Study On The Metabolic Regulatory Mechanism Related To Host Immune Response After Lymantria Dispar Multiple Nucleopolyhedrovirus(LdMNPV) Infection

Toll,IMD(Immune deficiency)and JNK pathways play important roles in insect innate immunity.However,there are few studies to investigate the roles of Toll,IMD and JNK pathways in the immunity of anti-baculovirus.The immunity is closely related to metabolism and the activation of immune response may lead to metabolic changes.The effect of baculovirus on host metabolism is not fully understood.Lymantria dispar multiple nucleopolyhedrovirus(Ld MNPV)belongs to Baculoviridae and specifically infect Lymantria dispar.In this study,L.dispar and Ld MNPV were used as materials to investigate the responses of Toll,IMD and JNK pathways and antimicrobial peptides(AMPs)to Ld MNPV infection and the effects of Ld MNPV infection on carbohydrate and lipid metabolism in L.dispar.The main results of this study are as follows:1.Ld MNPV could activate Toll pathway of L.dispar: 20 genes of Toll pathway of L.dispar were analyzed,including eleven Toll-like receptors genes(Ld TLR1-Ld TLR11),four Spaetzle genes(Ld Spz1,3,4 and 5)and one each for My D88,Pelle,Tube,Cactus and Dorsal genes.Eleven Toll-like receptors of L.dispar were highly expressed in head,hemolymph,fat body and Malpighian tubule.The expression levels of Ld Spz1 and Ld Spz5 were highest in hemolymph,while the expression levels of Ld Spz3 and Ld Spz4 were highest in head.Other genes of Toll pathway in L.dispar were highly expressed in different tissues.Expression levels of Ld TLR1,Ld TLR2,Ld TLR5,Ld TLR6,Ld TLR7,Ld TLR8,Ld Spz1,Ld Spz5 and other genes of Toll pathway in L.dispar were significantly up-regulated after Ld MNPV infection,indicating that Ld MNPV could activate the Toll pathway of L.dispar.2.Ld MNPV could activate the IMD and JNK pathways of L.dispar: Three genes of IMD pathway and three genes of JNK pathway in L.dispar were analyzed.Expression levels of three genes of the IMD pathway were highest in midgut,and the expression levels of three genes of the JNK pathway were highest in head.The expression levels of three genes of IMD pathway and three genes of JNK pathway of L.dispar were significantly up-regulated after Ld MNPV infection,indicating that Ld MNPV could activate the IMD and JNK pathways of L.dispar.JNK pathway is a branch of IMD pathway.The activation of JNK pathway by Ld MNPV again demonstrated that Ld MNPV could activate the IMD pathway of L.dispar.3.Five AMPs of L.dispar in response to Ld MNPV infection: Activation of the Toll and IMD pathways ultimately leads to the production of AMPs.Four cationic AMP genes and one anionic AMP gene of L.dispar were highly expressed in head,hemolymph and fat body.The expression of these five AMP genes were significantly induced by Ld MNPV infection.Compared to long-chain AMPs,the response speed of short-chain AMPs to Ld MNPV infection was faster.After the injection of gloverin ds RNA,the DNA replication levels of Ld MNPV in L.dispar was significantly increased,suggesting that gloverin could inhibit the DNA replication of Ld MNPV.The responses of five AMPs to Ld MNPV infection again demonstrated that Ld MNPV could activate Toll and IMD pathways of L.dispar.4.Effect of Ld MNPV infection on carbohydrate metabolism of L.dispar: The glycogen content of L.dispar decreased significantly at 48 and 72 h after Ld MNPV infection.The expression level of glycogen synthase gene was significantly up-regulated at 24 and 48 h post infection(hpi)and then significantly down-regulated at 72 hpi.The expression of glycogen synthase kinase 3 gene was significantly inhibited at 48 and 72 hpi.The trend of glycogen synthesis was present in L.dispar after Ld MNPV infection,which may be due to the insufficient glycogen content.The trehalose content of L.dispar significantly decreased at 48 and 72 h after Ld MNPV infection.The expression level of trehalose 6 phosphate synthase gene was significantly up-regulated only at 24 hpi,indicating that glycogen may not be converted to trehalose,but may be directly to glucose.The expression of the soluble trehalase gene was significantly inhibited while the expression level of membrane-bound trehalase gene was significantly up-regulated in L.dispar after Ld MNPV infection.Membrane-bound trehalase of L.dispar was mainly expressed in hemolymph,indicating that the decrease of trehalose content of L.dispar after Ld MNPV infection may be due to the degradation of trehalose in hemolymph into glucose.In addition,the glucose content of L.dispar significantly decreased after Ld MNPV infection.Two hexokinase genes were significantly up-regulated and the activity of hexokinase and the pyruvate content significantly increased in L.dispar after Ld MNPV infection,indicating that the glycolysis was enhanced after Ld MNPV infection.Three key genes involved in tricarboxylic acid cycle in L.dispar were significantly upregulated after Ld MNPV infection,indicating that the tricarboxylic acid cycle was enhanced after Ld MNPV infection.Therefore,the enhancement of glycolysis and tricarboxylic acid cycle may be important reasons for the decrease of carbohydrate content in L.dispar after Ld MNPV infection.Inhibition of glycolysis significantly reduced the expression levels of five AMP genes of L.dispar after Ld MNPV infection,indicating that the carbohydrate may provide energy for immune response of L.dispar through glycolysis after Ld MNPV infection.5.Effect of Ld MNPV infection on lipid metabolism of L.dispar: The change of phosphatidylcholine(PC)and lysophophatidylcholine(LPC)was the most obvious in glycerophospholipids of L.dispar after Ld MNPV infection.The PC content showed an increasing trend after Ld MNPV infection,while the LPC content showed a decreasing trend.Additionally,the expression level of phospholipase A2 of L.dispar was significantly upregulated after Ld MNPV infection,while the expression level of lysophospholipid acyltransferase was not changed,indicating that the conversion of PC to LPC may occurred after Ld MNPV infection.The contents of 20 kinds of triacylglycerol were significantly downregulated and the contents of 14 kinds of triacylglycerol were significantly up-regulated after Ld MNPV infection,while the total triacylglycerol content was not changed.Additionally,the unsaturation of up-regulated triacylglycerol was significantly lower than that of downregulated triacylglycerol.Therefore,Ld MNPV infection may lead to a tendency toward storage of triacylglycerol in L.dispar.The contents of four kinds of long chain fatty acids of L.dispar were significantly up-regulated after Ld MNPV infection.The content of cytoplasmic citric acid significantly decreased and the expression levels of genes related to fatty acid synthesis were significantly up-regulated in L.dispar after Ld MNPV infection,indicating that Ld MNPV infection led to enhanced fatty acid synthesis and the accumulation of long chain fatty acids.Enhanced fatty acid synthesis can promote de novo synthesis of ceramide.The contents of all differentially expressed ceramide and hexosylceramide in L.dispar after Ld MNPV infection were significantly up-regulated.The increase of the ceramide content in L.dispar after Ld MNPV infection was due to enhanced de novo synthesis rather than the degradation of sphingomyelin.In conclusion,Ld MNPV could activate the Toll,IMD and JNK pathways and the expression of AMP genes in L.dispar,and the carbohydrate may provide energy for the immune response through glycolysis.At the same time,viral infection increased carbon sources for fatty acid and lipid synthesis by enhancing glycolysis and tricarboxylic acid cycle and significantly altered lipid metabolism of L.dispar.