BmMD-2A responds to 20-hydroxyecdysone and regulates Bombyx mori silkworm innate immunity in larva-to-pupa metamorphosis.

20E-hydroxyecdysone (20E) plays important roles in larval molting and metamorphosis in insects and is also involved in the insect innate immune response. Insect metamorphosis is a highly successful strategy for environmental adaptation and is the most vulnerable stage during which the insect is susceptible to various pathogens. 20E regulates a series of antimicrobial peptides (AMPs) through the immunodeficiency (IMD) pathway activation in Drosophila; nevertheless, whether other immune pathways are involved in 20E-regulated insect immunity is unknown. Our previous studies showed that BmMD-2A is a member of the MD-2-related lipid recognition (ML) family of proteins that are involved in the Bombyx mori innate immunity Toll signaling pathway. In this study, we further demonstrate that BmMD-2A is also positively regulated by 20E, and the BmMD-2A neutralization experiment suggested that 20E activates some downstream immune effect factors, the AMP genes against Escherichia coli and Staphylococcus aureus, through the regulation of BmMD-2A in larval metamorphosis, implying that B. mori may use the Toll-ML signaling pathway to maintain innate immune balance in the larval-pupal metamorphosis stage, which is a different innate immunity pathway regulated by 20E compared to the IMD pathway in Drosophila.

P300/HDAC1 regulates the acetylation/deacetylation and autophagic activities of LC3/Atg8-PE ubiquitin-like system.

Protein acetylation plays potential roles in regulating autophagy occurrence. However, it varies greatly between yeast and mammals, and has not been thoroughly investigated in other organisms. Here, we reported that the components of BmAtg8-PE ubiquitin-like system (BmAtg3, BmAtg4, BmAtg7, and BmAtg8) in Bombyx mori were localized in the nucleus under nutrient-rich conditions, whereas they were exported to the cytoplasm upon autophagy induction. RNAi of BmP300 and inhibition of BmP300 activity resulted in nucleo-cytoplasmic translocation of BmAtg3 and BmAtg8, as well as premature induction of autophagy in the absence of stimulus. Conversely, RNAi of BmHDAC1 and inhibition of class I/II HADCs activities led to the nuclear accumulation of BmAtg3 and BmAtg8. In addition, acetylation sites in Atg proteins of BmAtg8-PE ubiquitin-like system were identified by mass spectrometry, and acetylation-site mutations caused nucleo-cytoplasmic translocation of BmAtg3, BmAtg4, and BmAtg8 along with autophagy promotion. Similarly, the subcellular localization of human ATG4b is determined by acetylation modification. In general, BmP300-mediated acetylation sequesters the components of BmAtg8-PE ubiquitin-like system in the nucleus, thus leading to the autophagy inhibition. Oppositely, BmHDAC1-mediated deacetylation leads to the nucleo-cytoplasmic translocation of the components of BmAtg8-PE ubiquitin-like system and promotes autophagy. This process is evolutionarily conserved between insects and mammals.

Toll9 from Bombyx mori functions as a pattern recognition receptor that shares features with Toll-like receptor 4 from mammals.

Toll/Toll-like receptors (TLRs) are key regulators of the innate immune system in both invertebrates and vertebrates. However, while mammalian TLRs directly recognize pathogen-associated molecular patterns, the insect Toll pathway is thought to be primarily activated by binding Spätzle cytokines that are processed from inactive precursors in response to microbial infection. Phylogenetic and structural data generated in this study supported earlier results showing that Toll9 members differ from other insect Tolls by clustering with the mammalian TLR4 group, which recognizes lipopolysaccharide (LPS) through interaction with myeloid differentiation-2 (MD-2)-like proteins. Functional experiments showed that BmToll9 from the silkmoth Bombyx mori also recognized LPS through interaction with two MD-2-like proteins, previously named BmEsr16 and BmPP, that we refer to in this study as BmMD-2A and BmMD-2B, respectively. A chimeric BmToll9-TLR4 receptor consisting of the BmToll9 ectodomain and mouse TLR4 transmembrane and Toll/interleukin-1 (TIR) domains also activated LPS-induced release of inflammatory factors in murine cells but only in the presence of BmMD-2A or BmMD-2B. Overall, our results indicate that BmToll9 is a pattern recognition receptor for LPS that shares conserved features with the mammalian TLR4-MD-2-LPS pathway.

Cholesterol derivatives induce dephosphorylation of the histone deacetylases Rpd3/HDAC1 to upregulate autophagy.

Histone deacetylases (HDACs) are important for global gene expression and contribute to numerous physiological events. Deacetylase Rpd3 in yeast and its conserved homolog HDAC1 in mammals oppositely regulate autophagy; however, how Rpd3/HDAC1 is regulated to mediate autophagy remains unclear. Here, we showed autophagy occurrence in silkworm (Bombyx mori) required BmRpd3, wherein steroid hormone 20-hydroxyecdysone (20E) signaling regulated its protein level and nuclear localization negatively. Inhibition of MTOR led to dephosphorylation and nucleo-cytoplasmic translocation of BmRpd3/HsHDAC1. Besides, cholesterol, 20E, and 27-hydroxycholesterol could all induce massive dephosphorylation and cytoplasmic localization of BmRpd3/HsHDAC1, and thus autophagy by affecting MTORC1 activity. In addition, three phosphorylation sites (Ser392, Ser421, and Ser423) identified in BmRpd3 were conserved in HsHDAC1. Single or triple phosphorylation-site mutation attenuated the phosphorylation levels of BmRpd3/HsHDAC1, leading to their cytoplasmic localization and autophagy activation. In general, cholesterol derivatives, especially hydroxylated cholesterol, caused dephosphorylation and nucleo-cytoplasmic shuttling of BmRpd3/HsHDAC1 through inhibition of MTOR signaling to facilitate autophagy in B. mori and mammals. These findings improve our understandings of BmRpd3/HsHDAC1-mediated autophagy induced by cholesterol derivatives and shed light on their potential as a therapeutic target for neurodegenerative diseases and autophagy-related studies.Abbreviations: 20E: 20-hydroxyecdysone; 27-OH: 27-hydroxycholesterol; ACTB: actin beta; AMPK: AMP-activated protein kinase; Atg: autophagy-related; BmSqstm1: Bombyx sequestosome 1; CQ: chloroquine; HDAC: histone deacetylase; LMNB: Lamin B1; MTOR: mechanistic target of rapamycin kinase; PE: phosphatidylethanolamine; SQSTM1/p62: sequestosome 1; TUBA1A: tubulin alpha 1a.

Clathrin-dependent endocytosis predominantly mediates protein absorption by fat body from the hemolymph in Bombyx mori.

During insect larval-pupal metamorphosis, proteins in the hemolymph are absorbed by the fat body for the maintenance of intracellular homeostasis; however, the type of proteins and how these proteins are internalized into the fat body are unclear. In Bombyx mori, the developmental profiles of total proteins in the hemolymph and fat body showed that hemolymph-decreased protein bands (55-100 kDa) were in accordance with those protein bands that increased in the fat body. Inhibition of clathrin-dependent endocytosis predominantly blocked the transportation of 55-100 kDa proteins from the hemolymph into the fat body, which was further verified by RNA interference treatment of Bmclathrin. Six hexamerins were shown to comprise ∼90% of the total identified proteins in both the hemolymph and fat body by mass spectrum (MS) analysis. In addition, hemolymph-specific proteins were mainly involved in material transportation, while fat body-specific proteins particularly participated in metabolism. In this paper, four hexamerins were found for the first time, and potential proteins absorbed by the fat body from the hemolymph through clathrin-dependent endocytosis were identified. This study sheds light on the protein absorption mechanism during insect metamorphosis.

Steroid hormone 20-hydroxyecdysone induces the transcription and complex assembly of V-ATPases to facilitate autophagy in Bombyx mori.

Vacuolar-type H + -adenosine triphosphatases (V-ATPases) are indispensable for lysosome acidification and participate in autophagic processes. The steroid hormone 20-hydroxyecdysone (20E) predominantly induces autophagy and regulates insect larval molting and metamorphosis; however, the specific mechanism of lysosome acidification regulation by 20E remains unclear. Here, we showed that the developmental profiles of Bombyx V-ATPases were in accordance with autophagy occurrence and lysosome acidification in the fat body during larval-pupal metamorphosis. BmV-ATPase-A and BmV-ATPase-B were required for lysosome acidification and autophagic flux. Both 20E treatment and starvation were able to induce lysosome acidification. Furthermore, BmV-ATPase transcription was induced by 20E treatment and reduced by RNAi targeting the 20E receptor BmUsp. On the one hand, 20E upregulated the transcription of BmV-ATPases through inducing Bombyx transcription factor EB (TFEB) and its nuclear translocation; on the other hand, 20E inhibited mTOR signaling to induce the transcription and assembly of BmV-ATPase subunits. Overall, 20E induces lysosome acidification by upregulating the transcription and assembly of V-ATPase subunits via activating BmTFEB and cooperating with nutrient signaling. These findings improve our understanding of the regulatory mechanisms underlying lysosome acidification and autophagic flux in Bombyx mori.

Regulation of antimicrobial peptide genes via insulin-like signaling pathway in the silkworm Bombyx mori.

Antimicrobial peptides (AMPs) are important effector molecules of insect humoral immunity, and expression of AMPs is mainly regulated by the Toll and immune deficiency (IMD) pathways. FoxO, a key downstream regulator of the insulin-like signaling (ILS) pathway, has been recently reported to be involved in the regulation of AMPs in Drosophila melanogaster. In the present study, we investigated AMP gene expression and the regulation pathway controlled by the starvation in the silkworm Bombyx mori. We discovered that antibacterial activity in the hemolymph of B. mori larvae was increased by starvation, and expression of AMP genes (BmCecB6, BmAtta1, BmLeb3 and BmDefB) as well as the ILS target genes (FoxO, InR and Brummer) were strongly activated in the fat body by starvation. Moreover, phosphorylation of Akt kinase was reduced in the Bm-12 cells after starvation, suggesting that the ILS pathway was inhibited by starvation. We then showed that more FoxO protein was present in the cytoplasm than in the nucleus of Bm-12 cells under normal conditions, but more FoxO was detected in the nucleus after cells were starved for 8 h, indicating that FoxO was activated by starvation. In summary, our results indicated that starvation can activate AMP gene expression in B. mori via the ILS/FoxO signaling pathway.

BmCalpains are involved in autophagy and apoptosis during metamorphosis and after starvation in Bombyx mori.

Apoptosis and autophagy play crucial roles during Bombyx mori metamorphosis and in response to various adverse conditions, including starvation. Recently, calpain, one of the major intracellular proteases, has been reported to be involved in apoptosis and autophagy in mammals. BmATG5 and BmATG6 have been identified to mediate apoptosis following autophagy induced by 20-hydroxyecdysone and starvation in B. mori. However, B. mori calpains and their functions remain unclear. In this study, phylogenetic analysis of calpains from B. mori, Drosophila melanogaster and Homo sapiens were performed and the results showed distinct close relationships of BmCalpain-A/B with DmCalpain-A/B, BmCalpain-C with DmCalpain-C, and BmCalpain-7 with HsCalpain-7. Then, the expression profiles of BmCalpains were analyzed by quantitative real-time polymerase chain reaction, and results showed that expression of BmCalpain-A/B, BmCalpain-C and BmCalpain-7 was significantly increased during B. mori metamorphosis and induced in the fat body and midgut of starved larvae, which is consistent with the expression profiles of BmAtg5, BmAtg6 and BmCaspase-1. Moreover, the apoptosis-associated cleavage of BmATG6 in Bm-12 cells was significantly enhanced when BmCalpain-A/B and BmCalpain-7 were induced by starvation, and was partially inhibited by the inhibitor of either calpain or caspase, but completely inhibited when both types of inhibitors were applied together. Our results indicated that BmCalpains, including BmCalpain-A/B, -C and -7, may be involved in autophagy and apoptosis during B. mori metamorphosis and after starvation, and may also contribute to the apoptosis-associated cleavage of BmATG6.

Molecular characterization, phylogeny analysis and pathogenicity of a Muscovy duck adenovirus strain isolated in China in 2014.

This study aimed to characterize a novel adenovirus (AdV) isolated from diseased Muscovy ducks in China. After the AdV was successfully propagated in duck embryo fibroblasts, the morphological and physicochemical properties of the virions were studied by electron microscopy and different tests. The results of the analyses were in conformity with AdV properties. The full genome sequence was determined and analyzed. The new isolate (named CH-GD-12-2014) shared over 91% sequence identity with duck AdV-2 representing the species Duck aviadenovirus B. The most important distinguishing feature between the two DAdV strains was the presence of a second fiber gene in the Chinese isolate. Phylogeny reconstruction confirmed the affiliation of the virus with goose and duck AdVs in the genus Aviadenovirus. Experimental infection resulted in embryo death, and intramuscular inoculation provoked morbidity and mortality among ducks and chickens.

BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20-hydroxyecdysone or starvation.

Autophagy and apoptosis, which could be induced by common stimuli, play crucial roles in development and disease. The functional relationship between autophagy and apoptosis is complex, due to the dual effects of autophagy. In the Bombyx Bm-12 cells, 20-hydroxyecdysone (20E) treatment or starvation-induced cell death, with autophagy preceding apoptosis. In response to 20E or starvation, BmATG8 was rapidly cleaved and conjugated with PE to form BmATG8-PE; subsequently, BmATG5 and BmATG6 were cleaved into BmATG5-tN and BmATG6-C, respectively. Reduction of expression of BmAtg5 or BmAtg6 by RNAi decreased the proportion of cells undergoing both autophagy and apoptosis after 20E treatment or starvation. Overexpression of BmAtg5 or BmAtg6 induced autophagy but not apoptosis in the absence of the stimuli, but promoted both autophagy and apoptosis induced by 20E or starvation. Notably, overexpression of cleavage site-deleted BmAtg5 or BmAtg6 increased autophagy but not apoptosis induced by 20E or starvation, whereas overexpression of BmAtg5-tN and BmAtg6-C was able to directly trigger apoptosis or promote the induced apoptosis. In conclusion, being cleaved into BmATG5-tN and BmATG6-C, BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20E or starvation in Bombyx Bm-12 cells, reflecting that autophagy precedes apoptosis in the midgut during Bombyx metamorphosis.