g/L favors the production of 20-hydroxyecdysone in suspension cultures of V. glabrata . Feeding of cholesterol at 5 mg/L, as precursor for biosynthesis of 20-hydroxyecdysone, yielded 1.11-fold higher accumulation of 20-hydroxyecdysone than the control cells. Increasing of cholesterol to 10 mg/L resulted in decreased production of 20-hydroxyecdysone. Key words : Vitex glabrata, 20-hydroxyecdysone, suspension culture, cholesterol.

Improvement of 20-hydroxyecdysone production in cell suspension cultures of Vitex glabrata R.Br. by precursor and elicitors feeding

Improvement of 20-hydroxyecdysone production in cell suspension cultures of Vitex glabrata R.Br. by precursor and elicitors feeding

Optimization of Cell Growth and 20-hydroxyecdysone Production in Cell Suspension Culture of Vitex glabrata R.Br.

The effects of the cultivation media, plant growth regulators and inoculum size on the cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata R.Br. were investigated. The cell growth and 20-hydroxyecdysone formation reach the highest when cells are cultured in the Gamborg's B5 medium supplemented with 2.0 mg/L BAP (6-benzylaminopurine) and 1.0 mg/L 2,4-D. The maximum 20-hydroxyecdysone productivity, of about 1.1 mg/L/day, was observed in the culture with 20% PCV (packed cell volume) of inoculum size. These data also show that the increment of the inoculum size to 20% PCV could increase the productivity in 7-folds.

Enhancement of 20-hydroxyecdysone production in cell suspension cultures of Vitex glabrata R.Br. by precursors feeding

The effect of ecdysteroid precursors feeding on cell growth and 20-hydroxyecdysone production of Vitex glabrata suspension cultures were studied. On the addition of cholesterol, there was no apparent increase of 20-hydroxyecdysone while growth was partially inhibited at higher levels. Feeding of 7-dehydrocholesterol and ergosterol did not affect the cell growth. Both precursors effectively increased production of 20-hydroxyecdysone. Feeding of 7-dehydrocholesterol as a precursor was most effective. The maximum 20-hydroxyecdysone productivity of about 1.31 mg/L/day was observed in culture with 10 mg/L 7-dehydrocholesterol. This data is the first indication that 7-dehydrocholesterol and ergosterol feeding are effective precursors for 20-hydroxyecdysone formation in plant cell suspension culture.

Genetic transformation ofAjuga multiflora bunge withAgrobacterium rhizogenes and 20-hydroxyecdysone production in hairy roots

An efficient transformation system forAjuga multiflora Bunge was established by usingAgrobacterium rhizogenes strain A4. After inoculation with the bacteria, we obtained a number of hairy-root clones from micro-calli of the explant petioles. One fast-growing line showed the highest production of 20-hydroxyecdysone (20-HE). PCR amplification of rooting locus (rol) genes revealed that the left hand-transferred DNA of the root-inducing plasmid was inserted into the genome of our transformedAjuga hairy roots. This integration was further confirmed by DNA-DNA hybridization. The 20-HE content in hairy roots was 10 times higher than that measured in the wild type.

Molting defective is required for ecdysone biosynthesis

20-hydroxyecdysone was discovered as the major biologically active insect steroid hormone half a century ago, yet much remains to be learned about its biosynthesis and its activities. 20-hydroxyecdysone controls many biological processes, including progression between larval stages, entry to pupariation and metamorphosis. A number of genes required for 20-hydroxyecdysone production have been identified, including those encoding enzymes that mediate four of the late steps of biosynthesis. A second smaller group of low ecdysone mutants do not encode enzymes. Here, we report identification of one such gene, which we call molting defective, on the basis of its lethal phenotype. molting defective encodes a nuclear zinc finger protein required for ecdysone biosynthesis.

The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels.

Ecdysteroids regulate a wide variety of cellular processes during arthropod development, yet little is known about the genes involved in the biosynthesis of these hormones. Previous studies have suggested that production of 20-hydroxyecdysone in Drosophila and other arthropods involves a series of cytochrome P450 catalyzed hydroxylations of cholesterol. In this report, we show that the disembodied (dib) locus of Drosophila codes for a P450-like sequence. In addition, we find that dib mutant embryos have very low titers of ecdysone and 20-hydroxyecdysone (20E) and fail to express IMP-E1 and L1, two 20E-inducible genes, in certain tissues of the embryo. In situ hybridization studies reveal that dib is expressed in a complex pattern in the early embryo, which eventually gives way to restricted expression in the prothoracic portion of the ring gland. In larval and adult tissues, dib expression is observed in the prothoracic gland and follicle cells of the ovaries respectively, two tissues known to synthesize ecdysteroids. Phenotypic analysis reveals that dib mutant embryos produce little or no cuticle and exhibit severe defects in many late morphogenetic processes such as head involution, dorsal closure and gut development. In addition, we examined the phenotypes of several other mutants that produce defective embryonic cuticles. Like dib, mutations in the spook (spo) locus result in low embryonic ecdysteroid titers, severe late embryonic morphological defects, and a failure to induce IMP-E1. From these data, we conclude that dib and spo likely code for essential components in the ecdysone biosynthetic pathway and that ecdysteroids regulate many late embryonic morphogenetic processes such as cell movement and cuticle deposition.

20-Hydroxyecdysone production in Ajuga hairy root controlling intracellular phosphate content based on kinetic model

After phosphate depletion in a batch culture medium, the growth rate of Ajuga hairy root decreased and the content of a secondary metabolite, 20-hydroxyecdysone (20-HE), increased. The 20-HE content in Ajuga hairy root increased when the intracellular phosphate content was kept less than 5 mg/g-dry weight. Indoleacetic acid (IAA) supplementation (0.1 mg/ l) increased the growth rate of Ajuga hairy root due to an increase in the number of root apical meristems although it led to a decrease in 20-HE content. From the above results, the culture was divided into two phases: a growth phase where all nutrient components in the medium are present above the critical level, and a secondary metabolite production phase where intracellular phosphate content is kept less than 5 mg/g-dry weight. A kinetic model was constructed to simulate the intracellular phosphate content during the culture. The fed-batch culture in which 0.1 mg/ l of IAA was supplemented only at the beginning of the culture, the intracellular phosphate content was kept below 5 mg/g-dry weight after 10 d, and then phosphate was eliminated from the medium after 20 d, was carried out in a turbine-blade reactor by controlling the medium condition according to the conductivity of the culture medium and the simulation data. The culture provided approximately 3-fold increase in 20-HE content, compared to the fed-batch culture without phosphate control.

Regenerants from Ajuga hairy roots with high productivity of 20-hydroxyecdysone

Regenerants derived from hairy roots of Ajuga reptans var. atropurpurea induced by infection with Agrobacterium rhizogenes MAFF03-01724 harboring pRi revealed a dwarfing response, i.e. decrease in leaf size, reduction in internode distance, and increase in leaf number. These morphogenic alterations were accompanied by an increase in root mass and lack of floral differentiation. In the pRi-transformed regenerants, the proportion of root mass to whole plant mass was higher than that of the untransformed ones, although both kinds of regenerants were comparable on a fresh weight basis. High capacity of rooting and 20-hydroxyecdysone production associated with the original hairy root line were stably maintained in clonal regenerants.