Search Results (105)

Background: 20-Hydroxyecdysone (20E) is the most ubiquitous ecdysteroid (Ecd) and plays critical roles during the life cycle of arthropods. To elucidate the metabolism pathway of 20E in the economically important species, Scylla paramamosain, we conducted a comprehensive exploration of the genes involved in the 20E metabolism pathway. Methods: A comprehensive exploration of genes involved in the 20E metabolism pathway was conducted, including gene annotation, local blast using the Drosophila ortholog as query, and TreeFam ortholog genes identification. Bioinformatics and expression profiling of the identified genes were performed to assess their roles in the 20E metabolism of green mud crabs. Results: This experiment indicated that, except for CYP306a1 and CYP314a1, all other ortholog genes involved in the Drosophila 20E metabolism can be found in the mud crab, suggesting that the function of these two genes might be replaced by other CYP genes or the “active” Ecd in mud crabs was not the 20E. All genes had the typical features of each gene family, clustered with the specific clade in the phylogenetic trees. In addition, all the identified genes had the highest expression level in the Y-organ, and sex-biased gene expression was observed in these genes. Conclusions: This study provided some valuable insights into the metabolism and diversity of ecdysteroids in crustaceans. Full article

20-hydroxyecdysone (20E) signaling plays an important role in regulating insect growth, development, and reproduction. However, the effect of 20E on reproductive diapause and its regulatory mechanisms have not been fully understood. Galeruca daurica is a new pest in the Inner Mongolia grasslands, and it aestivates in an obligatory reproductive diapause form. In this study, the complete open reading frame (ORF) sequence of the ecdysone receptor (EcR) was cloned from G. daurica. Application of 20E promoted the expression of EcR, nuclear hormone receptor (HR3), and vitellogenin (Vg), whereas it reduced the expression of fatty acid synthase (FAS) and lipid content, leading to delayed diapause entry in female adults. Silencing EcR or HR3 by RNAi increased the expression of FAS and lipid content, whereas it reduced the expression of Vg, inducing reproductive diapause. These results indicate that 20E may mediate reproductive diapause via a conserved EcR/HR3 cascade in G. daurica. Full article

The red palm weevil (RPW) is an invasive pest that causes devastating damage to a variety of palm plants, which exhibit specific immune priming to Bacillus thuringiensis (Bt). However, immune priming in RPW may incur a high fitness cost, and its molecular signaling pathways have not yet been reported. Here, we investigated the effect of Bt priming on RPW development and subsequently analyzed the hormonal and immune-related molecular pathways influencing the fitness cost induced by Bt priming. Bt priming delayed the body weight gain of fifth-instar larvae and prolonged their developmental duration. Bt priming significantly reduced the 20-hydroxyecdysone (20E) content in RPW hemolymph, and the expression levels of the 20E biosynthesis-related genes SHADOW and SHADE were significantly downregulated. Furthermore, we analyzed Toll pathway genes influencing Bt priming and found that only Spätzle (SPZ) transcription was significantly activated under Bt priming. After silencing SPZ expression, the negative effects of Bt priming on development, SHADOW expression, and 20E synthesis were eliminated, thereby suggesting that SPZ is a key molecular signal mediating developmental and immune trade-offs induced by Bt priming. Our results elucidate the molecular cascade pathway of immune priming and provide new targets for improving the efficiency of RPW biological controls. Full article

Spinacia oleracea L. cultivar platypus leaves are identified as a functional food due to their nutrient composition which promotes health beyond basic nutrition. Considering the increasing use of food supplements, S. oleracea baby leaves have been extracted by maceration, solid–liquid dynamic extraction (SLDE)-Naviglio, and ultrasound-assisted extraction (UAE) using EtOH and EtOH:H₂O mixtures. The analysis of the extracts by using LC-ESI/HRMSMS revealed 42 compounds (flavonoids, polar lipid derivatives, and 20-hydroxyecdysone), along with primary metabolites, detected by NMR analysis. A principal component analysis (PCA) of LC-ESI/HRMS and NMR data was performed, revealing how 20-hydroxyecdysone and flavonoids, the specialized metabolites mainly responsible for the biological activity of S. oleracea leaves, occurred in the highest amount in the EtOH and EtOH:H₂O (70:30, v/v) extracts obtained by SLDE-Naviglio extraction. 20-hydroxyecdysone was also quantified in all the extracts via LC-ESI/QTrap/MS/MS using the Multiple Reaction Monitoring (MRM) method. The EtOH extracts obtained by SLDE-Naviglio and maceration showed the highest content (82.16 and 81.27 mg/g extract, respectively). The total phenolic content (118.35–206.60 mg GAE/g), the flavonoid content (10.90–41.05 mg rutin/g), and the Trolox Equivalent Antioxidant Capacity (TEAC) (1.63–2.05 mM) of the extracts were determined. The EtOH:H₂O (70:30, v/v) extract analyzed by using SLDE-Naviglio showed the highest phenolic and flavonoid content and radical scavenging activity. Full article

RNA interference (RNAi)-based biotechnology has been previously implemented in decapod crustaceans. Unlike traditional RNAi methodologies that investigate single gene silencing, we employed a multigene silencing approach in decapods based on chimeric double-stranded RNA (dsRNA) molecules coined ‘gene blocks’. Two dsRNA constructs, each targeting three genes of the crustacean hyperglycaemic hormone (CHH) superfamily of neuropeptides, were produced: Type II construct targeting Cq-Molt-inhibiting hormone 1 (MIH1), Cq-MIH-like 1 (MIHL1), and Cq-MIHL2 isoforms and Type I construct targeting Cq-ion transport peptide (Cq-ITP; a putative hybrid of CHH and MIH) and Cq-CHH and Cq-CHH-like (CHHL) isoforms. Both constructs were injected into juvenile redclaw crayfish, Cherax quadricarinatus, to determine the effects of multigene knockdown on molting and developmental processes. A 20-Hydroxyecdysone (20E) enzyme-linked immunosorbent assay (ELISA) and glucose assay were used to determine the effects of RNAi on molting and hemolymph glycemic activities, respectively. Multigene silencing reduced the intermolt interval by 23%. Statistically significant elevated 20E was recorded in treated intermolt individuals, consistent with the reduced intermolt interval as well as unique and abnormal phenotypes related to the molting process, which indicates a shift in 20E-induced cascade. There was no effect of RNAi treatment on hemolymph glucose level or molt increment. Through multigene silencing and subsequent annotation of gene networks, gene blocks may provide a tailored approach to investigate complex polygenic traits with RNAi in a more efficient and scalable manner. Full article

20-Hydroxyecdysone (20E) is an arthropod steroid hormone that possesses a number of beneficial pharmacological activities in humans, including anabolic, antioxidant, hypoglycemic, cardioprotective, hepatoprotective, neuroprotective, and antineoplastic properties, etc. While several studies have explored the anabolic activity of 20E in muscle cells, they have concentrated on its effects on myofibril size, protein biosynthesis intensity, and myostatin expression, without assessing energy metabolism. In this research, we have demonstrated that 20E boosts both catabolism and anabolism, coupling energy-producing and biosynthetic metabolic processes in mouse myoblasts and fibroblasts in the same way. Using a transcriptomic approach, we identified the 20E-mediated up-regulation of genes involved in different metabolic processes. Further experiments revealed that 20E increased the levels of enzymes involved in glycolysis and one-carbon metabolism. It also increased the uptake of glucose, glycolysis, respiration, the production of ATP, and global protein biosynthesis in mouse myoblasts and fibroblasts. This phenomenon involves the PI3K/AKT/mTOR signaling pathway. Taken together, the observed 20E-dependent upregulation of energy metabolism may be the main reason for 20E’s well-known anabolic activity. Full article

A comprehensive metabolite profiling of the medicinal plant Silene viridiflora using an UHPLC-ESI-MS/MS method is described for the first time. A total of 71 compounds were identified and annotated, the most common of which were flavonoids, triterpene glycosides, and ecdysteroids. The three major compounds schaftoside, 26-hydroxyecdysone, and silviridoside can be chosen as the markers for the assessment of the quality of S. viridiflora preparations. The methanol extract and a variety of metabolites identified in S. viridiflora were screened for their cytotoxic and Wnt pathway-inhibiting activities against triple-negative breast cancer (TNBC), the deadliest form of cancer in women. 2-Deoxy-20-hydroxyecdysone with submicromolar IC₅₀ was identified as a result. The structure–activity relationship derived from the data from the in vitro proliferation assay showed that the hydroxyl group present at position C-2 of steroid core reduces the ecdysteroids’ cytotoxicity against cancer cells. Full article

Achyranthes japonica Nakai (AJN) is a medicinal plant known to be beneficial for the joints. Since it takes at least two years from sowing to harvesting in an open field, new AJN cultivation strategies are needed to shorten the production period and improve quality. In this study, high-quality AJN is produced as microgreens in a vertical farm using a commercial ginseng soil mix (Myeongpum-Insamsangto, Shinsung Mineral Co., Ltd., Goesan, Republic of Korea) and controlled environmental conditions. The cultivation conditions included a temperature of 23 ± 2 °C, relative humidity of 50 ± 10%, and a photosynthetic photon flux density of 170 ± 15 µmol·m⁻²·s⁻¹. Pre-harvest intermittent UV-B exposure, with an intensity of 1.0 ± 0.3 W/m⁻², was applied for one day before harvest to evaluate its effects, using controlled environmental conditions in the vertical farm. Ultraviolet-B (UV-B) irradiation increases secondary metabolite levels in plants; however, the effect of UV-B on 20-hydroxyecdysone (20E), an indicator of AJN, is unclear. Therefore, we aimed to investigate whether UV-B treatment of AJN microgreens affected growth and secondary metabolites. The treatment group was set to 12 h of continuous UV-B treatment during the day, two 6 h UV-B treatments, and four 3 h UV-B treatments to confirm the effectiveness of regular and intermittent treatment and recovery. Short-term UV-B treatment before harvesting increased phenols, flavonoids, antioxidant capacity, and 20E levels without affecting AJN biomass. The intermittent 6 h UV-B irradiation with a 6 h recovery time stimulated 20E content by approximately 1.4 times compared to the control. These study findings indicate that short-term UV-B treatment before harvesting, an appropriate recovery time, and intermittent UV-B exposure are more effective at increasing 20E content than continuous treatment. This approach provides a promising strategy for improving the nutritional and health benefits of AJN microgreens in vertical farming systems. Full article

microRNAs (miRNAs) function as vital regulators of diapause in insects through their ability to post-transcriptionally suppress target gene expression. In this study, the miRNA of Ostrinia furnacalis, an economically important global crop pest species, was characterized. For the included analyses, 9 small RNA libraries were constructed using O. furnacalis larvae in different diapause states (non-diapause, ND; diapause, D; diapause-termination, DT). The results identified 583 total miRNAs, of which 256 had previously been identified, whereas 327 were novel. Furthermore, comparison analysis revealed that 119 and 27 miRNAs were differentially expressed in the D vs. ND and DT vs. D, respectively. Moreover, the expression patterns of their miRNAs were also analyzed. GO and KEGG analysis of the target genes of differentially expressed miRNAs highlighted the importance of these miRNAs as diapause regulators in O. furnacalis, especially through metabolic processes, endocrine processes, 20-hydroxyecdysone, and circadian clock signaling pathways. In summary, this study highlighted the involvement of specific miRNAs in the control of diapause in O. furnacalis. To the best of our knowledge, this is the first study to identify miRNA expression patterns in O. furnacalis, thereby providing reference and novel evidence enhancing our current understanding of how small RNAs influence insect diapause. Full article

Sawfly species of the genus Monophadnus are specialised on Ranunculaceae plants from which the larvae can sequester furostanol saponins into the haemolymph, mainly (25R)-26-[(α-L-rhamnopyranosyl)oxy]-22α-methoxyfurost-5-en-3β-yl-O-β-D-glucopyranosyl-(1→3)-O-[6-acetyl-β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranoside (compound 1). In this work, TLC, GC-MS, and HPLC-DAD-ESI/MS analyses together with feeding, repeated simulated attacks, and ant deterrence bioassays were conducted to extend the chemoecological knowledge about two sawfly species specialised on H. foetidus L. (Monophadnus species A) and H. viridis L. (Monophadnus species B). Larvae of Monophadnus species B were mostly feeding on the squares treated with the n-butanol fraction from H. foetidus, compound 1 being its primary non-nutritional stimulant. In contrast, all H. viridis fractions stimulated feeding, with n-hexane marginally more active. β-sitosterol within n-hexane was determined as the nutritional stimulant. Quantitative analyses demonstrated that leaves of H. viridis but not H. foetidus contain the ecdysteroids 20-hydroxyecdysone and polypodine B. Moreover, the haemolymph of Monophadnus species B larvae reared on H. viridis contained the glycosides of polypodine B and 20-hydroxyecdysone at a concentration of 2.5 to 6.8 µmol/g fresh weight of haemolymph. This concentration is several thousand times higher than the concentration range of the aglycones in their host plant (3.63 × 10⁻⁴ to 2.23 × 10⁻⁴ µmol total ecdysteroids/g fresh weight of leaves), suggesting bioaccumulation. The larvae of both species fed on H. foetidus do not show any traces of ecdysteroids in their haemolymph, indicating a facultative role of these compounds in their defence as well as their inability to endogenously synthesise these compounds. The haemolymph containing ecdysteroids was a significant feeding deterrent against Myrmica rubra L. ant workers (one of their natural predators) at 0.8 mg/mL. The larvae kept effective deterrent levels of glycosylated ecdysteroids (≅175 mM) between simulated attacks on days 1 and 2, but the levels clearly decreased on day 3 (≅75 mM). Most larvae (89%) survived a first attack but only 23% a consecutive second one. As a conclusion, we report for the first time that two Monophadnus species feeding on H. viridis sequester phytoecdysteroids into the larval haemolymph in the form of glycosides. In addition, compound 1 possesses defensive and phagostimulant activities, and we present evidence for a combined effect of furostanol saponins and ecdysteroids as repellents against ants. Full article

Spodoptera frugiperda (J.E. Smith) is a crucial agricultural pest owing to its global impact on >300 crops. Among these, the corn strain of S. frugiperda causes significant damage to maize (Zea mays L.). However, limited research exists on the influence of maize nutrients on the metamorphosis of S. frugiperda and the underlying mechanisms. In this study, the effects of different growth stages of maize leaves, namely, tender leaves (tender) and mature leaves (mature), on various aspects of larval development, including body weight, body length, developmental age, pupation rate, and eclosion rate, were investigated. Additionally, we measured the levels of 20-hydroxyecdysone (20E) and three types of juvenile hormone (JH; i.e., JH I–III) in S. frugiperda larvae fed on tender or mature. The results revealed that larvae fed on Tender exhibited significantly prolonged instar duration, reduced body weight and length, and decreased pupation and eclosion rates, with the emergence of abnormal adults. Analysis of nutritional components in maize leaves revealed significantly higher levels of amino acids, soluble sugars, and sterols in mature than in tender. Hormone analysis in S. frugiperda larvae revealed higher 20E titers in individuals feeding on mature during prepupal and pupal stages. We demonstrated the crucial role of sterols in regulating the level of 20E and pupation rate of S. frugiperda. Based on these findings, we propose that isoleucine, arginine, glutamic acid, sucrose, campesterol, and β-sitosterol serve as key nutrients influencing the development of S. frugiperda. Moreover, β-sitosterol is a significant factor influencing the interaction between maize leaves and S. frugiperda. Our research results provide a reference for the control strategy of S. frugiperda based on breeding insect-resistant varieties by altering host nutrition. Full article

Furofuran lignans have been identified as the main substances responsible for the biological activities of the plant genus Phryma. Here, four new phrymarolin-type leptolignans A–D (7–10) and eight previously known lignans were isolated from P. leptostachya. Of these, nine exhibited significant antifeedant activity against armyworm (Mythimna separata) through a dual-choice bioassay, with the EC₅₀ values ranging from 0.58 to 10.08 μg/cm². In particular, the newly identified lignan leptolignan A (7) showed strong antifeedant activity, with an EC₅₀ value of 0.58 ± 0.34 μg/cm². Further investigation found that leptolignan A can inhibit the growth and nutritional indicators in the armyworm M. separata. The concentrations of two molting hormones, 20-hydroxyecdysone and ecdysone, were also found to decrease significantly following the treatment of the armyworms with the lignan, implying that the target of the P. leptostachya lignan may be involved in 20-hydroxyecdysone and ecdysone synthesis. These results enrich our knowledge of P. leptostachya metabolite structural diversity, and provide a theoretical basis for the control of armyworm using lignans. Full article

In holometabolous insects, such as Drosophila and Bombyx, prothoracicotropic hormone (PTTH) is well established to be critical in controlling developmental transitions and metamorphosis by stimulating the biosynthesis of ecdysone in the prothoracic glands (PGs). However, the physiological role of PTTH and the receptor Torso in hemimetabolous insects remains largely unexplored. In this study, homozygous PTTH- and Torso-null mutants of the brown planthopper (BPH), Nilaparvata lugens, were successfully generated by employing clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR–Cas9). Further characterization showed that both NlPTTH^−/− and NlTorso^−/− mutants exhibited prolonged nymphal duration and increased final adult size. Enzyme-linked immunosorbent assay (ELISA) revealed that NlPTTH^−/− and NlTorso^−/− mutants exhibited a significant reduction in 20-hydroxyecdysone (20E) in fifth-instar nymphs at 48 h post-ecdysis compared to Wt controls. Furthermore, our results indicated that both NlPTTH^−/− and NlTorso^−/− mutants had shortened lifespan, reduced female fecundity, and reduced egg hatching rates in adults. These findings suggest a conserved role for the PTTH–Torso signaling system in the regulation of developmental transitions by stimulating ecdysone biosynthesis in hemimetabolous insects. Full article

During choriogenesis in insects, chorion (eggshell) is formed by surrounding follicular epithelial cells in ovarioles. However, the regulatory endocrine factor(s) activating choriogenesis and the effect of chemical components on eggshell deserve further exploration. In two representative coleopterans, a coccinellid Henosepilachna vigintioctopunctata and a chrysomelid Leptinotarsa decemlineata, genes encoding the 20-hydroxyecdysone (20E) receptor heterodimer, ecdysone receptor (EcR) and ultraspiracle (USP), and two chitin biosynthesis enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP) and chitin synthase (ChS1), were highly expressed in ovaries of the young females. RNA interference (RNAi)-aided knockdown of either HvEcR or Hvusp in H. vigintioctopunctata inhibited oviposition, suppressed the expression of HvChS1, and lessened the positive signal of Calcofluor staining on the chorions, which suggests the reduction of a chitin-like substance (CLS) deposited on eggshells. Similarly, RNAi of LdEcR or Ldusp in L. decemlineata constrained oviposition, decreased the expression of LdUAP1 and LdChS1, and reduced CLS contents in the resultant ovaries. Knockdown of LdUAP1 or LdChS1 caused similar defective phenotypes, i.e., reduced oviposition and CLS contents in the L. decemlineata ovaries. These results, for the first time, indicate that 20E signaling activates choriogenesis in two coleopteran species. Moreover, our findings suggest the deposition of a CLS on the chorions. Full article

E-20-monooxygenase (E20MO) is an enzymatic product of the shade (shd) locus (cytochrome p450, E20MO). Initially discovered in Drosophila, E20MO facilitates the conversion of ecdysone (E) into 20-hydroxyecdysone (20E) and is crucial for oogenesis. Prior research has implicated 20E in growth, development, and insecticide resistance. However, little attention has been given to the association between the E20MO gene and DENV2 infection. The transcriptome of Ae. aegypti cells (Aag2 cells) infected with DENV2 revealed the presence of the E20MO gene. The subsequent quantification of E20MO gene expression levels in Aag2 cells post-DENV infection was carried out. A CRISPR/Cas9 system was utilized to create an E20MO gene knockout cell line (KO), which was then subjected to DENV infection. Analyses of DENV2 copies in KO and wild-type (WT) cells were conducted at different days post-infection (dpi). Plasmids containing E20MO were constructed and transfected into KO cells, with pre- and post-transfection viral copy comparisons. Gene expression levels of E20MO increased after DENV infection. Subsequently, a successful generation of an E20MO gene knockout cell line and the verification of code-shifting mutations at both DNA and RNA levels were achieved. Furthermore, significantly elevated DENV2 RNA copies were observed in the mid-infection phase for the KO cell line. Viral RNA copies were lower in cells transfected with plasmids containing E20MO, compared to KO cells. Through knockout and plasmid complementation experiments in Aag2 cells, the role of E20MO in controlling DENV2 replication was demonstrated. These findings contribute to our understanding of the intricate biological interactions between mosquitoes and arboviruses. Full article