Fig 1. Infection of silkworms with L. monocytogenes.

(A) Survival rate of silkworms. Fifteen insects per group were infected and monitored to determine the time required to kill 50% of the silkworms. Approximately 10⁴ bacteria belonging to the wild-type strain (WT, circles), LLO deletion mutant (Δhly, triangles), and LLO-complemented strain (Δhly::hly, squares) were sufficient to kill 50% (LD50) of the silkworms within 4 and 5 days. Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (B) The bacterial numbers of each strain detected in silkworms at 48 h post inoculation. The data represent averages based on triplicate samples from three identical experiments and the error bars represent the standard error of the mean (SEM) (n = 9). Significant differences were accepted at P < 0.05 and they are indicated by asterisks (*). (C) Hemocytes (red) containing L. monocytogenes strains (green, arrowheads) were observed by confocal laser scanning microscopy at 1 h and 24 h post inoculation. Scale bar represents 10 μm. (D) Immunoblot analysis of LLO expression. Samples were prepared from the wild-type strain, LLO deletion mutant, and LLO-complemented strain cultivated at 37°C or 25°C in BHI broth.