MARC dependent NO synthesis activates CanA-Relish-AMPs signal pathway in Eriocheir sinensis during nitrite stress

Abstract

As a signal molecule, nitric oxide (NO) can induce the production of antimicrobial peptides (AMPs) in invertebrate innate immunity and is produced through NO synthase (NOS) oxidation or nitrite reduction. Although the role of NOS-derived NO has been extensively studied, studies on nitrite-dependent NO are relatively scarce. In this study, we identified a mitochondrial amidoxime reducing component (mARC), a kind of nitrite reductase, in Eriocheir sinensis. Under nitrite stress, the expression level of EsmARC in the intestine of E. sinensis increased, and the production of NO increased. Furthermore, EsmARC knockdown resulted in a remarkable decrease in NO concentration. These findings indicate that nitrite stress induces the expression of mARC, which promotes the production of NO in E. sinensis. In addition, the expression levels of AMPs in the intestine were upregulated under nitrite stress. Moreover, EsmARC knockdown resulted in the downregulated expression of AMPs. EsmARC plays a positive role in the synthesis of AMPs under nitrite stress. Calcineurin subunit A (CanA) is a serine/threonine protein phosphatase involved in the process by which NO regulates the expression of AMPs. EsCanA knockdown significantly inhibited the transcription of EsRelish and the expression of AMPs under nitrite stress, and EsRelish silencing resulted in the downregulated expression levels of AMPs under nitrite stress. These results indicate that nitrite stress activates the CanA–Relish–AMP pathway in E. sinensis. In summary, mARC-dependent NO synthesis activates the CanA–Relish–AMP signal pathway in E. sinensis during nitrite stress. This research provides novel insights into the relationship between nitrite stress and NO-dependent immune signal activation in crustaceans.