Chemical Treatments for Insect Cell Differentiation: The Effects of 20-Hydroxyecdysone and Veratridine on Cultured Spodoptera frugiperda ( Sf 21) Insect Cell Ultrastructure

Previous studies have shown that insect cell cultures stop dividing, form clumps, and can be induced to grow processes reminiscent of axons, when the culture medium is supplemented with 20-hydroxyecdysone, insulin, or an agent that mimics their action, such as the ecdysone agonist, methoxyfenozide. Those cell growing processes resemble nerve cells, and the present study evaluates the ultrastructure of these cultures by transmission electron microscopy. <i>Sf</i>21 cells treated with 20-hydroxyecdysone (with or without veratridine amendment) and subjected to ultrastructural analysis had a similar somatic appearance to control cells, with slight changes in organelles and organization, such as a greater number of cytoplasmic vacuoles and mitochondrial granules.
Finger-like projections were observed between control and treated cells. However, no structural markers of synaptic contacts (e.g., vesicles or synaptic thickenings) were observed in controls, 20-hydroxyecdysone, or 20-hydroxyecdysone + veratridine treated cells. It is concluded that additional agents would be required to induce functional synaptogenesis in <i>Sf</i>21 cells.

Activation of the ROS/CncC and 20-Hydroxyecdysone Signaling Pathways Is Associated with Xanthotoxin-Induced Tolerance to λ-Cyhalothrin in Spodoptera litura

Adaptation to phytochemicals in herbivorous insects can influence tolerance to insecticides. However, it is unclear how insects use phytochemicals as cues to activate their metabolic detoxification systems. In this study, we found that dietary exposure to xanthotoxin enhanced tolerance of Spodoptera litura larvae to λ-cyhalothrin. Xanthotoxin ingestion significantly elevated the mRNA levels of 35 detoxification genes as well as the transcription factors Cap ‘n’ collar isoform-C (CncC) and its binding factor small muscle aponeurosis fibromatosis isoform-K (MafK). Additionally, xanthotoxin exposure increased the levels of reactive oxygen species (ROS), while ROS inhibitor N-acetylcysteine (NAC) treatment blocked xanthotoxin-induced expression of CncCMafK, and detoxification genes and also prevented xanthotoxin-enhanced larval tolerance to λ-cyhalothrin.
The 20-hydroxyecdysone (20E) signaling pathway was effectively activated by xanthotoxin, while blocking of 20E signaling transduction prevented xanthotoxin-enhanced larval tolerance to λ-cyhalothrin. Application of 20E induced the expression of multiple xanthotoxin-induced detoxification genes and enhanced λ-cyhalothrin tolerance in S. litura. NAC treatment blocked xanthotoxin-induced 20E synthesis, while the CncC agonist curcumin activated the 20E signaling pathway. These results indicate that the ROS/CncC pathway controls the induction of metabolic detoxification upon exposure to xanthotoxin, at least in part, through its regulation of the 20E signaling pathway.

Characterization of Heat Shock Protein 60 as an Interacting Partner of Superoxide Dismutase 2 in the Silkworm, Bombyx mori, and Its Response to the Molting Hormone, 20-Hydroxyecdysone

Oxidative stress promotes pupation in some holometabolous insects. The levels of superoxide, a reactive oxygen species (ROS), are increased and superoxide dismutase 1 (BmSod1) and superoxide dismutase 2 (BmSod2) are decreased during metamorphic events in silkworm (Bombyx mori). These observations strongly suggest that pupation is initiated by oxidative stress via the down-regulation of BmSod1 and BmSod2. However, the molecular mechanisms underlying ROS production during metamorphic events in silkworm remain unknown. To investigate these molecular mechanisms, the peripheral proteins of BmSod1 and BmSod2 were identified and characterized using dry and wet approaches in this study.
Based on the results, silkworm heat shock protein 60 (BmHsp60) was identified as an interacting partner of BmSod2, which belongs to the Fe/MnSOD family. Furthermore, the present study results showed that BmHsp60 mRNA expression levels were increased in response to oxidative stress caused by ultraviolet radiation and that BmHsp60 protein levels (but not mRNA levels) were decreased during metamorphic events, which are regulated by the molting hormone 20-hydroxyecdysone. These findings improve our understanding of the mechanisms by which holometabolous insects control ROS during metamorphosis.

RNA interference shows that Spook, the precursor gene of 20-hydroxyecdysone (20E), regulates the molting of Macrobrachium nipponense

  • The aim of this study was to explore the function of the Mn-Spook gene, which was found in the ovary transcriptome of the Oriental river prawn (Macrobrachium nipponense). The Spook gene, which is the precursor gene of 20-hydroxyecdysone (20E), plays an important role in the process of molting in many arthropods, but its function in M. nipponense is unclear.
  • We cloned the full-length Mn-Spook gene from the ovary of M. nipponense and found that it had the same conserved domains as the P450 gene of the Halloween family of genes. The Mn-Spook gene was highly expressed in ovary and gill tissue during the breeding period. During ovarian development, Mn-spook gene expression was highest at the nearly-ripe stage, and it also was highly expressed in the zoea developmental stage. Cellular localization analysis showed that Mn-Spook signals accumulated in the cytoplasmic membrane and nucleus of oocytes.
  • Finally, we used RNA interference to evaluate the function of the Mn-Spook gene. Compared with the control group, in vivo injection of Mn-Spook dsRNA effectively downregulated the expression of Mn-Spook and the content of 20E. The molting frequency of M. nipponense in the experimental group also was significantly inhibited. These results demonstrated that the Mn-Spook gene played an important role in the molting process of M. nipponense.


H918750 Toronto Research Chemicals 10g 293 EUR


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20-Hydroxyecdysone, 90%

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20-Hydroxyecdysone, 97%

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H918752 Toronto Research Chemicals 100mg 4500 EUR

CRAB 20-Hydroxyecdysone

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20-hydroxyecdysone (20E) signaling regulates amnioserosa morphogenesis during Drosophila dorsal closure: EcR modulates gene expression in a complex with the AP-1 subunit, Jun

  • Steroid hormones influence diverse biological processes throughout the animal life cycle, including metabolism, stress resistance, reproduction, and lifespan. In insects, the steroid hormone, 20-hydroxyecdysone (20E), is the central hormone regulator of molting and metamorphosis, and plays roles in tissue morphogenesis. For example, amnioserosa contraction, which is a major driving force in Drosophila dorsal closure (DC), is defective in embryos mutant for 20E biosynthesis.
  • Here, we show that 20E signaling modulates the transcription of several DC participants in the amnioserosa and other dorsal tissues during late embryonic development, including zipper, which encodes for non-muscle myosin. Canonical ecdysone signaling typically involves the binding of Ecdysone receptor (EcR) and Ultraspiracle heterodimers to ecdysone-response elements (EcREs) within the promoters of responsive genes to drive expression.
  • During DC, however, we provide evidence that 20E signaling instead acts in parallel to the JNK cascade via a direct interaction between EcR and the AP-1 transcription factor subunit, Jun, which together binds to genomic regions containing AP-1 binding sites but no EcREs to control gene expression. Our work demonstrates a novel mode of action for 20E signaling in Drosophila that likely functions beyond DC, and may provide further insights into mammalian steroid hormone receptor interactions with AP-1.

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