Effects Of Hydrogen Peroxide On The Immune Pathways In The Silkworm, Bombyx Mori

As an important economic insect, silkworm, Bombyx mori, has been domesticated for more than5,000years in China. Behind the great economic benefit, there are some limiting factors largely constrain the development of sericulture. The relatively low level of immunity in B. mori is one of these factors. Besides, the silkworm is also a good model insect of lepidoptera. Therfore the study of silkworm immunty is of great importance for sericulture practice and insect immunology.The innate immunity of insects is divided into humoral defenses, including the secretion of immune factors, and cellular defenses, such as phagocytosis that is mediated by hemocytes. Additionally, mitogen activated protein kinases (MAPKs) and Anus kinase/signal transducer and activator of transcription(JAK/STAT) signaling pathways are also associated with immunity. As an important molecule of reactive oxygen species (ROS), hydrogen peroxide (H2O2) is producted in the hosts and is essential for hosts defence. However, the role of H2O2in the silkworm immune system is still unclear. In this study, we detected the expression level of antimicrobial peptides (AMPs), mitogen activated protein kinases (JAK/STAT), activity of penoloxidase (PO), phosphorylation of mitogen activated protein kinases(MAPKs) and phagocytosis after H2O2injection. The main results are as follows:1. H2O2induced AMP genes expressionAfter H2O2injection, AMP genes expression in the fat body were detected by quantative PCR at four different time point from6to48h. Our results showed that in response to H2O2, Attacin2, CecropinB6, Cecropin D, Gloverin2and Morricin expression increased from6to48hpi continuously. The results demonstrated that H2O2could induce AMP genes expression.2. H2O2activated JAK/STAT pathwayAfter H2O2injection, the transcriptional levels of four members in JAK/STAT pathway were detected by semi-quantitative PCR at four different time points from6to48h. Our results showed that in response to increased H2O2in hemolymph, Dome, Hop, Socs expression increased from6to24hpi. Stat expression increased from12to48hpi. The results demonstrated that H2O2could activate JAK/STAT pathway.3. H2O2activated MAPK pathwayAfter H2O2injection, phosphorylation of three members (JNK、ERK、p38) in MAPK pathway was detected by Western blot. Our results showed that H2O2did not cause total levels of JNK and ERK changed, but caused p-JNK level increased slightly at6hpi and24hpi and p-ERK level increased from6to48hpi continuously. p-p38level showed an obvious increased at48hpi compared to controls. These results demonstrated that H2O2could induce MAPKs phosphorylation to activate MAPK pathway.4H2O2is involved in PPO cascade regulationPhenoloxidase activity in hemolymph was measured after the injection of H2O2. At12and24hpi, PO activity was higher in the H2O2treated samples. Western blot assays suggested the amount of PPO was higher in H2O2versus control plasma at12hpi but no significant difference was detected at other time points. We then assessed PPO activating enzyme (PPAE) transcriptional levels in hemocytes by quantitative PCR and the results showed that PPAE mRNA levels were higher in the H2O2treated samples at12-24hpi. All these results suggested that H2O2is involved in PPO cascade regulation.5. H2O2promoted phagocytosis of bacteria by hemocytesWe injected H2O2into larvae and two hours later injected dead, fluorescently-labeled S. aureus into the hemocoel, and then scored hemocytes for phagocytosis of these bacteria four hours later. We found that hemocytes from H2O2injected larvae displayed stronger fluorescence intensity than the control, suggesting that H2O2stimulated hemocytes to internalize more bacteria. To further confirm these results, fluorescently-labeled S. aureus were replaced with live bacteria (P. aeruginosa and S. aureus) in the above assays. Hemocytes was collected from treated silkworm larvae and counted the colony numbers of bacteria which released from the hemocytes. More bacterial colonies were recovered from hemocytes pre-treated with H2O2. Phagocytosis-related genes transcriptional level was measured after H2O2injection. Ykt6, Tim and Rab7expression were up-regulated promptly and remarkably by H2O2. These results demonstated H2O2promoted phagocytosis of bacteria by hemocytes.In summary, we draw a conclusion that H2O2acts as a messenger in silkworm immune system. It regulates immune response rapidly during infection and contributes to defending reactions to kill pathogens and parasites.