关键词: 家蝇血淋巴, 96孔酶标板, 黑化反应, 酚氧化酶

Abstract: Objective　To investigate the feasibility of 96-well microplate assay in determination of melanization responses of Musca domestica to different bacterial challenges. Methods　The hemolymph from third-instar larvae of M. domestica was stimulated in vitro with Escherichia coli, Staphylococcus aureus, Micrococcus luteus, or the mixture of E. coli and S. aureus. The phendoxidase activity of hemolymph was determined with a 96-well microplate reader, and the relationship between bacterial stimulation and housefly melanization was further analyzed. Results　Compared with the initial A490 values of normal control sample, those from the stimulated samples (numbered 1-3) constantly increased along with the increase in bacterial quantity. The fold changes in initial A490 values with stimulation by different bacteria were as follows: 1.33-1.38 times with E. coli, 1.30-2.40 times with S. aureus, 2.60-3.00 times with M. luteus, and 1.40-3.80 times with the mixture of E. coli and S. aureus. Obviously, stimulation with bacteria enhanced the phenoloxidase activity of hemolymph of M. domestica in vitro; meanwhile, the melanization responses of hemolymph got stronger with increase in bacterial quantity. The stimulating effects of single bacteria were as follows: M. luteus＞S. aureus＞E. coli, while the mixture of S. aureus and E. coli showed a better effect than single bacteria. Conclusion　 The 96-well microplate assay is a reliable method to determine the melanization responses in hemolymph of M. domestica during bacterial challenge.

Key words: Hemolymph of Musca domestica, 96-well microplate, Melanization response, Phenoloxidase