Nitric oxide (NO) at a high concentration is an effector to kill pathogens during insect immune responses, it also functions as a second messenger at a low concentration to regulate antimicrobial peptide (AMP) production in insects. Drosophila calcineurin subunit CanA1 is a ubiquitous serine/threonine protein phosphatase involved in NO-induced AMP production. However, it is unclear how NO regulates AMP expression. In this study, we used a lepidopteran pest Ostrinia furnacalis and Drosophila S2 cells to investigate how NO signaling affects the AMP production. Bacterial infections upregulated the transcription of nitric oxide synthase 1/2 (NOS1/2), CanA and AMP genes and increased NO concentration in larval hemolymph. Inhibition of NOS or CanA activity reduced the survival of bacteria-infected O. furnacalis. NO donor increased NO level in plasma and upregulated the production of CanA and certain AMPs. In S2 cells, killed Escherichia coli induced NOS transcription and boosted NO production, whereas knockdown of NOS blocked the NO level increase caused by E. coli. As in O. furnacalis larvae, supplementation of the NO donor increased NO level in the culture medium and AMP expression in S2 cells. Suppression of the key pathway genes showed that the IMD (but not Toll) pathway was involved in the upregulation of CecropinA1, Defensin, Diptericin, and Drosomycin by killed E. coli. Knockdown of NOS also reduced the expression of CanA1 and AMPs induced by E. coli, indicative of a role of NO in the AMP expression. Furthermore, CanA1 RNA interference and inhibition of its phosphatase activity significantly reduced NO-induced AMP expression, and knockdown of IMD suppressed NO-induced AMP expression. Together, these results suggest that NO-induced AMP production is mediated by CanA1 via the IMD pathway.

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Pathogen‐induced host immune responses reduce the efficacy of pathogens used to control pests. However, compared to the well‐deciphered immunity system of Drosophila melanogaster, the immunity system of agricultural pests is largely unconfirmed through functional analysis. Beginning to unveil mechanisms of transcription regulation of immune genes in the Asian corn borer, Ostrinia furnacalis, we cloned the complementary DNA (cDNA) of a transcription factor Relish by rapid amplification of cDNA ends. The 3164 bp cDNA, designated Of‐Relish, encodes a 956‐residue protein. Bioinformatic analysis showed that Of‐Relish had a Rel homology domain, a predicted cleavage site between Q409 and L410, six ankyrin repeats, and a death domain. The response of Of‐Relish expression to the Gram‐negative bacteria Pseudomonas aeruginosa was sooner and stronger than to the Gram‐positive Micrococcus luteus. The antimicrobial peptide genes Attacin and Gloverin had similar expression patterns in response to the infections. Knockdown of Of‐Relish led to a decrease in Attacin and Gloverin messenger RNA levels, suggesting that Attacin and Gloverin were regulated by Of‐Relish. Together, the results suggested that Of‐Relish is a key component of the IMD pathway in O. furnacalis, involved in defense against P. aeruginosa through activation of Attacin and Gloverin.

Matrix metalloproteinases (MMPs) are crucial for tissue remodeling and immune responses in insects, yet it remains unclear how MMPs affect the various immune processes against pathogenic infections and whether the responses vary among insects. In this study, we used the lepidopteran pest Ostrinia furnacalis larvae to address these questions by examining the changes of immune‐related gene expression and antimicrobial activity after the knockdown of MMP14 and bacterial infections. We identified MMP14 in O. furnacalis using the rapid amplification of complementary DNA ends (RACE), and found that it was conserved and belonged to the MMP1 subfamily. Our functional investigations revealed that MMP14 is an infection‐responsive gene, and its knockdown reduces phenoloxidase (PO) activity and Cecropin expression, while the expressions of Lysozyme, Attacin, Gloverin, and Moricin are enhanced after MMP14 knockdown. Further PO and lysozyme activity determinations showed consistent results with gene expression of these immune‐related genes. Finally, the knockdown of MMP14 decreased larvae survival to bacterial infections. Taken together, our data indicate that MMP14 selectively regulates the immune responses, and is required to defend against bacterial infections in O. furnacalis larvae. Conserved MMPs may serve as a potential target for pest control using a combination of double‐stranded RNA and bacterial infection.