Metamorphosis is a complex developmental process involving multiple pathways and a large number of genes that are regulated by juvenile hormone(JH)and 20-hydroxyecdysone(20E).Despite important progress in understanding various aspects of silkworm biology,the hormone signaling pathway in the silkworm remains poorly understood.Genome-wide screening using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-based libraries has recently emerged as a novel method for analyzing genome function,enabling further research into essential genes,drug targets,and virus-host interaction.Previously,we constructed a genome-wide CRISPR/Cas9-based library of the silkworm(Bombyx mori)and successfully revealed the genes involved in biotic or abiotic stress factor responses.In this study,we used our silkworm CRISPR library and large-scale genome-wide screening to analyze the key genes in the silkworm 20E signaling pathway and their mechanisms of action.Functional annotation showed that 20E regulates key proteins in processes that mainly occur in the cytoplasm and nucleus.Pathway enrichment analysis showed that 20E can activate phosphorylation and may affect innate immunity,interfere with intracellular nutrition and energy metabolism,and eventually cause cell apoptosis.The screening results were experimentally validated by generating cells with knockout alleles of the relevant genes,which had increased tolerance to 20E.Our findings provide a panoramic overview of signaling in response to 20E in the silkworm,underscoring the utility of genome-wide CRISPR mutant libraries in deciphering hormone signaling pathways and the mechanisms that regulate metamorphosis in insects.
The steroid hormone 20-hydroxyecdysone(20E)has been described to regulate fat body lipid metabolism in insects,but its accurate regulatory mechanism,especially the crosstalk between 20E-induced lipid metabolism and gluconeogenesis remains largely unclear.Here,we specially investigated the effect of 20E on lipid metabolism and gluconeogenesis in the fat body of Hyphantria cunea larvae,a notorious pest in forestry.Lipidomics analysis showed that a total of 1907 lipid species were identified in the fat body of H.cunea larvae assigned to 6 groups and 48 lipid classes.The differentially abundant lipids analysis showed a significant difference between 20E-treated and control samples,indicating that 20E caused a remarkable alteration of lipidomics profiles in the fat body of H.cunea larvae.Further studies demonstrated that 20E accelerated fatty acidβ-oxidation,inhibited lipid synthesis,and promoted lipolysis.Meanwhile,the activities of pyruvate carboxylase,phosphoenolpyruvate carboxykinase,fructose-1,6-bisphosphatase,and glucose-6-phosphatase were dramatically suppressed by 20E in the fat body of H.cunea larvae.As well,the transcriptions of genes encoding these 4 rate-limiting gluconeogenic enzymes were significantly downregulated in the fat body of H.cunea larvae after treatment with 20E.Taken together,our results revealed that 20E disturbed fat body lipid homeostasis,accelerated fatty acidβ-oxidation and promoted lipolysis,but negatively regulated gluconeogenesis in H.cunea larvae.The findings might provide a new insight into hormonal regulation of glucose and lipid metabolism in insect fat body.
20E-hydroxyecdysone(20E)plays important roles in larval molting and metamorphosis in insects and is also involved in the insect innate immune response.Insect metamorphosis is a highly successful strategy for environmental adaptation and is the most vulnerable stage during which the insect is susceptible to various pathogens.20E regulates a series of antimicrobial peptides(AMPs)through the immunodeficiency(IMD)pathway activation in Drosophila;nevertheless,whether other immune pathways are involved in 20E-regulated insect immunity is unknown.Our previous studies showed that BmMD-2A is a member of the MD-2-related lipid recognition(ML)family of proteins that are involved in the Bombyx mori innate immunity Toll signaling pathway.In this study,we further demonstrate that BmMD-2A is also positively regulated by 20E,and the BmMD-2A neutralization experiment suggested that 20E activates some downstream immune effect factors,the AMP genes against Escherichia coli and Staphylococcus aureus,through the regulation of BmMD-2A in larval metamorphosis,implying that B.mori may use the Toll-ML signaling pathway to maintain innate immune balance in the larval-pupal metamorphosis stage,which is a different innate immunity pathway regulated by 20E compared to the IMD pathway in Drosophila.
Ruonan ZhangXue ChenYuan WangXu BaiQiong YangYangjin ZhongXiao-Qiang YuFengliang JinWanying Yang
Juvenile hormone(JH) and 20-hydroxyecdysone(20 E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant(Met) and germ-cell expressed(Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases.Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C(PKC) which phosphorylated ultraspiracle(USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20 E through its nuclear receptor complex Ec RUSP. The usp;mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20 E signaling that delayed developmental timing. The usp;mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20 E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.
20-Hydroxyecdysone(20E)derived from plants has a wide range of physiological and pharmacological ef ects on animals and humans,and rapid and sensitive methods for screening of the endogenous 20E in plants are thus required.Herein,a matrixassisted laser desorption/ionization time-of-f ight tandem mass spectrometry(MALDI-TOF/TOF MS)method is described for rapid and sensitive determination of endogenous 20E in plants.It is based on the use of the(3-(acridin-9-ylamino)phenyl)boronic acid(AYPBA)as the mass tag to assist the MS and tandem MS(MS^(n))analysis of 20E on MALDI-TOF/TOF MS.Good linearity was obtained with a determination coef cient(R^(2))larger than 0.99 in the range of 0.025–2.5μΜ.The limit of detection(LOD)was 2.4 fmol.Acceptable precision and accuracy were gained by intra-and inter-day analysis with relative standard deviations less than 19.5%and relative recoveries ranging from 85.7 to 105.2%.In addition,the AYPBA labeled 20E produced abundant characteristic fragment ions under the high energy collision-induced dissociation,which facilitated the identif cation of 20E by MS^(2)analysis on MALDI-TOF/TOF MS.Using the method,we enabled the identif cation and quantif cation of endogenous 20E in four herbs including Cyanotis arachoidea,Achyranthes bidentata,Spinacia oleracea and Chenopodium quinoa willd.,demonstrating the feasibility of the proposed method for screening of the endogenous 20E in plants.
Objective The hippocampus is thought to be a vulnerable target of microwave exposure.The aim of the present study was to investigate whether 20-hydroxyecdysone(20E)acted as a fate regulator of adult rat hippocampal neural stem cells(NSCs).Furthermore,we investigated if 20E attenuated high power microwave(HMP)radiation-induced learning and memory deficits.Methods Sixty male Sprague-Dawley rats were randomly divided into three groups:normal controls,radiation treated,and radiation+20E treated.Rats in the radiation and radiation+20E treatment groups were exposed to HPM radiation from a microwave emission system.The learning and memory abilities of the rats were assessed using the Morris water maze test.Primary adult rat hippocampal NSCs were isolated in vitro and cultured to evaluate their proliferation and differentiation.In addition,hematoxylin&eosin staining,western blotting,and immunofluorescence were used to detect changes in the rat brain and the proliferation and differentiation of the adult rat hippocampal NSCs after HPM radiation exposure.Results The results showed that 20E induced neuronal differentiation of adult hippocampal NSCs from HPM radiation-exposed rats via the Wnt3a/β-catenin signaling pathway in vitro.Furthermore,20E facilitated neurogenesis in the subgranular zone of the rat brain following HPM radiation exposure.Administration of 20E attenuated learning and memory deficits in HPM radiation-exposed rats and frizzled-related protein(FRZB)reduced the 20E-induced nuclear translocation ofβ-catenin,while FRZB treatment also reversed 20E-induced neuronal differentiation of NSCs in vitro.Conclusion These results suggested that 20E was a fate regulator of adult rat hippocampal NSCs,where it played a role in attenuating HPM radiation-induced learning and memory deficits.
LIU Jing JingZHANG Hong YanCHEN XinZHANG Guang BinLIN Jiang KaiFENG HuaCHU Wei Hua
A wide array of biological events in the life history of insects are orchestrated by juvenile hormone (JH) and 20-hydroxyecdysone(20E)[1]. They control embryonic development, molting, metamorphosis, and reproduction. Some processes are governed by one of the two hormones while others rely on both. Larval development and metamorphosis in the tobacco hornworm, Manduca sexta, is a classic example manifesting the coordination of JH and20E. 20E triggers every molt in M. sexta, from larval-larval molt to larval-pupal and pupal-adult molts;the nature of molting is defined by JH [2]. High levels of JH in the early larva instars exert the anti-metamorphic function and 20E initiates a molt that leads to another larger larva. Once the hornworm reaches a critical weight (a body size "check-point"), the synthesis of JH is halted.In the absence of JH, 20E instigates the metamorphic program.
The insulin and 20-hydroxyecdysone(20E)pathways coordinately regulate in-sect vitellogenesis and ovarian development.However,the detailed molecular mechanisms such as the genes mediating the cooperation of the interaction of these 2 pathways in reg-ulating insect reproductive development are not well understood.In the present study,a small GTPase,Rab4oC,was identified from the notorious agricultural pest Bactrocera dorsalis.In addition to the well-known RAB domain,it also has a unique SOCS-box do-main,which is different from other Rab-GTPases.Moreover,we found that Rab40C was enriched in the ovaries of sexually mature females.RNA interference(RNAi)-mediated knockdown of BdRab40C resulted in a decrease in vitellogenin synthesis,underdevel-oped ovaries,and low fertility.Furthermore,depletion of insulin receptor InR or the het-erodimer receptor of 20E(EcRor USP)by RNAi significantlydecreased the transcription of BdRab40C and resulted in lower fecundity.Further studies revealed that the transcrip-tion of BdRab40C could be upregulated by the injection of insulin or 20E.These results indicate that Rab40C participates in the insulin and 20E pathways to coordinately regulate reproduction in B.dorsalis.Our results not only provide new insights into the insulin-and 20E-stimulated regulatory pathways controlling female reproduction in insects but also contribute to the development of potential eco-friendly strategies for pest control.
