Functional Analysis Of3-dehydroecdysone3β-reductase Gene In Immune System Of Silkworm, Bombyx Mori

Ecdysone is one of the most important hormones in insects, which controls the growth, development, reproduction and decay of insects. At present, many studies have shown that ecdysone also plays a role in insect innate immunity, but how ecdysone regulate insect innate immune has not been elucidated. Different studies obtained different conclusions, even in silkworm, the role of ecdysone on the immune system has not been determined. In addition, previous studies of the relationship between ecdyson and insect immunity were through in vitro experiments, or injected exogenous ecdysone to observe the impact on immunity. After being infected, whether the insects will take the initiative to regulate ecdysone titers to affect the autoimmune is still unknown. In this study, we take the silkworm as a model to study the role of ecdysone on the silkworm immune system. We found that after infected by exogenous microorganisms, the silkworm significantly upregulated the expression of3-dehydroecdysteroid3beta-reductase(3DE-3β-redutase) gene which encodes ecdysone hormone synthesis enzyme, suggesting that it may play a role on the innate immunity of silkworm. In addition, we confirmed the function of this gene in innate immunity by RNAi interference. The results are as follows:(1) When infected, injection of a certain concentration of20E can significantly improve the survival rate of the silkworm. Further, after injection of FITC-labeled bacteria, we observed that the number of bacteria in hemolymph with20E was significantly lower than the number of bacteria in hemolymph without20E. These results suggest that20E enhances the cellular immune in silkworm. (2) Insect hemocyte will spread when infected. Different types of hemocyte have effect on pathogens, such as phagocytosis, encapsulation and nodule formation. Therefore, hemocyte-spreading reflects the strength of the cellular immunity. Treating hemolymph of silkworm with different concentrations of20E, hemocyte-spreading rates have changed significantly, the higher concentration of20E, the higher hemocyte-spreading rates.(3) When infected,3DE-3β-reductase gene was up-regulated expression at different time points. This suggests that silkworm may upregulate3DE-3β-reductase gene expression to increase ecdysteroids titer.(4) After successful interference with3DE-3β-reductase gene of silkworm, when injected exogenous microorganisms, silkworm survival was significantly lower than the control group (not to interfere in3DE-3β-reductase gene). In addition, after knocked down3DE-3β-reductase gene, expressions of most antimicrobial peptide genes were down-regulated when infected with bacteria. This shows that3DE-3β-reductase gene may participate in the immune system of the silkworm.In summary, our results showed that ecdysone may enhance the cellular immune in silkworm. In addition, when infected by exogenous microorganisms, the silkworm upregulates3DE-3β-reductase gene expression to increase ecdysteroids titer to enhance the innate immunity.