Study On The Links Between Toll Pathway And IMD Pathway In Innate Immunity Of Litopenaeus Vannamei

The two important signaling pathways in innate immunity of Litopenaeus vannamei: Toll pathway and IMD pathway, is very important to activate the immune related genes,expression of antibacterial peptides(AMPs), and resistance to pathogen infection. But,compared with other vertebrates, the research of innate immune mechanism in shrimp is still slow. Therefore, it is necessary to study the immune mechanism of defencing the pathogenic bacteria in shrimp, and that can provide more theoretical basis for preventing of shrimp disease professional and efficiently.In this study, we use the way of real-time fluorescence quantitative polymerase chain reaction(RT-qPCR), and tested the change of expression of eight key innate immunity genes(Toll and IMD, Pelle, IAP1, TRAF6, ALF, crustin, Penaeidin3) in four tissues of Litopenaeus vannamei,and detected their change expression after challenge with two kinds of bacterial. After that,we use the RNA interference technology, silenced the Toll gene upstream of the Toll pathway and the IMD gene upstream of the IMD pathway, detected the change of relative mRNA expression of key genes that downstream of two pathway,which post Vibrio anguillarum challenge and Micrococcus lysodeikticus challenge,and try to analyse the potential relationship between Toll pathway and IMD pathway. The results showed that:1. Through the analysis of the tissue distribution:the expression of genes in hemocytes, intestine, gill are similar: TRAF6, Toll, Pelle and IAP1 are highly expressed; the expression of IMD and ALF are slightly lower; Crustin and Pen3 expressed low. While in the hepatopancreas, IMD gene was expressed highly which different from other tissues;and TRAF6, Toll, Pelle expressed slightly lower; ALF, Pen3, IAP1 and Crustin have a low expression.While in the hepatopancreas showed a significant higher relative expression than other tissues(hemocytes, intestine, gill),that in three kinds of antimicrobial peptides(ALF, Crustin,and Pen3), Toll and IMD.2. Analysis of the key gene expression changes after infected with Vibrio anguillarum and Micrococcus lysodeikticus:The expression of Toll gene were significantly up regulated in hemocytes, but downregulated significantly in hepatopancreas; the expression of TRAF6 were down regulated,except that in hepatopancreas post Micrococcus lysodeikticus challenge;Pelle were down regulated, only up-regulated in hemocytes post Vibrio anguillarum challenge; Pen3 were up-regulated, only down regulated in hemocytes post Vibrio anguillarum challenge; IMD gene were up-regulated in both group; IAP ere down regulated in both group; ALF were only significantly up-regulated in Vibrio anguillarum challenge group; crustin were up regulated in hepatopancreas and were down regulated in hemocytes post Vibrio anguillarum challenge.3. Silenced Toll gene with RNA interference technology by double stranded RNA(dsRNA), in hemocytes: IMD, Akirin was significantly upregulated in Vibrio anguillarum group; TRAF6, Pelle,IAP1 were raised in both infected groups.After the IMD gene were silenced, Akrin expression were significantly up-regulated in Vibrio anguillarum group; Toll, Pelle,IAP1 were raised in both infected groups; and TRAF6 relative expression didnot changes.Therefore, we speculate that:1. IMD has a higher expression in hepatopancreas than hemocytes、intestine、gill; The expression of IMD is influenced by Toll;2. Hepatopancreas is a main location for the Toll pathway, IMD pathway and the production of antimicrobial peptides; The immune response of the hepatopancreas and hemocytes is different after infection;3.Toll pathway could be activated by Gram positive bacteria and Gram negative bacteria,and usually more easily activated by Gram positive bacteria; IMD pathway is mainly activated by Gram negative bacteria;Toll pathway and IMD pathway are not independent in shrimp, which are associated with to resistant pathogen infection;4. Akirin could involve in the antibacterial reaction from other ways include IMD pathway; Crustin and Pen3 could be regulated simultaneously by Toll pathway and IMD pathway.