NO Synthesis in Immune-Challenged Locust Hemocytes and Potential Signaling to the CNS

SIMPLE SUMMARY: Insects, in the same way as vertebrates, are exposed to a broad variety of pathogens but lack their adaptive immune system. Relying on their innate immune system, they respond to pathogens by phagocytosis, melanization, and the synthesis of antimicrobial or cytotoxic compounds. In this study, we evaluated the production of the cytotoxic gaseous radical nitric oxide (NO) in hemocytes, the immune cells of the model insect Locusta migratoria in response to various immune stimuli. Both sessile and circulating hemocytes responded to gram-negative Escherichia coli and gram-positive Streptococcus suis injection with a strong increase in NO production. In contrast, the gram-positive bacterium Staphylococcus aureus elicited only a minor response. In addition, bacteria were encapsulated by hemocytes. Since NO is an important neurotransmitter, NO-producing hemocytes were tested on the locust central nervous system (CNS) in an embryo culture model. CNS neurons responded with a distinct increase in production of the second messenger, cGMP. This is indicative of the influence of the immune response on the CNS. Our findings show that NO production in hemocytes and capsule formation need complex stimuli and contribute to the understanding of neuroimmune interactions in insects. ABSTRACT: Similar to vertebrates, insects are exposed to a broad variety of pathogens. The innate insect immune system provides several response mechanisms such as phagocytosis, melanization, and the synthesis of antimicrobial or cytotoxic compounds. The cytotoxic nitric oxide (NO), which is also a neurotransmitter, is involved in the response to bacterial infections in various insects but has rarely been shown to be actually produced in hemocytes. We quantified the NO production in hemocytes of Locusta migratoria challenged with diverse immune stimuli by immunolabeling the by-product of NO synthesis, citrulline. Whereas in untreated adult locusts less than 5% of circulating hemocytes were citrulline-positive, the proportion rose to over 40% after 24 hours post injection of heat-inactivated bacteria. Hemocytes surrounded and melanized bacteria in locust nymphs by forming capsules. Such sessile hemocytes also produced NO. As in other insect species, activated hemocytes were found dorsally, close to the heart. In addition, we frequently observed citrulline-positive hemocytes and capsules near the ventral nerve cord. Neurites in the CNS of sterile locust embryos responded with elevation of the second messenger cGMP after contact with purified adult NO-producing hemocytes as revealed by immunofluorescence. We suggest that hemocytes can mediate a response in the CNS of an infected animal via the NO/cGMP signaling pathway.