RNA N6-methyladenosine of DHAPAT and PAP involves in regulation of diapause of Bombyx mori via the lipid metabolism pathway.

Environment-induced epigenetics are involved in diapause regulation, but the molecular mechanism that epigenetically couples nutrient metabolism to diapause regulation remains unclear. In this study, we paid special attention to the significant differences in the level of N6-adenosine methylation (m6A) of dihydroxyacetone phosphate acyltransferase (DHAPAT) and phosphatidate phosphatase (PAP) genes in the lipid metabolism pathway of the bivoltine silkworm (Bombyx mori) strain Qiufeng developed from eggs incubated at a normal temperature (QFHT, diapause egg producer) compared to those from eggs incubated at a low temperature (QFLT, non-diapause egg producer). We knocked down DHAPAT in the pupal stage of the QFLT group, resulting in the non-diapause destined eggs becoming diapausing eggs. In the PAP knockdown group, the colour of the non-diapause destined eggs changed from light yellow to pink 3 days after oviposition, but they hatched as normal. Moreover, we validated that YTHDF3 binds to m6A-modified DHAPAT and PAP mRNAs to promote their stability and translation. These results suggest that RNA m6A methylation participates in the diapause regulation of silkworm by changing the expression levels of DHAPAT and PAP and reveal that m6A epigenetic modification can be combined with a lipid metabolism signal pathway to participate in the regulation of insect diapause traits, which provides a clearer image for exploring the physiological basis of insect diapause.

BmINR and BmAC6 genes involve in diapause regulation via the insulin/IGF signaling pathway in the silkworm (Bombyx mori).

Diapause of the silkworm (Bombyx mori) is an important ecological adaptation strategy regulated by multiple signaling pathways. As an evolutionarily conserved signaling pathway, the insulin/IGF signaling (IIS) pathway is essential in regulating lifespan, energy accumulation, and stress resistance in diapause insects. However, the regulatory mechanism of IIS on diapause in B. mori is still not fully understood. To investigate the role of the IIS pathway in regulating diapause, we first analyzed the transcription levels of the insulin receptor (BmINR) and its downstream gene adenylate cyclase 6 (BmAC6). The diapause-terminated eggs of a bivoltine strain QiuFeng (V2-QF) were incubated at 25 °C in natural room light for preparing diapause egg producers (DEPs) and at 17 °C in total darkness for preparing non-diapause egg producers (NDEPs), respectively. Then we investigated the effects of BmINR and BmAC6 on diapause phenotype and expression of diapause-related genes by RNA interference (RNAi) and overexpression techniques. The results showed that the mRNA expression levels of BmINR and BmAC6 in the head and ovary of NDEPs were higher than those in DEPs during the early and middle pupal stages. Furthermore, when BmINR was knocked down in the NDEPs, approximately 14.43% of eggs were in light red color and subsequently changed into gray-purple color after 48 h post-oviposition, then stayed in a diapause state. On the other hand, overexpression of BmINR or BmAC6 via recombinant baculoviruses did not cause any obvious phenotypic alterations in NDEPs, but it upregulated the expression of genes related to carbohydrate metabolism, which provides energy for embryonic growth and development. Therefore, it can be concluded that BmINR and BmAC6 genes regulate embryonic diapause in bivoltine B. mori.

m6A-dependent mevalonate kinase in juvenile hormone synthesis pathway regulates the diapause process of bivoltine silkworm (Bombyx mori).

BACKGROUND: Research has shown that epigenetic modification are involved the regulation of diapause in bivoltine silkworms (Bombyx mori), but it remains unclear how epigenetic modification in response to environmental signals precisely to regulate the diapause processing of bivoltine B. mori. METHODS AND RESULTS: In this study, the diapause terminated eggs of bivoltine B. mori, Qiufeng (QF) were divided into two groups: a QFHT group incubated at 25 °C with a natural day/night cycle to produce diapause eggs, and a QFLT group incubated at 16.5 °C in darkness to produce non-diapause eggs. On the 3rd day of the pupal stage, the total RNAs of the eggs were extracted and their N6-adenosine methylation (m6A) abundances were analyzed to explore the effects of m6A methylation on diapause in the silkworm. The results showed that 1984 m6A peaks are shared, 1563 in QFLT and 659 in QFHT. The m6A methylation level of the QFLT group was higher than that of the QFHT one in various signaling pathways. The m6A methylation rate of mevalonate kinase (MK) in the insect hormone synthesis pathway was significantly different between the two groups. The knockdown of MK by RNA interference in the pupae of QFLT resulted in females laying diapause eggs rather than non-diapause eggs after mating. CONCLUSIONS: m6A methylation involves in the diapause regulation of bivoltine B. mori by changing the expression levels of MK. This result provides a clearer image of the environmental signals on the regulation of diapause in bivoltine silkworms.

Metabolomics analysis of dietary restriction results in a longer lifespan due to alters of amino acid levels in larval hemolymph of Bombyx mori.

Dietary restriction (DR) has been a very important discovery in modern aging biology research. Its remarkable anti-aging effect has been proved in a variety of organisms, including members of Lepidoptera, but mechanisms by which DR increases longevity are not fully understood. By using the silkworm (Bombyx mori), a model of lepidopteran insect, we established a DR model, isolated hemolymph from fifth instar larvae and employed LC-MS/MS metabolomics to analyze the effect of DR on the endogenous metabolites of silkworm, and tried to clarify the mechanism of DR to prolong lifespan. We identified the potential biomarkers by analyzing the metabolites of the DR and control groups. Then, we constructed relevant metabolic pathways and networks with MetaboAnalyst. DR significantly prolonged the lifespan of silkworm. The differential metabolites between the DR and control groups were mainly organic acids (including amino acid), and amines. These metabolites are involved in metabolic pathways such as amino acid metabolism. Further analysis showed that, the levels of 17 amino acids were significantly changed in the DR group, indicating that the prolonged lifespan was mainly due to changes in amino acid metabolism. Furthermore, we identified 41 and 28 unique differential metabolites in males and females, respectively, demonstrating sex differences in biological responses to DR. The DR group showed higher antioxidant capacity and lower lipid peroxidation and inflammatory precursors, with differences between the sexes. These results provide evidence for various DR anti-aging mechanisms at the metabolic level and novel reference for the future development of DR-simulating drugs or foods.

Determination of Key Components in the Bombyx mori p53 Apoptosis Regulation Network Using Y2H-Seq.

The apoptosis pathway is highly conserved between invertebrates and mammals. Although genes encoding the classical apoptosis pathway can be found in the silkworm genome, the regulatory pathway and other apoptotic network genes have yet to be confirmed. Consequently, characterizing these genes and their underlying mechanisms could provide critical insights into the molecular basis of organ apoptosis and remodeling. A homolog of p53, a key apoptosis regulator in vertebrates, has been identified and cloned from Bombyx mori (Bmp53). This study confirmed via gene knockdown and overexpression that Bmp53 directly induces cell apoptosis and regulates the morphology and development of individuals during the metamorphosis stage. Furthermore, yeast two-hybrid sequencing (Y2H-Seq) identified several potential apoptotic regulatory interacting proteins, including the MDM2-like ubiquitination regulatory protein, which may represent an apoptosis factor unique to Bmp53 and which differs from that in other Lepidoptera. These results provide a theoretical basis for analyzing the various biological processes regulated by Bmp53 interaction groups and thus provide insight into the regulation of apoptosis in silkworms. The global interaction set identified in this study also provides a basic framework for future studies on apoptosis-dependent pupation in Lepidoptera.

YTHDF3 Is Involved in the Diapause Process of Bivoltine Bombyx mori Strains by Regulating the Expression of Cyp307a1 and Cyp18a1 Genes in the Ecdysone Synthesis Pathway.

The variable diapause features of bivoltine silkworm (Bombyx mori) strains regulated by environmental signals in the embryonic stage are closely related to epigenetics. Previously, we showed that the expression of YTHDF3 is significantly different in the pupae of the bivoltine silkworm Qiufeng developed from eggs incubated at a normal temperature (QFHT, diapause egg producer) compared to those from eggs incubated at a low temperature (QFLT, nondiapause egg producer), indicating that the expression of diapause-associated genes is regulated by the m6A modification level. However, how YTHDF3 regulates the expression of diapause-related genes remains unclear. In this study, we observed that the knockdown of B. mori YTHDF3 resulted in delayed embryo development, while the overexpression of YTHDF3 resulted in the transformation of nondiapause-destined eggs into a mixture of diapause and nondiapause eggs. Further studies showed that YTHDF3, as a reading protein, can recognize the m6A site of Cyp307a1 and Cyp18a1 genes in the ecdysone synthesis pathway (ESP), and the overexpression of YTHDF3 affects the diapause traits of the silkworm by decreasing the stabilities of mRNAs of Cyp307a1 and Cyp18a1 and inhibiting their translation. The above results demonstrate that m6A modification mediates YTHDF3 to affect the expression levels of its target genes, Cyp307a1 and Cyp18a1, in the ESP to regulate diapause in bivoltine B. mori. This is the first report of the m6A methylation regulation mechanism in diapause in B. mori and provides new experimental data for clarifying the diapause regulation network.

Development of a fast and ultrasensitive black phosphorus-based colorimetric/photothermal dual-readout immunochromatography for determination of norfloxacin in tap water and river water.

A rapid and ultrasensitive method for colourimetric/photothermal dual-readout detection was developed using an 808 nm NIR laser and a thermal imaging app on mobile phone. Norfloxacin was used as a model contaminant to demonstrate this universal rapid detection method. It is innovatively, to use the advanced two-dimensional material black phosphorus as a colourimetric/photothermal reagent for the first time. The samples were added to the strip, and the analytes were selectively captured on the conjugate pad by monoclonal antibody-modified magnetic/upconversion nanocomposites. The samples flowed through the strips by capillary action until reaching the control line, where immune complex formation occurred due to the presence of secondary antibody. The added black phosphorus could be captured by the the antigens which were directly exposed to the test line and a brown band could be observed by naked eye. Upon illumination by NIR light for 1 min, the real-time temperature is obtained for quantitative analysis through the thermal imaging performed by mobile phone camera. This method can achieve the detection of norfloxacin in water samples within 20 min, and the detection limits of colorimetric and photothermal readout can reach 45 pg mL-1. Compared with conventional strips, this method provided an increased sensitivity by about two orders of magnitude, with a integrated portable laser and a mobile phone. It is a valuable method for rapid detection and can be applied to other environmental contaminants as well.

Surface Siloxane-Modified Silica Materials Combined with Metal-Organic Frameworks as Novel MALDI Matrixes for the Detection of Low-MW Compounds.

Surface siloxane (3-aminopropyl triethoxysilane hydrolyzates)-modified silica materials were used as "initiators", which resulted in the release and desorption of intact molecules adsorbed on the surface of a matrix. A covalently cross-linked MIL-53(Al) material was used to enhance the ionization of analytes. Herein, we have provided an efficient matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) matrix strategy, which responded to both ion and laser irradiation with low background interference in the low-molecular-weight (MW) region. The matrixes MIL-53(Al), SBA-15@APTES, SiO2@APTES, SBA-15@APTES@MOF, and SiO2@APTES@MOF were synthetized and used for the analysis of a series of low-MW compounds to verify the effectiveness of the strategies. Compared to conventional matrixes, the surface-modified SBA-15@APTES@MOF and SiO2@APTES@MOF had low background, high sensitivity, extensive applicability, good stability, and ultrahigh tolerance of salt concentrations. The detection limits of standard analytes were determined to range from 0.1 to 1 × 10-5 mg/mL for 16 amino acids as well as citric acid, reserpine, tetraethylammonium chloride, melamine, bisphenol A, and malachite green. These results could help in designing more efficient nanostructure-initiator materials and further promote the application of MALDI-TOF MS.

The spatial filtering method of particle velocity measurement based on linear electrostatic sensor matrix.

An electrostatic sensor is a powerful tool for measuring flow parameters of gas-solid flow. In this study, a linear electrostatic sensor matrix (LESM) is designed to measure local particle average velocity within pipeline. The 3D simulation model of the LESM is built, and its spatial filtering effect is analyzed by the finite element method. The quantitative relational expression between peak frequency of the LESM and particle velocity is obtained. Then, the LESM-based particle velocimeter is further developed and its performance is verified on a gravity transport test rig. Simulation and experimental results demonstrate that the measurement method is capable of eliminating the effect of particle size and particle spatial position on velocity measurement and measuring velocity with a relative error not greater than 7.5%, while the standard deviation of the consecutive velocity measurement is within ±9.96% over the velocity range of 2.34-4.75 m/s.