The in vitro potential of non-thermal atmospheric pressure plasma against Nosema ceranae infection in honeybees (Apis mellifera)

Nosema ceranae is a widespread microsporidian parasite that primarily affects adult honeybees (Apis mellifera) but has also been reported to infect honeybee larvae. It has been considered as a main cause of colony losses. This study evaluated the anti-microsporidian activity of atmospheric non-thermal plasma against N. ceranae, which could provide a new technology for controlling Nosema disease. Argon and helium plasma substantially decreased N. ceranae spores, with the maximal reductions of 96.55% and 96.05%, respectively. Also, plasma activated water (PAW) generated by argon gas affected spores by a 50.51-77.14% of reduction, with H₂O₂ concentrations ranging from 50 to 200 mg/L, depending on the plasma exposure time. Therefore, the effect of H₂O₂ on the inactivation of N. ceranae spores was evaluated. The results showed that H₂O₂ concentration up to 100 mg/L reduced spore numbers by approximately 42.64-49.75%. H₂O₂ levels remained stable for up to 6 days, followed by a reduction of 50-80% in argon-generated PAW and 33.33-80% in helium-generated PAW by day 14. Moreover, this study demonstrated that both direct plasma and PAW treatments resulted in a significant reduction in the number of N. ceranae spores infecting adult bees, especially on day 14 after inoculation (71.88-87.42% of reduction). To elucidate the anti-N. ceranae activity of gas plasma and PAW, we performed an integrity of cell membrane and scanning electron microscopy analysis. The results of the cell membrane tests and microscopy reveal much about how these two methods could work, both plasma discharge and PAW. However, the development of plasma devices and PAW, as well as their safety for honeybees should be further investigated for effective control of N. ceranae and other pathogens in honeybees.