Scavenger receptor B2, a type III membrane pattern recognition receptor, senses LPS and activates the IMD pathway in crustaceans.

The immune deficiency (IMD) pathway is critical for elevating host immunity in both insects and crustaceans. The IMD pathway activation in insects is mediated by peptidoglycan recognition proteins, which do not exist in crustaceans, suggesting a previously unidentified mechanism involved in crustacean IMD pathway activation. In this study, we identified a Marsupenaeus japonicus B class type III scavenger receptor, SRB2, as a receptor for activation of the IMD pathway. SRB2 is up-regulated upon bacterial challenge, while its depletion exacerbates bacterial proliferation and shrimp mortality via abolishing the expression of antimicrobial peptides. The extracellular domain of SRB2 recognizes bacterial lipopolysaccharide (LPS), while its C-terminal intracellular region containing a cryptic RHIM-like motif interacts with IMD, and activates the pathway by promoting nuclear translocation of RELISH. Overexpressing shrimp SRB2 in Drosophila melanogaster S2 cells potentiates LPS-induced IMD pathway activation and diptericin expression. These results unveil a previously unrecognized SRB2-IMD axis responsible for antimicrobial peptide induction and restriction of bacterial infection in crustaceans and provide evidence of biological diversity of IMD signaling in animals. A better understanding of the innate immunity of crustaceans will permit the optimization of prevention and treatment strategies against the arising shrimp diseases.

Lipases are differentially regulated by hormones to maintain free fatty acid homeostasis for insect brain development.

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.

The homotetramerization of a GPCR transmits the 20-hydroxyecdysone signal and increases its entry into cells for insect metamorphosis.

Animal steroid hormones initiate signaling by passive diffusion into cells and binding to their nuclear receptors to regulate gene expression. Animal steroid hormones can initiate signaling via G protein-coupled receptors (GPCRs); however, the underlying mechanisms are unclear. Here, we show that a newly discovered ecdysone-responsive GPCR, ErGPCR-3, transmits the steroid hormone 20-hydroxyecdysone (20E) signal by binding 20E and promoting its entry into cells in the lepidopteran insect Helicoverpa armigera Knockdown of ErGPCR-3 in larvae caused delayed and abnormal pupation, inhibited remodeling of the larval midgut and fat body, and repressed 20E-induced gene expression. Also, 20E induced both the interaction of ErGPCR-3 with G proteins and rapid intracellular increase in calcium, cAMP and protein phosphorylation. ErGPCR-3 was endocytosed by GPCR kinase 2-mediated phosphorylation, and interacted with ß-arrestin-1 and clathrin, to terminate 20E signaling under 20E induction. We found that 20E bound to ErGPCR-3 and induced the ErGPCR-3 homodimer to form a homotetramer, which increased 20E entry into cells. Our study revealed that homotetrameric ErGPCR-3 functions as a cell membrane receptor and increases 20E diffusion into cells to transmit the 20E signal and promote metamorphosis.

Mechanistic insights into diversified photoluminescence behaviours of BF2 complexes of N-benzoyl 2-aminobenzothiazoles.

Many BF2 complexes of heteroaromatics are well known for their dual-state emission (DSE) properties. However, AIE and ACQ effects have also been observed in certain cases. To date, no rational explanations have been proposed for these uncommon photoluminescence (PL) behaviours. The current research prepared four BF2 complexes of N-benzoyl 2-aminobenzothiazoles with diversified photoluminescence (PL) properties as model compounds and utilized quantum chemical calculation tools to address this issue. Theoretical calculations revealed that the electron-donating groups (EDGs) at the para-position of the exocyclic phenyl ring exert significant influence on their ground-state electronic structures and vertical excitation features. Potential energy curve (PEC) analysis showed that the exocyclic phenyl ring and NMe2 could not function as effective rotors due to elevated energy barriers. Only the NPh2 of BFBB-3 could spontaneously rotate ∼60° to induce the formation of an emissive twisted intramolecular charge transfer (TICT) state. The two-channel model involving both vibronic relaxation and S0/S1 surface crossing revealed that the drastic narrowing of the S1/S0 energy gap in the region approaching minimun energy conical intersection (MECI) led to the generation of a dark state in BFBB-1. The small energy barrier to access the dark-state region makes the resulting fast internal conversion a competitive channel for excited-state deactivation. In contrast, the presence of EDGs in BFBB-2 and 4 inhibits this pathway, thereby resulting in intense fluorescence emissions in solution. In addition, crystallographic analysis illustrated that the F atoms perpendicular to the polyheterocycle promoted a slipped face-to-face packing mode and enhanced intermolecular interactions. The efficiencies of their solid-state emissions are mainly affected by the degree of π-π overlaps.

20-Hydroxyecdysone counteracts insulin to promote programmed cell death by modifying phosphoglycerate kinase 1.

BACKGROUND: The regulation of glycolysis and autophagy during feeding and metamorphosis in holometabolous insects is a complex process that is not yet fully understood. Insulin regulates glycolysis during the larval feeding stage, allowing the insects to grow and live. However, during metamorphosis, 20-hydroxyecdysone (20E) takes over and regulates programmed cell death (PCD) in larval tissues, leading to degradation and ultimately enabling the insects to transform into adults. The precise mechanism through which these seemingly contradictory processes are coordinated remains unclear and requires further research. To understand the coordination of glycolysis and autophagy during development, we focused our investigation on the role of 20E and insulin in the regulation of phosphoglycerate kinase 1 (PGK1). We examined the glycolytic substrates and products, PGK1 glycolytic activity, and the posttranslational modification of PGK1 during the development of Helicoverpa armigera from feeding to metamorphosis. RESULTS: Our findings suggest that the coordination of glycolysis and autophagy during holometabolous insect development is regulated by a balance between 20E and insulin signaling pathways. Glycolysis and PGK1 expression levels were decreased during metamorphosis under the regulation of 20E. Insulin promoted glycolysis and cell proliferation via PGK1 phosphorylation, while 20E dephosphorylated PGK1 via phosphatase and tensin homolog (PTEN) to repress glycolysis. The phosphorylation of PGK1 at Y194 by insulin and its subsequent promotion of glycolysis and cell proliferation were important for tissue growth and differentiation during the feeding stage. However, during metamorphosis, the acetylation of PGK1 by 20E was key in initiating PCD. Knockdown of phosphorylated PGK1 by RNA interference (RNAi) at the feeding stage led to glycolysis suppression and small pupae. Insulin via histone deacetylase 3 (HDAC3) deacetylated PGK1, whereas 20E via acetyltransferase arrest-defective protein 1 (ARD1) induced PGK1 acetylation at K386 to stimulate PCD. Knockdown of acetylated-PGK1 by RNAi at the metamorphic stages led to PCD repression and delayed pupation. CONCLUSIONS: The posttranslational modification of PGK1 determines its functions in cell proliferation and PCD. Insulin and 20E counteractively regulate PGK1 phosphorylation and acetylation to give it dual functions in cell proliferation and PCD.

20-hydroxyecdysone reprograms amino acid metabolism to support the metamorphic development of Helicoverpa armigera.

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.

Effect of Astragalus polysaccharides on the cryopreservation of goat semen.

During cryopreservation, sperm encounters oxidative stress induced by excessive reactive oxygen species (ROS), destroying the sperm plasma membrane structure and reducing its physiological functions. The present study aimed to evaluate the effect of Astragalus polysaccharides (APS) on the cryopreservation of dairy goat semen. Semen was collected from six goats, and then qualified semen with movement >80% was selected after preliminary evaluation. The semen was divided into six aliquots, diluted with dairy goat semen extender (1:10) at 37 °C, containing 0 g/L (control), 0.1 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L and 0.5 g/L APS, cryopreserved, and stored in liquid nitrogen (-196 °C). Sperm quality was assessed after freeze-thawing. The highest sperm motility, motion performance, plasma membrane integrity, acrosome integrity, and antioxidant properties (total antioxidant capacity and levels of antioxidant enzymes) were recorded (P < 0.05) in the 0.2 g/L APS group after the semen was freeze-thawed. The control and the optimal group (0.2 g/L) were then selected to analyze the effects of APS on sperm energy metabolism (mitochondrial membrane potential [MMP] and adenosine triphosphate [ATP]), sperm apoptosis, and the expression of the AMPK signaling pathway. The results showed that treatment with 0.2 g/L APS increased sperm MMP and ATP content after freeze-thawing, reduced sperm apoptosis by regulating apoptosis-related proteins, and promoted AMPK phosphorylation by activating the AMPK signaling pathway. The cleavage rate of frozen goat sperm during in vitro fertilization (IVF) was also observed to increase. These findings suggest meaningful ways to improve cryopreservation of dairy goat semen and provide new insights into the mechanism by which APS protects sperm from oxidative damage via AMPK activation.

Juvenile hormone induces methoprene-tolerant 1 phosphorylation to increase interaction with Taiman in Helicoverpa armigera.

Methoprene-tolerant 1 (Met1) is a basic-helix-loop-helix Per/Arnt/Sim (bHLH-PAS) protein identified as the intracellular receptor of juvenile hormone (JH). JH induces phosphorylation of Met1; however, the phosphorylation site and outcomes of phosphorylation are not well characterized. In the present study, using the lepidopteran insect and serious agricultural pest Helicoverpa armigera (cotton bollworm) as a model, we showed that JH III induced threonine-phosphorylation of Met1 at threonine 393 (Thr393) in the Per-Arnt-Sim (PAS) B domain. Thr393-phosphorylation was necessary for Met1 binding to the JH response element (JHRE) to promote the transcription of Kr-h1 (encoding transcription factor Krüppel homolog 1) because Thr393-phosphorylated Met1 increased its interaction with Taiman (Tai) and prevented the Met1-Met1 association. However, JH III could not prevent Met1-Met1 association after Met1-Thr393 was mutated, suggesting that Thr393-phosphorylation is an essential mechanism by which JH prevents Met1-Met1 association. The results showed that JH induces Met1 phosphorylation on Thr393, which prevents Met1-Met1 association, enhances Met1 interaction with Tai, and promotes the binding of Met1-Tai transcription complex to the E-box in the JHRE to regulate Kr-h1 transcription.

Identification and Functional Analysis of G Protein-Coupled Receptors in 20-Hydroxyecdysone Signaling From the Helicoverpa armigera Genome.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in animals and humans, which transmit various signals from the extracellular environment into cells. Studies have reported that several GPCRs transmit the same signal; however, the mechanism is unclear. In the present study, we identified all 122 classical GPCRs from the genome of Helicoverpa armigera, a lepidopteran pest species. Twenty-four GPCRs were identified as upregulated at the metamorphic stage by comparing the transcriptomes of the midgut at the metamorphic and feeding stages. Nine of them were confirmed to be upregulated at the metamorphic stage. RNA interference in larvae revealed the prolactin-releasing peptide receptor (PRRPR), smoothened (SMO), adipokinetic hormone receptor (AKHR), and 5-hydroxytryptamine receptor (HTR) are involved in steroid hormone 20-hydroxyecdysone (20E)-promoted pupation. Frizzled 7 (FZD7) is involved in growth, while tachykinin-like peptides receptor 86C (TKR86C) had no effect on growth and pupation. Via these GPCRs, 20E regulated the expression of different genes, respectively, including Pten (encoding phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase), FoxO (encoding forkhead box O), BrZ7 (encoding broad isoform Z7), Kr-h1 (encoding Krüppel homolog 1), Wnt (encoding Wingless/Integrated) and cMyc, with hormone receptor 3 (HHR3) as their common regulating target. PRRPR was identified as a new 20E cell membrane receptor using a binding assay. These data suggested that 20E, via different GPCRs, regulates different gene expression to integrate growth and development.