Generation Of Nitric Oxide Assay And Antimicrobial Peptides Genes Expression After Oral Infection With Bacteria In The Silkworm, Bombyx Mori

Insects get infection mainly via food intake, as human getting diseases through the diet.Bombyx mori is a typical model insect of Lepidoptera and also an economic insect, and thereare lots of researches based on it. We studied the expression of NOS (Nitric Oxide Synthase)and the concentration of NO (Nitric Oxide), which affected the immunity of insects byinducing the expression of related antimicrobial peptide (AMP) after the silkworm infectedby Pseudomonas aeruginosa (P. aeruginosa) and Staphyloccocus aureus (S. aureus). In thisstudy, we clarified the relationship between NO and AMPs in the silkworm intestinalimmunity.We tested NO concentrations in the haemolymph and intestinal digestive fluid anddetected the expression of AMP genes in midgut and fat body after feeding withgram-negative bacteria (P. aeruginosa), gram-positive bacteria (S. aureus), NO donor(S-nitroso-N-acetyl-penicillamine, SNAP; Diethylamine-NO complex, NOC) and NOSinhibitor (NG-Nitro-L-arginine methylester hydrochloride, L-NAME). After infecting with P.aeruginosa, we found levels of NO concentration elevated in the gut content at16h and in thehaemolymph at20h. When the silkworm was infected by S. aureus, NO concentration risedsharply in the haemolymph but it didn’t change in the gut content. It suggested that thecharacteristic of NO concentration’s increasing depends on the bacterial species and the timeafter infection.We found the expression of NOS and AMP genes increased in the midgut after infection.The infection of S. aureus induced the expression of Cecropin B6, ecropin D, Moricin andAttacin2. Cecropin B6and Moricin also expressed after infected by P. aeruginosa, but theexpression of this two AMPs was lower than the inducing of infection by S. aureus. We alsofound that the expression of Gloverin2and Lebocin was induced by both P. aeruginosa and S.aureus, and the induction by P. aeruginosa is lower than S. aureus. Researchers had found four negative regulators of the Imd pathway: Caspar、Dusp36、PIMS and CYLD. We detected their expression in the midgut and found that Caspar、Dusp36and PIMS could be induced at8h、16h、24h after infected by P. aeruginosa and S.aureus,which was the same as the sharply decreasing expression of NOS2、Gloverin2andLebocin at16h and24h after infection. It suggested that the silkworm could produce someimmune factors to prevent the infection, and some negative regulators can also be expressedto prevent excessive activity.Feeding the silkworms with L-NAME2hours after intestinal infection by P.aeruginosa, we found the expression of some antimicrobial peptides Gloverin2, Attacin2Cecropin D were suppressed. After feeding the uninfected silkworm with SNAP and NOC,we found the expression of some antimicrobial peptides Gloverin2, Lebocin, Cecropin B6,Cecropin D, Moricin, Attacin2, defensin in the midgut and Lebocin, Cecropin B6, CecropinD, Moricin, defensin in the fatbody increased. We found intestinal infection causes thesynthesis of NO and the induction of some antibacterial peptide transcription in silkworm.Inhibit NO expression could restrain the transcription of some antibacterial peptide; inverselyincreasing the NO expression would lead to some antibacterial peptide genes expressionsuggesting that NO can causes some AMPs’ transcription.