Nitric oxide plays a crucial role in midgut immunity under microsporidian infection in Antheraea pernyi

Highlights

• NO level and NOS activity were influenced by microsporidia immune stimulation.

• AMP and immune genes can be influenced by NO content in Antheraea pernyi gut.

• Microsporidia infection rate change under NOS inhibitor treatment.

Abstract

The insect gut participates in initial local immune responses by producing reactive oxygen and nitrogen species as well as anti-microbial peptides to resist pathogenic invasions. Nitric oxide (NO), a signaling and an immune effector molecule synthesized by the enzyme NO synthase (NOS), mediates an early step of the signal transduction pathway. In this study, we evaluated NO levels after Nosema pernyi infection in Antheraea pernyi gut. NOS activity was higher in the microsporidia-infected gut of A. pernyi than in that of control. Three NOS-related genes were cloned, and their spatio-temporal expression patterns were evaluated. ApNOS2 was expressed quickly in the midgut after N. pernyi infection. Sodium nitroprusside, dihydrate (SNP), or Nω-_L-nitro-arginine methyl ester, hydrochloride (_L-NAME), altered the NO content in A. pernyi midgut. Anti-microbial peptides (AMPs) in the groups exposed to N. pernyi plus SNP and N. pernyi plus _L-NAME exhibited higher and lower expression, respectively, relative to the control. These results indicate that microsporidia infection triggers short-term activation of NO and NOS genes in the A. pernyi gut that is downregulated after 24 h. Notably, infection rates can be influenced by a NOS inhibitor. Furthermore, NO can be induced by pathogens. Similarly, NO content in the A. pernyi gut also influences AMPs in humoral immunity and some immune-related genes. Our results suggest that nitric oxide plays a vital role in A. pernyi gut immunity.

Introduction

The gut is one of the largest organs in the insect body cavity that serves to digest and absorb nutrients. It is also the first line of defense against a wide variety of orally transmitted pathogens. The insect intestinal epithelium, which functions as a physical barrier to combat harmful pathogens, also deploys efficient defense mechanisms to counteract infections. Redox molecules, including nitric oxide (NO) or NO-mediated reactive nitrogen species (RNS) and reactive oxygen species (ROS), are key regulators of immunity (Wink et al., 2011). Both ROS and NO/RNS are released by cells such as macrophages during immune responses (Forman and Torres, 2001). Along with anti-microbial peptides (AMPs) in humoral immunity, these molecules are the initial defense-line against pathogen infections (Levy, 2004).

NO is a small membrane-permeable signal molecule that functions as a cell signaling and immune effector in invertebrates (Alderton et al., 2001). NO is also critical in eradicating pathogens (Davies, 2000). NO synthase (NOS) has three isoforms; NOS-1 and NOS-3 are constitutively expressed in endothelial cells and neurons, whereas inducible type NOS-2 can generate high levels of NO following transcriptional induction (Colasanti et al., 2012; Sadekuzzaman et al., 2018). The NOS isoforms are also named according to their tissue of origin, i.e., eNOS (endothelial NOS, NOS1), nNOS (neuronal NOS, NOS3), and iNOS (inducible, macrophage NOS, NOS2).

In insects, NO is produced in response to microbial infection in several species of lepidopterans, dipterans, and hemipterans (Eleftherianos et al., 2010; Hao et al., 2003; Whitten et al., 2007). In Drosophila, NO is induced in the midgut and hemocytes after immune challenge with bacteria or parasitoids, and acts as a cytotoxic and signaling molecule that triggers the production of the AMP diptericin (Carton et al., 2009; Nappi et al., 2000). In blood-sucking mosquitoes, NOS expression is rapidly upregulated after blood feeding, leading to the elevated NO concentrations, which directly limit the development of the protozoan malaria parasites through the inducible synthesis of NO (Dimopoulos et al., 1998; Lim et al., 2005; Luckhart et al., 1998). Inhibition of NOS activity in Anopheles gambiae using chemical inhibitors significantly increases the number of Plasmodium oocysts formed in the midgut, suggesting that NO is essential in intestinal defense (Gupta et al., 2009). Not only does the incoming immune signal initiate the production of NO to intercept and kill pathogens, but also downstream signaling pathways that lead to the full expression of the immune response are modulated by NO in complex manners (Sadekuzzaman and Kim, 2018; Valko et al., 2007).

The Chinese oak silkworm Antheraea pernyi (Lepidoptera: Saturniidae) is a well-known wild moth that produces silk and high-protein foods commercially cultivated in China (Liu et al., 2010; Wang et al., 2015). As wild living insects, A. pernyi are inevitably infected by kinds of pathogenic microorganisms in the outdoor environment, including bacteria, fungi, virus and microsporidia (Wang et al., 2019a). Nosema pernyi is a common microsporidium parasite in A. pernyi and is a lethal pathogen that causes microsporidiosis through horizontal and vertical transmission (Wang et al., 2015). Microsporidia can infect the A. pernyi gut through polar tube and spore wall proteins (Ma et al., 2017; Wang et al., 2019b). However, NO content and related gene expressions during microsporidia infection are unclear. Here, we report the spatiotemporal dynamics of NO or NOS under microsporidia infection and relationships between NO contents and AMPs expression in A. pernyi. It is anticipated that changes in NO content can indirectly influence the microsporidia infection rate.