ABSTRACT
BACKGROUND: Free fatty acids (FFAs) play vital roles as energy sources and substrates in organisms; however, the molecular mechanism regulating the homeostasis of FFA levels in various circumstances, such as feeding and nonfeeding stages, is not fully clarified. Holometabolous insects digest dietary triglycerides (TAGs) during larval feeding stages and degrade stored TAGs in the fat body during metamorphosis after feeding cessation, which presents a suitable model for this study. RESULTS: This study reported that two lipases are differentially regulated by hormones to maintain the homeostasis of FFA levels during the feeding and nonfeeding stages using the lepidopteran insect cotton bollworm Helicoverpa armigera as a model. Lipase member H-A-like (Lha-like), related to human pancreatic lipase (PTL), was abundantly expressed in the midgut during the feeding stage, while the monoacylglycerol lipase ABHD12-like (Abhd12-like), related to human monoacylglycerol lipase (MGL), was abundantly expressed in the fat body during the nonfeeding stage. Lha-like was upregulated by juvenile hormone (JH) via the JH intracellular receptor methoprene-tolerant 1 (MET1), and Abhd12-like was upregulated by 20-hydroxyecdysone (20E) via forkhead box O (FOXO) transcription factor. Knockdown of Lha-like decreased FFA levels in the hemolymph and reduced TAG levels in the fat body. Moreover, lipid droplets (LDs) were small, the brain morphology was abnormal, the size of the brain was small, and the larvae showed the phenotype of delayed pupation, small pupae, and delayed tissue remodeling. Knockdown of Abhd12-like decreased FFA levels in the hemolymph; however, TAG levels increased in the fat body, and LDs remained large. The development of the brain was arrested at the larval stage, and the larvae showed a delayed pupation phenotype and delayed tissue remodeling. CONCLUSIONS: The differential regulation of lipases expression by different hormones determines FFAs homeostasis and different TAG levels in the fat body during the feeding larval growth and nonfeeding stages of metamorphosis in the insect. The homeostasis of FFAs supports insect growth, brain development, and metamorphosis.
Subject(s)
Brain , Fatty Acids, Nonesterified , Homeostasis , Animals , Brain/metabolism , Brain/growth & development , Fatty Acids, Nonesterified/metabolism , Lipase/metabolism , Lipase/genetics , Moths/growth & development , Moths/physiology , Moths/metabolism , Larva/growth & development , Larva/metabolism , Juvenile Hormones/metabolism , Insect Proteins/metabolism , Insect Proteins/genetics , Metamorphosis, Biological/physiology , Ecdysterone/metabolismABSTRACT
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.
Subject(s)
Lignans , Animals , Lignans/pharmacology , Lignans/chemistry , Ecdysterone/pharmacology , Ecdysterone/metabolism , Moths/drug effects , Moths/growth & development , Moths/metabolism , Ecdysone/metabolism , Molting/drug effects , Plant Extracts/pharmacology , Plant Extracts/chemistryABSTRACT
Ecdysteroid molting hormones coordinate arthropod growth and development. Binding of 20-hydroxyecdysone (20E) to ecdysteroid receptor EcR/RXR activates a cascade of nuclear receptor transcription factors that mediate tissue responses to hormone. Insect ecdysteroid responsive and Forkhead box class O (FOXO) transcription factor gene sequences were used to extract orthologs from blackback land crab (Gecarcinus lateralis) Y-organ (YO) transcriptome: Gl-Ecdysone Receptor (EcR), Gl-Broad Complex (Br-C), Gl-E74, Gl-Hormone Receptor 3 (HR3), Gl-Hormone Receptor 4 (HR4), Gl-FOXO, and Gl-Fushi tarazu factor-1 (Ftz-f1). Quantitative polymerase chain reaction quantified mRNA levels in tissues from intermolt animals and in YO of animals induced to molt by multiple limb autotomy (MLA) or eyestalk ablation (ESA). Gl-EcR, Gl-Retinoid X Receptor (RXR), Gl-Br-C, Gl-HR3, Gl-HR4, Gl-E74, Gl-E75, Gl-Ftz-f1, and Gl-FOXO were expressed in all 10 tissues, with Gl-Br-C, Gl-E74, Gl-E75, and Gl-HR4 mRNA levels in the YO lower than those in most of the other tissues. In MLA animals, molting had no effect on Gl-Br-C, Gl-E74, and Gl-Ftz-f1 mRNA levels and little effect on Gl-EcR, Gl-E75, and Gl-HR4 mRNA levels. Gl-HR3 and Gl-FOXO mRNA levels were increased during premolt stages, while Gl-RXR mRNA level was highest during intermolt and premolt stages and lowest at postmolt stage. In ESA animals, YO mRNA levels were not correlated with hemolymph ecdysteroid titers. ESA had no effect on Gl-EcR, Gl-E74, Gl-HR3, Gl-HR4, Gl-Ftz-f1, and Gl-FOXO mRNA levels, while Gl-RXR, Gl-Br-C, and Gl-E75 mRNA levels were decreased at 3 days post-ESA. These data suggest that transcriptional up-regulation of Gl-FOXO and Gl-HR3 contributes to increased YO ecdysteroidogenesis during premolt. By contrast, transcriptional regulation of ecdysteroid responsive genes and ecdysteroidogenesis were uncoupled in the YO of ESA animals.
Subject(s)
Ecdysteroids , Molting , Animals , Molting/genetics , Ecdysteroids/metabolism , Ecdysteroids/genetics , Receptors, Steroid/genetics , Receptors, Steroid/metabolism , Ecdysterone/metabolism , Brachyura/genetics , Brachyura/metabolism , Brachyura/growth & development , Endocrine Glands/metabolismABSTRACT
In crustaceans, the steroid hormone 20-hydroxyecdysone (20E) initiates molting, and the molting process is also regulated by energy metabolism. AMPK is an energy sensor and plays a critical role in systemic energy balance. Here, the regulatory mechanism in the interaction between 20E and AMPK was investigated in Chinese mitten crab, Eriocheir sinensis. The results showed that the 20E concentration and the mRNA expression levels of 20E receptors in hepatopancreas were down-regulated post AMPK activator (AICAR) treatment, and were up-regulated after AMPK inhibitor (Compound C) injection in crabs. Besides, the molt-inhibiting hormone (MIH) gene expression in eyestalk showed the opposite patterns in response to the AICAR and Compound C treatment, respectively. Further investigation found that there was a significant reduction in 20E concentration post PI3K inhibitor (LY294002) treatment, and the phosphorylation level of PI3K was increased in hepatopancreas after AMPK inhibitor injection. On the other hand, the positive regulation of PI3K-mediated activation of AMPK was also observed, the phosphorylation levels of AMPKα, AMPKß and PI3K in hepatopancreas were significantly increased post 20E injection. In addition, the phosphorylation levels of AMPKα and AMPKß induced by 20E were decreased after the injection of PI3K inhibitor. Taken together, these results suggest that the regulatory cross-talk between 20E and AMPK is likely to act through PI3K pathway in E. sinensis, which appeared to be helpful for a better understanding in molting regulation.
Subject(s)
AMP-Activated Protein Kinases , Brachyura , Ecdysterone , Hepatopancreas , Molting , Phosphatidylinositol 3-Kinases , Animals , Brachyura/immunology , Ecdysterone/metabolism , AMP-Activated Protein Kinases/metabolism , Hepatopancreas/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Invertebrate Hormones/metabolism , Chromones/pharmacology , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Ribonucleotides/pharmacology , Morpholines/pharmacology , Arthropod Proteins/metabolism , Arthropod Proteins/genetics , Phosphorylation , Energy MetabolismABSTRACT
Nuclear receptors (NRs) are ligand-regulated transcription factors that are important for the normal growth and development of insects. However, systematic function analysis of NRs in the molting process of Lasioderma serricorne has not been reported. In this study, we identified and characterized 16 NR genes from L. serricorne. Spatiotemporal expression analysis revealed that six NRs were mainly expressed in 3-d-old 4th-instar larvae; five NRs were primarily expressed in 5-d-old adults and four NRs were predominately expressed in prepupae. All the NRs were highly expressed in epidermis, fat body and foregut. RNA interference (RNAi) experiments revealed that knockdown of 15 NRs disrupted the larva-pupa-adult transitions and caused 64.44-100 % mortality. Hematoxylin-eosin staining showed that depletion of 12 NRs prevented the formation of new cuticle and disrupted apolysis of old cuticle. Silencing of LsHR96, LsSVP and LsE78 led to newly formed cuticle that was thinner than the controls. The 20E titer and chitin content significantly decreased by 17.67-95.12 % after 15 NR dsRNA injection and the gene expression levels of 20E synthesis genes and chitin metabolism genes were significantly reduced. These results demonstrated that 15 NR genes are essential for normal molting and metamorphosis of L. serricorne by regulating 20E synthesis and chitin metabolism.
Subject(s)
Coleoptera , Gene Expression Regulation, Developmental , Metamorphosis, Biological , Molting , Receptors, Cytoplasmic and Nuclear , Animals , Molting/genetics , Metamorphosis, Biological/genetics , Coleoptera/genetics , Coleoptera/growth & development , Coleoptera/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Larva/genetics , Larva/growth & development , Chitin/metabolism , RNA Interference , Insect Proteins/genetics , Insect Proteins/metabolism , Phylogeny , Ecdysterone/metabolismABSTRACT
In insects, the expression of 20E response genes that initiate metamorphosis is triggered by a pulse of 20-hydroxyecdysone (20E). The 20E pulse is generated through two processes: synthesis, which increases its level, and inactivation, which decreases its titer. CYP18A1 functions as an ecdysteroid 26-hydroxylase and plays a role in 20E removal in several representative insects. However, applying 20E degradation activity of CYP18A1 to other insects remains a significant challenge. In this study, we discovered high levels of Hvcyp18a1 during the larval and late pupal stages, particularly in the larval epidermis and fat body of Henosepilachna vigintioctopunctata, a damaging Coleopteran pest of potatoes. RNA interference (RNAi) targeting Hvcyp18a1 disrupted the pupation. Approximately 75% of the Hvcyp18a1 RNAi larvae experienced developmental arrest and remained as stunted prepupae. Subsequently, they gradually turned black and eventually died. Among the Hvcyp18a1-depleted animals that successfully pupated, around half became malformed pupae with swollen elytra and hindwings. The emerged adults from these deformed pupae appeared misshapen, with shriveled elytra and hindwings, and were wrapped in the pupal exuviae. Furthermore, RNAi of Hvcyp18a1 increased the expression of a 20E receptor gene (HvEcR) and four 20E response transcripts (HvE75, HvHR3, HvBrC, and HvαFTZ-F1), while decreased the transcription of HvßFTZ-F1. Our findings confirm the vital role of CYP18A1 in the pupation, potentially involved in the degradation of 20E in H. vigintioctopunctata.
Subject(s)
Coleoptera , Insect Proteins , Animals , Insect Proteins/genetics , Insect Proteins/metabolism , Coleoptera/genetics , Larva/genetics , Larva/metabolism , Insecta/metabolism , Metamorphosis, Biological , Ecdysterone/metabolism , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , RNA Interference , Pupa/genetics , Pupa/metabolismABSTRACT
Selective gene expression in cells in physiological or pathological conditions is important for the growth and development of organisms. Acetylation of histone H4 at K16 (H4K16ac) catalyzed by histone acetyltransferase 8 (KAT8) is known to promote gene transcription; however, the regulation of KAT8 transcription and the mechanism by which KAT8 acetylates H4K16ac to promote specific gene expression are unclear. Using the lepidopteran insect Helicoverpa armigera as a model, we reveal that the transcription factor FOXO promotes KAT8 expression and recruits KAT8 to the promoter region of autophagy-related gene 8 (Atg8) to increase H4 acetylation at that location, enabling Atg8 transcription under the steroid hormone 20-hydroxyecdysone (20E) regulation. H4K16ac levels are increased in the midgut during metamorphosis, which is consistent with the expression profiles of KAT8 and ATG8. Knockdown of Kat8 using RNA interference results in delayed pupation and repression of midgut autophagy and decreases H4K16ac levels. Overexpression of KAT8-GFP promotes autophagy and increases H4K16ac levels. FOXO, KAT8, and H4K16ac colocalized at the FOXO-binding region to promote Atg8 transcription under 20E regulation. Acetylated FOXO at K180 and K183 catalyzed by KAT8 promotes gene transcription for autophagy. 20E via FOXO promotes Kat8 transcription. Knockdown or overexpression of FOXO appeared to give similar results as knockdown or overexpression of KAT8. Therefore, FOXO upregulates KAT8 expression and recruits KAT8 to the promoter region of Atg8, where the KAT8 induces H4 acetylation to promote Atg8 transcription for autophagy under 20E regulation. This study reveals the mechanism that KAT8 promotes transcription of a specific gene.
Subject(s)
Autophagy , Ecdysterone , Helicoverpa armigera , Histone Acetyltransferases , Histones , Protein Processing, Post-Translational , Acetylation , Autophagy/genetics , Ecdysterone/metabolism , Promoter Regions, Genetic , Helicoverpa armigera/genetics , Helicoverpa armigera/metabolism , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Histones/metabolismABSTRACT
Insecticide mode of action studies provide insights into how new insecticidal actives function and contribute to assessing safety to humans and nontarget organisms. Insect cell lines that express potential target sites can serve as valuable tools in this effort. In this paper, we report on the influence of two signaling molecules on protein expression in a nervous system cell line established from Spodoptera frugiperda (Bayer/BCIRL-SfNS2-0714-TR). We selected this line because we established it in our laboratory and we are experienced in using it. Cells were exposed to the insect developmental hormone (1 µg/mL 20-hydroxyecdysone, 20E) and/or a cyclooxygenase (COX) inhibitor (25 µM indomethacin, INDO; inhibits prostaglandin [PG] biosynthesis) for 24 h (Day 2), 72 h (Day 4), or 120 h (Day 6). We selected a PG biosynthesis inhibitor because PGs act in many aspects of insect biology, such as embryonic development, immunity, and protein phosphorylation. We selected the developmental hormone, 20E, because it also acts in fundamental aspects of insect biology. We identified specific proteins via in silico analysis. Changes in protein expression levels were determined using liquid chromatography-mass spectrometry (MS) + MS-MS. The largest number of changes in protein expression occurred on Day 2. The combination of 20E plus INDO led to 222 differentially expressed proteins, which documents the deep significance of PGs and 20E in insect biology. 20E and, separately, INDO led to changes in 30 proteins each (p value < 0.01; >2X or <0.5X-fold changes). We recorded changes in the expression of 9 or 12 proteins (20E), 10 or 6 proteins (INDO), and 21 or 20 proteins (20E + INDO) on D4 and D6, respectively. While the cell line was established from neuronal tissue, the differentially expressed proteins act in a variety of fundamental cell processes. In this paper, we moved beyond a list of proteins by providing detailed, Gene Ontology term analyses and enrichment, which offers an in-depth understanding of the influence of these treatments on the SfNS2 cells. Because proteins are active components of cell physiology in their roles as enzymes, receptors, elements of signaling transduction pathways, and cellular structures, changes in their expression levels under the influence of signaling molecules provide insights into their function in insect cell physiology.
Subject(s)
Ecdysterone , Indomethacin , Humans , Animals , Ecdysterone/pharmacology , Ecdysterone/metabolism , Spodoptera/metabolism , Insecta/metabolism , Cell Line , Hormones , Nervous System/metabolism , Insect Proteins/metabolismABSTRACT
Parasitoids are important components of the natural enemy guild in the biological control of insect pests. They depend on host resources to complete the development of a specific stage or whole life cycle and thus have evolved towards optimal host exploitation strategies. In the present study, we report a specific survival strategy of a fly parasitoid Exorista sorbillans (Diptera: Tachinidae), which is a potential biological control agent for agricultural pests and a pest in sericulture. We found that the expression levels of nitric oxide synthase (NOS) and nitric oxide (NO) production in host Bombyx mori (Lepidoptera: Bombycidae) were increased after E. sorbillans infection. Reducing NOS expression and NO production with an NOS inhibitor (NG-nitro-L-arginine methyl ester hydrochloride) in infected B. mori significantly impeded the growth of E. sorbillans larvae. Moreover, the biosynthesis of 20-hydroxyecdysone (20E) in infected hosts was elevated with increasing NO production, and inhibiting NOS expression lowered 20E biosynthesis. More importantly, induced NO synthesis was required to eliminate intracellular bacterial pathogens that presumably competed for shared host resources. Inhibiting NOS expression down-regulated the transcription of antimicrobial peptide genes and increased the number of bacteria in parasitized hosts. Collectively, this study revealed a new perspective on the role of NO in host-parasitoid interactions and a novel mechanism for parasitoid regulation of host physiology to support its development.
Subject(s)
Bombyx , Diptera , Ecdysterone , Host-Parasite Interactions , Nitric Oxide , Animals , Bombyx/genetics , Bombyx/microbiology , Bombyx/parasitology , Diptera/physiology , Ecdysterone/metabolism , Larva/growth & development , Larva/parasitology , Larva/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase/geneticsABSTRACT
In the present study, we tried to clarify when and how pupal commitment (PT) better to use PC occurs and what is involved in the PT of Bombyx mori. To clarify this, we examined the responsiveness of a wing disc to ecdysone, referring to metamorphosis-related BR-C, development-related Myc and Wnt, and chromatin remodeling-related genes at around the predicted PT stage of the Bombyx wing disc. Wing disc responsiveness to juvenile hormone (JH) and ecdysone was examined using Methoprene and 20-hydroxyecdysone (20E) in vitro. The body weight of B. mori increased after the last larval ecdysis, peaked at Day 5 of the fifth larval instar (D5L5), and then decreased. The responsiveness of the wing disc to JH decreased after the last larval ecdysis up to D3L5. Bmbr-c (the Broad Complex of B. mori) showed enhanced expression in D4L5 wing discs with 20E treatment. Some chromatin remodeler and histone modifier genes (Bmsnr1, Bmutx, and Bmtip60) showed upregulation after being cultured with 20E in D4L5 wing discs. A low concentration of 20E is suggested to induce responsiveness to 20E in D4L5 wing discs. Bmbr-c, Bmsnr1, Bmutx, and Bmtip60 were upregulated after being cultured with a low concentration of 20E in D4L5 wing discs. The expression of Bmmyc and Bmwnt1 did not show a change after being cultured with or without 20E in D4L5 wing discs, while enhanced expression was observed with 20E in D5L5 wing discs. From the present results, we concluded that PT of the wing disc of B. mori occurred beginning on D4L5 with the secretion of low concentrations of ecdysteroids. Bmsnr1, Bmutx, Bmtip60, and BR-C are also involved.
Subject(s)
Bombyx , Ecdysone , Animals , Bombyx/metabolism , Chromatin Assembly and Disassembly , Pupa/genetics , Pupa/metabolism , Histone Code , Insect Proteins/genetics , Insect Proteins/metabolism , Ecdysterone/pharmacology , Ecdysterone/metabolism , Metamorphosis, Biological/physiology , Juvenile Hormones/pharmacology , Juvenile Hormones/metabolism , Larva/genetics , Larva/metabolism , Gene Expression , Gene Expression Regulation, DevelopmentalABSTRACT
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.
Subject(s)
Bombyx , Ecdysterone , Animals , Ecdysterone/metabolism , Bombyx/metabolism , Metamorphosis, Biological/genetics , Juvenile Hormones/metabolism , Signal Transduction , Insect Proteins/genetics , Insect Proteins/metabolismABSTRACT
The Dpp signaling, as one of the branches within the TGF-ß superfamily, plays a crucial role in regulating various biological processes in insects. However, its impact on female reproduction through vitellogenesis remains unclear. In this study, the expression profiles implied that the Dpp signaling genes, including Dpp, Punt, Mad, and Medea, were up-regulated during reproductive development in the ovary of Colaphellus bowringi. Knockdown of these five Dpp signaling genes revealed significant effects of Dpp, Tkv, Mad, and Medea on ovarian development through vitellogenesis in the fat body. Our finding further indicated that Dpp signaling influences the expression of 20-hydroxyecdysone (20E) receptor and responsive genes in the fat body. Additionally, knockdown of 20E receptor EcR resulted in similar phenotypes as observed in the Dpp pathway genes knockdown, implying a regulatory role for Dpp signaling via EcR in vitellogenesis. Furthermore, knocking down Dpp, Tkv, and EcR in female adults led to a reduction in total dry weight and protein content, as well as the expression of mTOR, a factor linked to protein intake. These results suggest that the Dpp signaling pathway modulates vitellogenesis by impacting the AA/TOR-mediated 20E pathway in the fat body, providing novel insights into the network governing insect reproduction and offering potential targets for controlling female pest reproduction.
Subject(s)
Brassica , Coleoptera , Animals , Female , Coleoptera/genetics , Coleoptera/metabolism , Ecdysterone/metabolism , Brassica/metabolism , Vitellogenesis , Signal Transduction , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Insect Proteins/metabolismABSTRACT
In Daphnia magna, 20-hydroecdysone (20E) is the main molting hormone and its metabolism is of interest to identify new biomarkers of exposure to contaminants. The present study aimed to (i) assess baseline levels of 20E and transcription levels of four related-genes (shade, neverland, ultraspiracle, and ecdysteroid receptor); and (ii) evaluate effects in D. magna after 21 days of exposure to fenarimol (anti-ecdysteroid) and a mixture of gemfibrozil and clofibric acid (lipid-lowering drugs) at sublethal concentrations. Endpoints included transcription of the target genes and quantification of 20E, mortality, and reproduction of daphnids. Baseline results showed that average responses were relatively similar and did not vary more than 2-fold. However, intra-day variation was generally high and could be explained by sampling individuals with slightly different stages of their development. Exposure tests indicated a significant decrease in daphnid reproduction following chronic exposure to a concentration of 565 µg/L of fenarimol. However, no difference was observed between the control and exposed groups for any of the investigated genes, nor for the levels of 20E after 21 days of exposure. Following exposition to gemfibrozil and clofibric acid at 1 µg/L, no changes were observed for the measured parameters. These results suggest that changes in transcription levels of the target genes and concentrations of 20E may not be sensitive endpoints that can be used as biomarkers of sublethal exposure to the target compounds in D. magna. Measuring multiple time points instead of a single measure as well as additional molecular endpoints obtained from transcriptomic and metabolomic studies could afford more insights on the changes occurring in exposed daphnids to lipid-altering compounds and identify efficient biomarkers of sublethal exposure.
Subject(s)
Ecdysterone , Water Pollutants, Chemical , Humans , Animals , Ecdysterone/metabolism , Ecdysterone/pharmacology , Molting/genetics , Gemfibrozil/toxicity , Reproduction , Biomarkers/metabolism , Clofibric Acid/metabolism , Clofibric Acid/pharmacology , Daphnia , Water Pollutants, Chemical/metabolismABSTRACT
Estrogen-related receptor (ERR) is a key regulator of insect growth, development, and metabolic processes in insects; however, the molecular mechanisms underlying its effects are not fully understood. We investigated roles of 20-hydroxyecdysone (20E) and insulin/insulin-like signaling/target of rapamycin (IIS/TOR) signaling pathways in the effects of PvERR on larval development, metamorphosis, and adult growth in ant Polyrhachis vicina Roger. PvFOXO expression levels depended on caste and developmental stage. PvERR RNAi significantly reduced the expression levels of IIS/TOR signaling pathway genes and 20E signaling pathway genes in fourth-instar larvae, pupae, females, and workers and significantly increased the expression levels of IIS/TOR signaling pathway genes PvFOXO and PvAkt in males. PvFOXO RNAi resulted in developmental defects and increased mortality. After PvFOXO RNAi, the expression of PvERR, 20E signaling pathway genes, and IIS/TOR signaling pathway genes decreased significantly in pupae, females, and workers and increased significantly in fourth-instar larvae. Exogenous 20E attenuated expression changes induced by PvFOXO RNAi in a sex- and stage-specific manner. These results indicate that ERR interacts with 20E and IIS/TOR signaling pathways to regulate caste determination, metamorphosis, and male fertility in P. vicina and that correlations between PvERR and PvFOXO are caste- and stage-specific.
Subject(s)
Ants , Animals , Female , Male , Ants/genetics , Ants/metabolism , Insulin/metabolism , Ecdysterone/metabolism , Receptors, Estrogen/metabolism , Larva/metabolism , Insecta , Signal Transduction , Metamorphosis, Biological/genetics , Pupa/genetics , Estrogens/metabolism , Insect Proteins/genetics , Insect Proteins/metabolismABSTRACT
Bruceine D (BD) is an effective insecticidal compound found in the Chinese herb Brucea javanica (L.) Merr. BD inhibits the growth and metamorphosis of Plutella xylostella and Drosophila melanogaster; however, its target protein and the molecular mechanism of insecticidal activity remain unclear. In this study, proteins with high affinity for BD were screened using surface plasmon resonance and high-performance liquid chromatography coupled with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, revealing the ecdysone receptor (EcR) is the main target of BD. In vivo results showed that BD inhibited insect growth and metamorphosis through inhibition of the expression of 20E response genes. In vitro dual luciferase and enhanced green fluorescent protein (EGFP) fluorescence experiments indicated that BD suppressed the transcriptional activation activity of EcR by blocking the ecdysone response element (EcRE)-triggered transcriptional cascade, suggesting that BD inhibits the formation of the 20E-EcR-USP-EcRE complex. Moreover, molecular docking demonstrated that BD bound well to EcR. Elucidating the insecticidal mechanism of BD will be helpful in the development of green pesticides to control pests.
Subject(s)
Drosophila Proteins , Insecticides , Animals , Ecdysone/metabolism , Drosophila melanogaster/metabolism , Insecticides/pharmacology , Molecular Docking Simulation , Signal Transduction , Drosophila Proteins/metabolism , Ecdysterone/metabolism , Ecdysterone/pharmacologyABSTRACT
Ecdysteroids are crucial in regulating the growth and development of insects. In the fruit fly Drosophila melanogaster, both C27 and C28 ecdysteroids have been identified. While the biosynthetic pathway of the C27 ecdysteroid 20-hydroxyecdysone (20E) from cholesterol is relatively well understood, the biosynthetic pathway of C28 ecdysteroids from C28 or C29 dietary sterols remains unknown. In this study, we found that different dietary sterols (including the C27 sterols cholesterol and 7-dehydrocholesterol, the C28 sterols brassicasterol, campesterol, and ergosterol, and the C29 sterols ß-sitosterol, α-spinasterol, and stigmasterol) differentially affected the expression of 20E biosynthetic genes to varying degrees, but similarly activated 20E primary response gene expression in D. melanogaster Kc cells. We also found that a single dietary sterol was sufficient to support D. melanogaster growth and development. Furthermore, the expression levels of some 20E biosynthetic genes were significantly altered, whereas the expression of 20E signaling primary response genes remained unaffected when flies were reared on lipid-depleted diets supplemented with single sterol types. Overall, our study provided preliminary clues to suggest that the same enzymatic system responsible for the classical C27 ecdysteroid 20E biosynthetic pathway also participated in the conversion of C28 and C29 dietary sterols into C28 ecdysteroids.
Subject(s)
Drosophila melanogaster , Sterols , Animals , Sterols/metabolism , Drosophila melanogaster/metabolism , Ecdysteroids/metabolism , Ecdysterone/pharmacology , Ecdysterone/metabolism , Cholesterol/metabolismABSTRACT
Ecdysone signaling in Drosophila remains a popular model for investigating the mechanisms of steroid action in eukaryotes. The ecdysone receptor EcR can effectively bind ecdysone-response elements with or without the presence of a hormone. For years, EcR enhancers were thought to respond to ecdysone via recruiting coactivator complexes, which replace corepressors and stimulate transcription. However, the exact mechanism of transcription activation by ecdysone remains unclear. Here, we present experimental data on 11 various coregulators at ecdysone-responsive loci of Drosophila S2 cells. We describe the regulatory elements where coregulators reside within these loci and assess changes in their binding levels following 20-hydroxyecdysone treatment. In the current study, we detected the presence of some coregulators at the TSSs (active and inactive) and boundaries marked with CP190 rather than enhancers of the ecdysone-responsive loci where EcR binds. We observed minor changes in the coregulators' binding level. Most were present at inducible loci before and after 20-hydroxyecdysone treatment. Our findings suggest that: (1) coregulators can activate a particular TSS operating from some distal region (which could be an enhancer, boundary regulatory region, or inactive TSS); (2) coregulators are not recruited after 20-hydroxyecdysone treatment to the responsive loci; rather, their functional activity changes (shown as an increase in H3K27 acetylation marks generated by CBP/p300/Nejire acetyltransferase). Taken together, our findings imply that the 20-hydroxyecdysone signal enhances the functional activity of coregulators rather than promoting their binding to regulatory regions during the ecdysone response.
Subject(s)
Drosophila Proteins , Receptors, Steroid , Animals , Drosophila/genetics , Drosophila/metabolism , Ecdysone , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Ecdysterone/pharmacology , Ecdysterone/metabolism , Receptors, Steroid/genetics , Receptors, Steroid/metabolism , Transcriptional Activation , Drosophila melanogaster/metabolism , Microtubule-Associated Proteins/metabolism , Nuclear Proteins/metabolismABSTRACT
A heterodimeric complex of two nuclear receptors, the ecdysone receptor (ECR) and ultraspiracle (USP), transduces 20-hydroxyecdysone (20E) signaling to modulate insect growth and development. Here, we aimed to determine the relationship between ECR and 20E during larval metamorphosis and also the specific roles of ECR during larval-adult transition in Apis mellifera. We found that ECR gene expression peaked in the 7-day-old larvae, then decreased gradually from the pupae stage. 20E slowly reduced food consumption and then induced starvation, resulting in small-sized adults. In addition, 20E induced ECR expression to regulate larval development time. Double-stranded RNAs (dsRNAs) were prepared using common dsECR as templates. After dsECR injection, larval transition to the pupal stage was delayed, and 80% of the larvae showed prolonged pupation beyond 18 h. Moreover, the mRNA levels of shd, sro, nvd, and spo, and ecdysteroid titers were significantly decreased in ECR RNAi larvae compared with those in GFP RNAi control larvae. ECR RNAi disrupted 20E signaling during larval metamorphosis. We performed rescuing experiments by injecting 20E in ECR RNAi larvae and found that the mRNA levels of ECR, USP, E75, E93, and Br-c were not restored. 20E induced apoptosis in the fat body during larval pupation, while RNAi knockdown of ECR genes reduced apoptosis. We concluded that 20E induced ECR to modulate 20E signaling to promote honeybee pupation. These results assist our understanding of the complicated molecular mechanisms of insect metamorphosis.
Subject(s)
Ecdysterone , Receptors, Steroid , Bees/genetics , Animals , Ecdysterone/pharmacology , Ecdysterone/metabolism , Ecdysone/metabolism , Insect Proteins/genetics , Insect Proteins/metabolism , Receptors, Steroid/genetics , Receptors, Steroid/metabolism , Metamorphosis, Biological/physiology , Larva/geneticsABSTRACT
Amino acid metabolism is regulated according to nutrient conditions; however, the mechanism is not fully understood. Using the holometabolous insect cotton bollworm (Helicoverpa armigera) as a model, we report that hemolymph metabolites are greatly changed from the feeding larvae to the wandering larvae and to pupae. Arginine, alpha-ketoglutarate (α-KG), and glutamate (Glu) are identified as marker metabolites of feeding larvae, wandering larvae, and pupae, respectively. Arginine level is decreased by 20-hydroxyecdysone (20E) regulation via repression of argininosuccinate synthetase (Ass) expression and upregulation of arginase (Arg) expression during metamorphosis. α-KG is transformed from Glu by glutamate dehydrogenase (GDH) in larval midgut, which is repressed by 20E. The α-KG is then transformed to Glu by GDH-like in pupal fat body, which is upregulated by 20E. Thus, 20E reprogrammed amino acid metabolism during metamorphosis by regulating gene expression in a stage- and tissue-specific manner to support insect metamorphic development.
Subject(s)
Ecdysterone , Moths , Animals , Ecdysterone/pharmacology , Ecdysterone/metabolism , Larva/metabolism , Metamorphosis, Biological , Amino Acids/metabolism , Insect Proteins/metabolismABSTRACT
The plant mirid bug Apolygus lucorum is an omnivorous pest that can cause considerable economic damage. The steroid hormone 20-hydroxyecdysone (20E) is mainly responsible for molting and metamorphosis. The adenosine monophosphate-activated protein kinase (AMPK) is an intracellular energy sensor regulated by 20E, and its activity is regulated allosterically through phosphorylation. It is unknown whether the 20E-regulated insect's molting and gene expression depends on the AMPK phosphorylation. Herein, we cloned the full-length cDNA of the AlAMPK gene in A. lucorum. AlAMPK mRNA was detected at all developmental stages, whereas the dominant expression was in the midgut and, to a lesser extent, in the epidermis and fat body. Treatment with 20E and AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AlCAR) or only AlCAR resulted in activation of AlAMPK phosphorylation levels in the fat body, probed with an antibody directed against AMPK phosphorylated at Thr172, enhancing AlAMPK expression, whereas no phosphorylation occurred with compound C. Compared to compound C, 20E and/or AlCAR increased the molting rate, the fifth instar nymphal weight and shortened the development time of A. lucorum in vitro by inducing the expression of EcR-A, EcR-B, USP, and E75-A. Similarly, the knockdown of AlAMPK by RNAi reduced the molting rate of nymphs, the weight of fifth-instar nymphs and blocked the developmental time and the expression of 20E-related genes. Moreover, as observed by TEM, the thickness of the epidermis of the mirid was significantly increased in 20E and/or AlCAR treatments, molting spaces began to form between the cuticle and epidermal cells, and the molting progress of the mirid was significantly improved. These composite data indicated that AlAMPK, as a phosphorylated form in the 20E pathway, plays an important role in hormonal signaling and, in short, regulating insect molting and metamorphosis by switching its phosphorylation status.