Ecdysone Titer Research Articles

Bruceine D inhibits the growth of Spodoptera litura by inducing cell apoptosis in the midgut via an oxidative burst.

Spodoptera litura is one of the most harmful lepidoptera pests in China, and is difficult to control due to its strong resistance to the current frequently used insecticide species. The requirement to develop pesticides with novel toxicology mechanisms to control S. litura is urgent. The quassinoid of bruceine D display outstanding systemic properties and strong insecticidal activity against S. litura, which possess notable application potential for integrative management of S. litura, but the mechanism of toxicity remains unclear. In this study, we found that bruceine D exerts potent growth inhibitory activity against S. litura, disrupting the ecdysone and juvenile hormone titers, and causing long-term adverse effects. Association analysis between transcriptomics and metabolomics suggested that bruceine D affected the digestion and absorption capacity of S. litura larvae by inducing a strong oxidative stress response and cell apoptosis in the intestine. Further analysis demonstrated that bruceine D can inhibit the activities of digestive and antioxidant enzymes and induce malondialdehyde (MDA)and reactive oxygen species (ROS) overaccumulation in the midgut. Moreover, the protein level of Bax, cleavage caspase 3, and cytochrome c expressed in cytoplasm (cyto) were up-regulated by bruceine D, while Bcl-2 and cytochrome c expressed in mitochondria (mito) were down-regulated. In addition, there was a noticeable increase in caspase-3 protease activity. Histopathological observations revealed that bruceine D damages the structure of midgut epithelial cells and activates lysosomes, which subsequently disrupts the midgut tissue. Overall, our findings suggested that bruceine D induced excessive ROS accumulation in midgut epithelial cells. The resulting cell apoptosis disrupted midgut tissue, leading ultimately to reduced nutrient digestion and absorption in the midgut and the inhibition of larval growth. © 2024 Society of Chemical Industry.

The inhibition of ecdysone signal pathway was the key of pyriproxyfen poisoning for silkworm, Bombyx mori

Pyriproxyfen is a juvenile hormone-like pesticide. Once intake occurs, it leads to a series of poisoning characters consequences in silkworm, Bombyx mori (ID: 7091, Lepidoptera), such as non- cocooning, non-pupation, production of low-active eggs, and extended stages. However, the poisoning mechanism is still unclear. Here, silkworms were fed mulberry leaves soaked with different pyriproxyfen concentrations, and the heads were dissected for transcriptome analysis, while the hemolymph was used for determinations of ecdysone and juvenile hormone titers. As a result, after conjoint analysis of 3 feeding groups and a control group, 555 differentially expressed genes (DEGs) were obtained, which were mainly involved in hormone metabolism, glycometabolism and protein metabolism. Meanwhile, 119 genes were significantly correlated with the pyriproxyfen concentrations, and they were mainly involved in drug metabolism and glycometabolism. The ecdysone titers in several feeding groups were significantly lower than those of the control group, while juvenile hormone was not detected in all groups, including the control and feeding groups. Correspondingly, due to activation of the juvenile hormone signaling pathway by pyriproxyfen, key genes in the ecdysone synthesis pathway were downregulated, and a large number of downstream genes were up- or downregulated. In addition, nearly all genes in the detoxification pathway were upregulated. These results suggested that, affected by the juvenile hormone signaling pathway, ecdysone titers decreased and further affected a series of downstream processes, and this was the key reason for pyriproxyfen poisoning in silkworm, B. mori, which could lay a foundation for the study of pyriproxyfen resistance in silkworm.

Diallyl trisulfide reduced the reproductive capacity of male Sitotroga cerealella via the regulation of juvenile and ecdysone hormones

Environmental pollution and resistance in animals are major concerns for the application of synthetic pesticides. Diallyl trisulfide (DAT), an active compound in garlic essential oil, is a novel tool for active and safe control of agricultural insect pests. In this study, we analysed the effects of DAT (0.01 μL/L) on the protein content in male reproductive tissues (accessory glands, ejaculatory ducts, and testis), and juvenile hormone (JH) and ecdysone titres in a highly detrimental pest of stored products, Sitotroga cerealella. Evaluation of the expression profile of JH and ecdysone pathway-related genes in various tissues indicated that the accessory gland protein and ecdysone titres were markedly decreased after DAT fumigation, whereas the testis protein content and JH titre were increased. However, the protein content of the ejaculatory ducts remained unchanged between the treated and control groups. Further investigation revealed that DAT disrupted the mRNA expression of key enzymes involved in JH and ecdysone pathways. While increased mRNA levels of juvenile hormone acid O-methyltransferase (JHMAT) and Kruppel homologue 1 (Kr-h1) were observed after 4 and 7 h of DAT fumigation, the levels of juvenile hormone epoxide hydrolase (JHEH) were substantially reduced 3 h post-fumigation. mRNA levels of the ecdysone-responsive gene, FTZF1, and cytochrome P450 enzyme, CYP315A1, were notably decreased at 7 h and 4 h, respectively, post-fumigation, whereas CYP314A1 and CYP302A1 mRNA levels decreased after 3 h and 4 h, respectively. While DAT fumigation disrupted sperm number in the testis, ejaculatory ducts, and seminal vesicles, topical application of the 20-hydroxyecdysone (20E) analogue also lowered sperm number in the ejaculatory ducts. Topical application of methoprene, a JH analogue, increased the protein content in the testes, but not in the accessory glands or ejaculatory ducts. However, the survival rate was not affected by the topical application of methoprene or 20E. These data suggest that DAT regulates JH and ecdysone via its molecular pathway genes and modulates endocrine secretion during the male reproductive process.

Exorista sorbillans parasitism induces early maturation by altering ecdysone and juvenile hormone titers in silkworm

Exorista sorbillans parasitism induces early maturation by altering ecdysone and juvenile hormone titers in silkworm

Toxicological effects of trace amounts of pyriproxyfen on the midgut of non-target insect silkworm

Pyriproxyfen is an insect growth regulator that is widely used in public health and pest control in agriculture. Our previous studies have shown that trace amounts of pyriproxyfen in the environment can cause serious toxic effects in the non-target insect silkworm, including failing to pupate, metamorphose and spin cocoons. However, it is unknown why pyriproxyfen not only has no lethal effects on fifth instar larvae but also tend to increase their body weight. The midgut is the main digestive organs of the silkworm, our results showed that the residual of pyriproxyfen in the silkworm at 24 h after 1 × 10−4 mg/L pyriproxyfen treatment caused severe damage to the midgut microvilli, goblet cells, and nuclei of the silkworm, but body weight and digestibility of the larval were both increased. In addition, pyriproxyfen significantly (p < 0.05) increased the activities of digestive enzymes (α-amylase, trehalase, trypsin and lipase) in the midgut of silkworm. However, it caused down-regulation of ecdysone synthesis-related genes at the end of the fifth instar silkworm, decreased ecdysone titer, and prolonged larval instar. At the same time, pyriproxyfen also activated transcription of detoxification enzymes-related genes such as the cytochrome P450 enzyme genes Cyp9a22 and Cyp15C1, the carboxylesterase genes CarE-8 and CarE-11, and the glutathione S-transferase gene GSTo2. This study elucidated a novel toxicological effect of pyriproxyfen to insects, which not only expands the understanding of the effects of juvenile hormone pesticides on lepidopteran insects but also provides a reference for exploring the ecological security of non-target organisms.

Opportunistic binding of EcR to open chromatin drives tissue-specific developmental responses

Steroid hormones perform diverse biological functions in developing and adult animals. However, the mechanistic basis for their tissue specificity remains unclear. In Drosophila, the ecdysone steroid hormone is essential for coordinating developmental timing across physically separated tissues. Ecdysone directly impacts genome function through its nuclear receptor, a heterodimer of the EcR and ultraspiracle proteins. Ligand binding to EcR triggers a transcriptional cascade, including activation of a set of primary response transcription factors. The hierarchical organization of this pathway has left the direct role of EcR in mediating ecdysone responses obscured. Here, we investigate the role of EcR in controlling tissue-specific ecdysone responses, focusing on two tissues that diverge in their response to rising ecdysone titers: the larval salivary gland, which undergoes programmed destruction, and the wing imaginal disc, which initiates morphogenesis. We find that EcR functions bimodally, with both gene repressive and activating functions, even at the same developmental stage. EcR DNA binding profiles are highly tissue-specific, and transgenic reporter analyses demonstrate that EcR plays a direct role in controlling enhancer activity. Finally, despite a strong correlation between tissue-specific EcR binding and tissue-specific open chromatin, we find that EcR does not control chromatin accessibility at genomic targets. We conclude that EcR contributes extensively to tissue-specific ecdysone responses. However, control over access to its binding sites is subordinated to other transcription factors.

Cyromazine Effects the Reproduction of Drosophila by Decreasing the Number of Germ Cells in the Female Adult Ovary.

Simple SummaryCyromazine, an insect growth regulator, is used to control the Dipteran pest population. Previous findings observed that treatment with cyromazine increased the larval mortality, by interfering with the ecdysone signaling. In addition, the application of exogenous 20E significantly reduced the mortality caused by cyromazine. Many studies have also supported the role of ecdysone signaling in the maintenance of germline stem cells (GSCs), where mutations in ecdysone signaling-related genes significantly decreased the number of GSCs. However, to date, no study has reported the effect of cyromazine on the GSCs of Drosophila melanogaster. In the present study, we observed that cyromazine significantly reduced the number of both GSCs and cystoblasts (CBs) in the ovary of adult female. To further understand the effect of cyromazine on germ cells, we selected some key genes related to the ecdysone signaling pathway and evaluated their expression through RT-qPCR. Additionally, we measured the ecdysone titer from the cyromazine-treated ovaries. Our results indicated a significant decrease in the expression of ecdysone signaling-related genes and also in the ecdysone titer. These results further supported our findings that cyromazine reduced the number of germ cells by interfering with the ecdysone signaling pathway.In the present study, we observed a 58% decrease in the fecundity of Drosophila melanogaster, after treatment with the cyromazine. To further elucidate the effects of cyromazine on reproduction, we counted the number of both germline stem cells (GSCs) and cystoblasts (CBs) in the ovary of a 3-day-old adult female. The results showed a significant decrease in the number of GSCs and CBs as compared to the control group. The mode of action of cyromazine is believed to be through the ecdysone signaling pathway. To further support this postulate, we observed the expression of key genes involved in the ecdysone signaling pathway and also determined the ecdysone titer from cyromazine-treated ovaries. Results indicated a significant decrease in the expression of ecdysone signaling-related genes as compared to the control group. Furthermore, the titer of the ecdysone hormone was also markedly reduced (90%) in cyromazine-treated adult ovaries, suggesting that ecdysone signaling was directly related to the decrease in the number of GSCs and CBs. However, further studies are required to understand the mechanism by which cyromazine affects the GSCs and CBs in female adult ovaries.

Precocious Metamorphosis of Silkworm Larvae Infected by BmNPV in the Latter Half of the Fifth Instar.

The mulberry silkworm (Bombyx mori) is a model organism, and BmNPV is a typical baculovirus. Together, these organisms form a useful model to investigate host–baculovirus interactions. Prothoracic glands (PGs) are also model organs, used to investigate the regulatory effect of synthetic ecdysone on insect growth and development. In this study, day-4 fifth instar silkworm larvae were infected with BmNPV. Wandering silkworms appeared in the infected groups 12 h earlier than in the control groups, and the ecdysone titer in infected larvae was significantly higher than that of the control larvae. We then used RNA sequencing (RNA-seq) to analyze silkworm PGs 48 h after BmNPV infection. We identified 15 differentially expressed genes (DEGs) that were classified as mainly being involved in metabolic processes and pathways. All 15 DEGs were expressed in the PGs, of which Novel01674, BmJing, and BmAryl were specifically expressed in the PGs. The transcripts of BmNGDN, BmTrypsin-1, BmACSS3, and BmJing were significantly increased, and BmPyd3, BmTitin, BmIGc2, Novel01674, and BmAryl were significantly decreased from 24 to 72 h in the PGs after BmNPV infection. The changes in the transcription of these nine genes were generally consistent with the transcriptome data. The upregulation of BmTrypsin-1 and BmACSS3 indicate that these DEGs may be involved in the maturation process in the latter half of the fifth instar of silkworm larvae. These findings further our understanding of silkworm larval development, the interaction between BmNPV infection and the host developmental response, and host–baculovirus interactions in general.

Polished rice mediates ecdysone-dependent control of Drosophila embryonic organogenesis.

In many animals, progression of developmental stages is temporally controlled by steroid hormones. In Drosophila, the level of ecdysone titer oscillates and developmental stage transitions, such as larval molting and metamorphosis, are induced at each of ecdysone peaks. Ecdysone titer also peaks at the stage of mid-embryogenesis and the embryonic ecdysone is necessary for morphogenesis of several organs, although the regulatory mechanisms of embryonic organogenesis dependent on ecdysone signaling are still open questions. In this study, we find that absence or interruption of embryonic ecdysone signaling caused multiple defects in the tracheal system, including decrease in luminal protein deposition, uneven dilation of the dorsal trunk and loss of terminal branches. We also reveal that an ecdysone-inducible gene polished rice (pri) is essential for tip cell fate decision in dorsal branches. As over-expression of pri can restore the defects caused by disturbance of ecdysone biosynthesis, pri functions as one of the major mediators of embryonic ecdysone signal in tracheogenesis. These results demonstrate that ecdysone and its downstream target pri play essential roles in tracheal development by modulating cell fate decision.

Manipulation of host ecdysteroid hormone levels facilitates infection by the fungal insect pathogen, Metarhizium rileyi.

Entomopathogenic fungi such as Metarhizium rileyi and Beauveria bassiana are widely used insect biological control agents. Little, however, is known concerning genetic or enzymatic factors that differentiate the mechanisms employed by these two fungal pathogens to infect target hosts. Infection by either of these organisms is known to increase levels of the growth and molting hormone, ecdysone, which also regulates the expression of a number of innate immune pathways. M. rileyi, but not B. bassiana, has apparently evolved an ecdysteroid-22-oxidase (MrE22O) that inactivate ecdysone. We show that deletion of MrE22O impaired virulence compared with the wild-type strain, with an increase in ecdysone titer seen in hosts that was coupled to an increase in the expression of antimicrobial genes. An M. rileyi strain engineered to overexpress MrE22O (MrE22OOE ), as well as trans-expression in B. bassiana (Bb::MrE220OE ) resulted, in strains displaying enhanced virulence and dampening of host immune responses compared with their respective wild-type parental strains. These results indicate that ecdysone plays an important role in mediating responses to fungal infection and that some insect pathogenic fungi have evolved mechanisms for targeting this hormone as a means for facilitating infection.

A New Role for Neuropeptide F Signaling in Controlling Developmental Timing and Body Size in Drosophila melanogaster.

As juvenile animals grow, their behavior, physiology, and development need to be matched to environmental conditions to ensure they survive to adulthood. However, we know little about how behavior and physiology are integrated with development to achieve this outcome. Neuropeptides are prime candidates for achieving this due to their well-known signaling functions in controlling many aspects of behavior, physiology, and development in response to environmental cues. In the growing Drosophila larva, while several neuropeptides have been shown to regulate feeding behavior, and a handful to regulate growth, it is unclear if any of these play a global role in coordinating feeding behavior with developmental programs. Here, we demonstrate that Neuropeptide F Receptor (NPFR), best studied as a conserved regulator of feeding behavior from insects to mammals, also regulates development in Drosophila Knocking down NPFR in the prothoracic gland, which produces the steroid hormone ecdysone, generates developmental delay and an extended feeding period, resulting in increased body size. We show that these effects are due to decreased ecdysone production, as these animals have reduced expression of ecdysone biosynthesis genes and lower ecdysone titers. Moreover, these phenotypes can be rescued by feeding larvae food supplemented with ecdysone. Further, we show that NPFR negatively regulates the insulin signaling pathway in the prothoracic gland to achieve these effects. Taken together, our data demonstrate that NPFR signaling plays a key role in regulating animal development, and may, thus, play a global role in integrating feeding behavior and development in Drosophila.

Wing patterning in faster developing Drosophila is associated with high ecdysone titer and wingless expression

‘Developmental robustness’ is the ability of biological systems to maintain a stable phenotype despite genetic, environmental or physiological perturbations. In holometabolous insects, accurate patterning and development is guaranteed by alignment of final gene expression patterns in tissues at specific developmental stage such as molting and pupariation, irrespective of individual rate of development. In the present study, we used faster developing Drosophila melanogaster populations that show reduction of ~22% in egg to adult development time. Flies from the faster developing population exhibit phenotype constancy, although significantly small in size. The reduction in development time in faster developing flies is possibly due to coordination between higher ecdysteroid release and higher expression of developmental genes. The two together might be ensuring appropriate pattern formation and early exit at each development stage in the populations selected for faster pre-adult development compared to their ancestral controls. We report that apart from plasticity in the rate of pattern progression, alteration in the level of gene expression may be responsible for pattern integrity even under reduced development time.

Characterization of G-protein coupled receptors from the blackback land crab Gecarcinus lateralis Y organ transcriptome over the molt cycle

BackgroundG-protein coupled receptors (GPCRs) are ancient, ubiquitous, constitute the largest family of transducing cell surface proteins, and are integral to cell communication via an array of ligands/neuropeptides. Molt inhibiting hormone (MIH) is a key neuropeptide that controls growth and reproduction in crustaceans by regulating the molt cycle. It inhibits ecdysone biosynthesis by a pair of endocrine glands (Y-organs; YOs) through binding a yet uncharacterized GPCR, which triggers a signalling cascade, leading to inhibition of the ecdysis sequence. When MIH release stops, ecdysone is synthesized and released to the hemolymph. A peak in ecdysone titer is followed by a molting event. A transcriptome of the blackback land crab Gecarcinus lateralis YOs across molt was utilized in this study to curate the list of GPCRs and their expression in order to better assess which GPCRs are involved in the molt process.ResultsNinety-nine G. lateralis putative GPCRs were obtained by screening the YO transcriptome against the Pfam database. Phylogenetic analysis classified 49 as class A (Rhodopsin-like receptor), 35 as class B (Secretin receptor), and 9 as class C (metabotropic glutamate). Further phylogenetic analysis of class A GPCRs identified neuropeptide GPCRs, including those for Allatostatin A, Allatostatin B, Bursicon, CCHamide, FMRFamide, Proctolin, Corazonin, Relaxin, and the biogenic amine Serotonin. Three GPCRs clustered with recently identified putative CHH receptors (CHHRs), and differential expression over the molt cycle suggests that they are associated with ecdysteroidogenesis regulation. Two putative Corazonin receptors showed much higher expression in the YOs compared with all other GPCRs, suggesting an important role in molt regulation.ConclusionsMolting requires an orchestrated regulation of YO ecdysteroid synthesis by multiple neuropeptides. In this study, we curated a comprehensive list of GPCRs expressed in the YO and followed their expression across the molt cycle. Three putative CHH receptors were identified and could include an MIH receptor whose activation negatively regulates molting. Orthologs of receptors that were found to be involved in molt regulation in insects were also identified, including LGR3 and Corazonin receptor, the latter of which was expressed at much higher level than all other receptors, suggesting a key role in YO regulation.

Analysis of the Drosophila melanogaster anti-ovarian response to honey bee queen mandibular pheromone.

Queen mandibular pheromone (QMP) is a potent reproductive signal to which honey bee workers respond by suppressing their ovaries and adopting alloparental roles within the colony. This anti-ovarian effect of QMP on workers can, surprisingly, be induced in other insects, including fruit flies, in which females exposed to synthetic QMP develop smaller ovaries with fewer eggs. In this study, we use the Drosophila melanogaster model to identify the components of synthetic QMP required for the anti-ovarian effect. We found that virgin females respond strongly to 9-oxo-2-decenoic acid and 10-hydroxy-2-decenoic acid (10HDA), suggesting that the decenoic acid components of QMP are essential for the anti-ovarian response. Further, a nuclear factor of activated T-cells reporter system revealed neurones expressing the olfactory receptors Or-56a, Or-49b and Or-98a are activated by QMP in the antenna. In addition, we used olfactory receptor GAL4 drivers and a neuronal activator (a neuronal activating bacterial sodium channel) to test whether the candidate neurones are potential labelled lines for a decenoic acid response. We identified Or-49b as a potential candidate receiver of the 10HDA signal. Finally, the anti-ovarian response to synthetic QMP is not mediated by decreasing the titre of the reproductive hormones ecdysone and juvenile hormone.

Transcriptome Analysis of Newly Emerged Honeybees Exposure to Sublethal Carbendazim During Larval Stage.

There are increasing concerns regarding the impact of agrochemical pesticides on non-target organisms. Pesticides could cause honeybee abnormal development in response to neurotoxins such as neonicotinoid. However, knowledge of carbendazim, a widespread fungicide in beekeeping practice, influencing on honeybee (Apis mellifera L.) brain development is lacking. Large-scale transcriptome approaches were applied to determine the changes in global gene expression in the brains of newly emerged honeybees after carbendazim exposure during the larval stage. To further understand the effects of carbendazim on the brain development of honeybees, the functions of differentially expressed genes were compared between the treatment and control groups. We found that neuroregulatory genes were down-regulated after carbendazim exposure, which suggest the neurotoxic effects of this fungicide on honeybee nervous system. Carbendazim exposure also altered the expression of genes implicated in metabolism, transport, sensor, and hormone. Notably, larvae in the carbendazim-treated group observed longer time to shift into the dormant pupal state than the control group. Moreover, a low juvenile hormone and high ecdysone titers were found in the treatment group compared to control group. The data is the first report of neurotoxic effects on honeybee caused by carbendazim, and the sublethal carbendazim may disturb honeybee development and is a potential chemical threating the honeybee colonies.

TITER EKDISON LOBSTER HIJAU PASIR (Panulirus homarus L.) PADA FASE PREMOLTING AKHIR

Spiny Lobster, Panulirus homarus , farming is very potential to be developed in Lombok Island, but physiological aspects such as nutrient needs and growth hormone, especially ecdysone titer, were little known. This research aimed to determine the ecdysone titer of Panulirus homarus during the last pre-molting phase. This study was conducted as a quasi-experiment with one group post-test design. The lobsters used in this experiment have the length of carapacs and the body weight of 1.5–2.0 cm and 4.33–5.77 g respectively. The lobster fed for 42 days with gel pellets three times a day before ecdysone titer measurement. The ecdysone hormone was then isolated from the second segment of the abdomen on the basal of the fifth pereiopod. Ecdyson titer was measured by using High-Performance Liquid Chromatography (HPLC). The result showed that the titer of ecdysone was 5.27 µg/ml hemolymph. The titer of ecdysone was found higher compared to previous research on Homarus americanus ; this result suggested that the spiny lobster Panulirus homarus needs a higher amount of ecdysone hormone to initiate the molting process.

Gonadal ecdysone titers are modulated by protein availability but do not impact protein appetite

How animals survey internal nutrient availability to modulate specific appetites is currently largely unknown. Dietary proteins have a profound impact on the reproductive capacity and the selection of food sources in insects. When deprived of dietary proteins, insects stop producing eggs and develop strong protein appetites. In many adult insects, the ovaries are the site of synthesis of the ecdysone hormone. Therefore, an attractive hypothesis is that protein availability changes the gonadal production of ecdysone, which instructs the brain to increase its preference for yeast. We combine quantitative feeding assays, dietary manipulations, hormonal measurements, and genetic germline manipulations to test this hypothesis in Drosophila melanogaster. Our results show that upon yeast deprivation mated adult female Drosophila develop a strong yeast appetite and strongly reduce their egg production. This dietary manipulation also leads to a drastic reduction in ecdysone titers. However, the drop in ecdysone is not linked to the increase in yeast appetite as mutants with impaired oogenesis are able to adapt yeast intake to their nutrient state while displaying a constitutive low ecdysone titer. Interestingly, a low ecdysone titer is correlated with a lower level of overall food intake. Our data therefore show that in mated females the level of ecdysone reflects the level of protein in the diet and the physiological state of the ovaries. While the ovaries and ecdysone are unlikely to instruct the brain to develop a yeast appetite upon protein deprivation, they seem to be able to control overall food intake.

Differentially expressed genes in the head of the 2nd instar pre-molting larvae of the nm2 mutant of the silkworm, Bombyx mori.

Molting is an important physiological process in the larval stage of Bombyx mori and is controlled by various hormones and peptides. The silkworm mutant that exhibits the phenotype of non-molting in the 2nd instar (nm2) is incapable of molting in the 2nd instar and dies after seven or more days. The ecdysone titer in the nm2 mutant is lower than that in the wildtype, and the mutant can be rescued by feeding with 20E and cholesterol. The results of positional cloning indicated that structural alteration of BmCPG10 is responsible for the phenotype of the nm2 mutant. To explore the possible relationship between BmCPG10 and the ecdysone titer as well as the genes affected by BmCPG10, digital gene expression (DGE) profile analysis was conducted in the nm2 mutant, with the wildtype strain C603 serving as the control. The results revealed 1727 differentially expressed genes, among which 651 genes were upregulated and 1076 were downregulated in nm2. BLASTGO analysis showed that these differentially expressed genes were involved in various biological processes, cellular components and molecular functions. KEGG analysis indicated an enrichment of these differentially expressed genes in 240 pathways, including metabolic pathways, pancreatic secretion, protein digestion and absorption, fat digestion and absorption and glycerolipid metabolism. To verify the accuracy of the DGE results, quantitative reverse transcription PCR (qRT-PCR) was performed, focusing on key genes in several related pathways, and the results were highly consistent with the DGE results. Our findings indicated significant differences in cuticular protein genes, ecdysone biosynthesis genes and ecdysone-related nuclear receptors genes, but no significant difference in juvenile hormone and chitin biosynthesis genes was detected. Our research findings lay the foundation for further research on the formation mechanism of the nm2 mutant.

Severe developmental timing defects in the prothoracicotropic hormone (PTTH)-deficient silkworm, Bombyx mori

The insect neuropeptide prothoracicotropic hormone (PTTH) triggers the biosynthesis and release of the molting hormone ecdysone in the prothoracic gland (PG), thereby controlling the timing of molting and metamorphosis. Despite the well-documented physiological role of PTTH and its signaling pathway in the PG, it is not clear whether PTTH is an essential hormone for ecdysone biosynthesis and development. To address this question, we established and characterized a PTTH knockout line in the silkworm, Bombyx mori. We found that PTTH knockouts showed a severe developmental delay in both the larval and pupal stages. Larval phenotypes of PTTH knockouts can be classified into three major classes: (i) developmental arrest during the second larval instar, (ii) precocious metamorphosis after the fourth larval instar (one instar earlier in comparison to the control strain), and (iii) metamorphosis to normal-sized pupae after completing the five larval instar stages. In PTTH knockout larvae, peak levels of ecdysone titers in the hemolymph were dramatically reduced and the timing of peaks was delayed, suggesting that protracted larval development is a result of the reduced and delayed synthesis of ecdysone in the PG. Despite these defects, low basal levels of ecdysone were maintained in PTTH knockout larvae, suggesting that the primary role of PTTH is to upregulate ecdysone biosynthesis in the PG during molting stages, and low basal levels of ecdysone can be maintained in the absence of PTTH. We also found that mRNA levels of genes involved in ecdysone biosynthesis and ecdysteroid signaling pathways were significantly reduced in PTTH knockouts. Our results provide genetic evidence that PTTH is not essential for development, but is required to coordinate growth and developmental timing.

Transcription factor E74A affects the ecdysone titer by regulating the expression of the EO gene in the silkworm, Bomby mori

The formation of ecdysone pulse in insects is synergistically controlled by its biosynthesis and degradation. Previous studies have revealed the feedback regulation of the prothoracic gland (PG) activity to affect the hormone synthesis. However, the molecular regulatory mechanism of the ecdysone degradation is still unclear. In this study, we showed that ecdysone oxidase (EO) gene encoding a hormone metabolism enzyme was also induced by hormone itself in the domestic silkworm, Bombyx mori. Furthermore, luciferase reporter, chromatin immunoprecipitation and electrophoretic mobility shift assays showed that ecdysone inducible transcription factor E74A could bind to the cis-regulatory elements of the EO gene. Then, down-regulating the expression of the E74A by RNA interference (RNAi) decreased the expression of the EO gene and caused a higher ecdysone titer compared with the control. Thus, our results demonstrated a new feedback regulation degradation (EO) pathway controlled by ecdysone itself through transcription factor E74A, expanding the knowledge about the regulatory system that determines the formation of ecdysone pulse.