GC-MS Analysis and Antimicrobial Properties of Defensive Secretions from the Millipede Coxobolellus saratani (Diplopoda: Spirobolida: Pseudospirobolellidae).

The defensive secretions of the millipede, Coxobolellus saratani Pimvichai, Enghoff & Backeljau, 2022 were analyzed by gas chromatography-mass spectrometry to provide the very first data on the composition of the defensive secretions of the family Pseudospirobolellidae (Diplopoda: superorder Juliformia, order Spirobolida). This unveiled at least 12 identifiable compounds, including six quinones, two phenols, and four fatty acid esters. The three most prevalent identifiable compounds were 2,3-dimethoxy-1,4-benzoquinone (25.52%), hexyl pentadecanoate (11.57%) (the first report of a fatty acid ester compound in the order Spirobolida and tentatively indicating that this may be a shared feature of the Juliformia), and 3,4-dimethoxyphenol (10.51%). The antimicrobial activity of the defensive secretions was evaluated against three gram-positive bacteria (Bacillus cereus, Staphylococcus aureus, and S. aureus DMST20654), four gram-negative bacteria (Escherichia coli, E. coli ATCC25922, Pseudomonas aeruginosa, and Salmonella ser. Typhi ATCC16122), and two yeast strains (Candida albicans and C. albicans ATCC10231). The antibiotic kanamycin and the antifungal drug fluconazole were employed as positive controls. Paper disc diffusion assays demonstrated that the fresh, undiluted, secretions inhibited the growth of all tested microorganisms. Furthermore, broth microdilution analysis revealed Minimum Inhibitory Concentrations (MIC) ranging from 40 to 20,000 µg/mL and Minimum Bactericidal/Fungicidal Concentrations (MBC/MFC) ranging from 1,250 to > 20,000 µg/mL. The MIC values indicated that the defensive secretions of C. saratani are notably more efficient than kanamycin and fluconazole in inhibiting the growth of S. aureus DMST20654, E. coli ATCC25922 and C. albicans, but inhibit less effectively the visible growth of the six other microbial taxa tested. Finally, the MBC/MFC values revealed that the secretions of C. saratani may show less potent antimicrobial activity against the nine microbial taxa tested than kanamycin and fluconazole. Nevertheless, these results suggest once more that millipede defensive secretions may not only deter predators, but may also provide millipedes with a chemical defense against pathogens and parasites.