Universal sample preparation method for proteome analysis

In insects, 20-hydroxyecdysone (20E) limits the growth period by triggering developmental transitions; 20E also modulates the growth rate by antagonizing insulin/insulin-like growth factor signaling (IIS). Previous work has shown that 20E cross-talks with IIS, but the underlying molecular mechanisms are not fully understood. Here we found that, in both the silkworm Bombyx mori and the fruit fly Drosophila melanogaster, 20E antagonized IIS through the AMP-activated protein kinase (AMPK)-protein phosphatase 2A (PP2A) axis in the fat body and suppressed the growth rate. During Bombyx larval molt or Drosophila pupariation, high levels of 20E activate AMPK, a molecular sensor that maintains energy homeostasis in the insect fat body. In turn, AMPK activates PP2A, which further dephosphorylates insulin receptor and protein kinase B (AKT), thus inhibiting IIS. Activation of the AMPK-PP2A axis and inhibition of IIS in the Drosophila fat body reduced food consumption, resulting in the restriction of growth rate and body weight. Overall, our study revealed an important mechanism by which 20E antagonizes IIS in the insect fat body to restrict the larval growth rate, thereby expanding our understanding of the comprehensive regulatory mechanisms of final body size in animals.

https://www.pnas.org/content/pnas/117/17/9292.full.pdf

The AMPK-PP2A axis in insect fat body is activated by 20-hydroxyecdysone to antagonize insulin/IGF signaling and restrict growth rate.

Annotation of metabolites remains a major challenge in liquid chromatography–mass spectrometry (LC–MS) based untargeted metabolomics. The current gold standard for metabolite identification is to match the detected feature with an authentic standard analyzed on the same equipment and using the same method as the experimental samples. However, there are substantial practical challenges in applying this approach to large data sets. One widely used annotation approach is to search spectral libraries in reference databases for matching metabolites; however, this approach is limited by the incomplete coverage of these libraries. An alternative computational approach is to match the detected features to candidate chemical structures based on their mass and predicted fragmentation pattern. Unfortunately, both of these approaches can match multiple identities with a single feature. Another issue is that annotations from different tools often disagree. This paper presents a novel LC–MS data annotation method, term...

Biologically Consistent Annotation of Metabolomics Data

This paper reports the first successful fabrication of an ITO/silkworm hemolymph/Al bio-memristor using silkworm hemolymph as the active layer. Experiments demonstrate that the silkworm hemolymph bio-memristor is a nonvolatile rewritable bipolar memory device with a current switching ratio exceeding 103. The state of the bio-memristor can be retained for more than 104 seconds and remains stable for at least 500 cycles. Tests of 1/f noise have shown that the resistance switching characteristics of the silkworm hemolymph bio-memristor are related to the formation and breaking of conductive filaments, which result from the migration of oxygen ions and the oxidation and reduction of metal cations in the silkworm hemolymph film. The naturally non-toxic silkworm hemolymph offers advantages for human health, environmental protection, and biocompatibility. The proposed nonvolatile rewritable bio-memristor based on silkworm hemolymph possesses great application potential.

/pdf/nonvolatile-bio-memristor-based-on-silkworm-hemolymph-5fyc7sgi90.pdf

Nonvolatile Bio-Memristor Based on Silkworm Hemolymph Proteins

Corazonin (Crz) is a neuropeptide hormone, but also a neuropeptide modulator that is internally released within the CNS, and it has a widespread distribution in insects with diverse physiological functions. Here, we identified and cloned the cDNAs of Bactrocera dorsalis that encode Crz and its receptor CrzR. Mature BdCrz has 11 residues with a unique Ser11 substitution (instead of the typical Asn) and a His in the evolutionary variable position 7. The BdCrzR cDNA encodes a putative protein of 608 amino acids with 7 putative transmembrane domains, typical for the structure of G-protein-coupled receptors. When expressed in Chinese hamster ovary (CHO) cells, the BdCrzR exhibited a high sensitivity and selectivity for Crz (EC50 ≈ 52.5 nM). With qPCR, the developmental stage and tissue-specific expression profiles in B. dorsalis demonstrated that both BdCrz and BdCrzR were highly expressed in the larval stage, and BdCrzR peaked in 2-day-old 3rd-instar larvae, suggesting that the BdCrzR may play an important role in the larval-pupal transition behavior. Immunochemical localization confirmed the production of Crz in the central nervous system (CNS), specifically by a group of three neurons in the dorso-lateral protocerebrum and eight pairs of lateral neurons in the ventral nerve cord. qPCR analysis located the BdCrzR in both the CNS and epitracheal gland, containing the Inka cells. Importantly, dsRNA-BdCrzR-mediated gene-silencing caused a delay in larval-pupal transition and pupariation, and this phenomenon agreed with a delayed expression of tyrosine hydroxylase and dopa-decarboxylase genes. We speculate that CrzR-silencing blocked dopamine synthesis, resulting in the inhibition of pupariation and cuticular melanization. Finally, injection of Crz in head-ligated larvae could rescue the effects. These findings provide a new insight into the roles of Crz signaling pathway components in B. dorsalis and support an important role of CrzR in larval-pupal transition and pupariation behavior.

https://biblio.ugent.be/publication/8511406/file/8511407.pdf

A Role of Corazonin Receptor in Larval-Pupal Transition and Pupariation in the Oriental Fruit Fly Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).

The silkworm is a lepidopteran insect that has an open circulatory system with hemolymph consisting of blood and lymph fluid. Hemolymph is not only considered as a depository of nutrients and energy, but it also plays a key role in substance transportation, immunity response, and proteolysis. In this study, we used LC-MS/MS to analyze the hemolymph proteins of four developmental stages during metamorphosis. A total of 728 proteins were identified from the hemolymph of the second day of wandering stage, first day of pupation, ninth day of pupation, and first day as an adult moth. GO annotations and categories showed that silkworm hemolymph proteins were enriched in carbohydrate metabolism, proteolysis, protein binding, and antibacterial humoral response. The levels of nutrient, immunity-related, and structural proteins changed significantly during development and metamorphosis. Some, such as cuticle, odorant-binding, and chemosensory proteins, showed stage-specific expression in the hemolymph. In addition, the expression of several antimicrobial peptides exhibited their highest level of abundance in the hemolymph of the early pupal stage. These findings provide a comprehensive proteomic insight of the silkworm hemolymph and suggest additional molecular targets for studying insect metamorphosis.

Comparative proteomics analysis of silkworm hemolymph during the stages of metamorphosis via liquid chromatography and mass spectrometry.

[Analysis of molting fluid in silkworm (Bombyx mori) by two-dimensional electrophoresis and mass spectrometry].

Before metamorphosis, most holometabolous insects, such as the silkworm studied here, undergo a special phase called the wandering stage. Insects in this stage often display enhanced locomotor activity (ELA). ELA is vital because it ensures that the insect finds a safe and suitable place to live through the pupal stage. The physiological mechanisms of wandering behavior are still unclear. Here, we integrated proteomics and metabolomics approaches to analyze the brain of the lepidopteran insect, silkworm, at the feeding and wandering stages. Using LC–MS/MS and GC–MS, in all we identified 3004 proteins and 37 metabolites at these two stages. Among them, 465 proteins and 22 metabolites were changed. Neural signal transduction proteins and metabolites, such as neurofilament, dopaminergic synapse related proteins, and glutamic acid, were significantly altered, which suggested that active neural conduction occurred in the brain at the wandering stage. We also found decreased dopamine degradation at the wanderin...

Integrative Proteomics and Metabolomics Analysis of Insect Larva Brain: Novel Insights into the Molecular Mechanism of Insect Wandering Behavior

Identification and expression pattern of cathepsin family in silkworm (Bombyx mori)

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Comparative proteomics analysis of silkworm hemolymph during the stages of metamorphosis via liquid chromatography and mass spectrometry.

[Analysis of molting fluid in silkworm (Bombyx mori) by two-dimensional electrophoresis and mass spectrometry].

Integrative Proteomics and Metabolomics Analysis of Insect Larva Brain: Novel Insights into the Molecular Mechanism of Insect Wandering Behavior

Identification and expression pattern of cathepsin family in silkworm (Bombyx mori)