Single-cell analysis reveals the effects of glutaraldehyde and formaldehyde on individual Nosema bombycis spores

Abstract

Nosema bombycis (Nb) is the pathogen that causes pebrine in silkworms. Aldehydes are effective disinfectants commonly used in sericulture. However, the precise mechanism of their action on Nb spores remains unclear. Here, we used laser tweezers Raman spectroscopy to investigate the effects of glutaraldehyde and formaldehyde on individual Nb spores, as well as phase contrast microscopy imaging to monitor the germination dynamics of individual treated spores, to acquire a deeper understanding of the mechanism of action of aldehydes and to provide a theoretical reference for establishing an effective strategy for disease control in sericulture. The positions of the Raman peaks remained constant during treatment. The Raman intensity was enhanced and the germination rate of the spores significantly decreased with treatment time. T_lag, the time when individual spores begin to germinate, and T_germ, the time for complete germination, increased with enhanced treatment. The germination time (ΔT_germ) showed no significant difference from that for untreated spores. Heterogeneity was shown, which is relevant to the resistance of Nb spores to aldehydes. The results indicate that glutaraldehyde and formaldehyde do not destroy the spore wall and plasma membrane, do not cause the leakage of intracellular components, and might not damage the extrusion apparatus. The effects of aldehydes on Nb spores are mainly on the spore coat. They may block the external factors that stimulate spore germination. Single-cell analysis based on novel optical techniques reveals the action of chemical sporicides on microsporidia spores in real time and explains the heterogeneity of cell stress resistance. These applications of new techniques offer new insight into traditional disinfectants.