Nitric oxide production in celomocytes of the earthworm Eisenia hortensis following bacterial challenge

Abstract

In this in vitro investigation, nitric oxide (NO) production was induced within celomocytes of the earthworm Eisenia hortensis following microbial challenge. Celomocytes were pre-loaded with the fluorescent indicator 4-amino-5-methylamino-2’, 7’-difluorofluorescein diacetate (DAF-FM DA) in order to detect the presence of intracellular nitric oxide subsequent to a 16 h incubation with chemicallyfixed soil bacteria including Bacillus megaterium, Arthrobacter globiformis, Pseudomonas stutzeri, and Azotobacter chroococcum at a range of multiplicities of infection (MOIs). Flow cytometric analysis measuring increases in relative fluorescence intensity (RFI), which is directly proportional to the amount of intracellular NO produced, permitted determination of statistical significance (p < 0.05) of exposed celomocytes compared to baseline controls. Significant increases in NO were detected reproducibly in celomocytes treated with all bacterial species used. The most prominent results were observed after exposure to Gram positive B. megaterium and A. globiformis where 100 % of earthworms tested exhibited statistically significant increases of RFI at MOIs of 100:1 and 500:1, respectively. Furthermore, significant decreases in NO production in bacteria-stimulated earthworm celomocytes incubated with the NOS inhibitor aminoguanidine hydrochloride were observed. These results demonstrate microbial induction of NO synthesis in earthworms and provide evidence of an antimicrobial role of NO in the innate immune system.