# Effects of cold plasma generated ozone on development of Galleria mellonella induced alterations in hemolymph protein and biochemistry of beeswax

## TL;DR

Cold plasma-generated ozone effectively kills wax moth larvae and alters their protein levels and beeswax chemistry.

## Contribution

The study introduces cold plasma ozone as a novel, non-thermal method for controlling Galleria mellonella with specific effects on developmental stages and biochemistry.

## Key findings

- Ozone exposure at 800 ppmv for 20 minutes completely inhibited adult emergence from pupae.
- Larvae showed higher tolerance to ozone compared to eggs and pupae.
- Ozone treatment increased hemolymph protein content and altered beeswax hydrocarbons.

## Abstract

Greater wax moth Galleria mellonella is a serious pest in apiculture, invading honeybee colonies and inflicting widespread damage. Ozone gas generated by the non-thermal atmospheric pressure plasma produced with a dielectric barrier discharge (DBD) reactor is an attractive solution for insect control. The effect of ozone gas generated by cold plasma on the egg, larva, and pupae stages of G. mellonella was investigated by using two concentrations, 400 and 800 ppmv, at different exposure times of 5, 10, 20, 40, 60, and 80 min. The mortality rate of the larvae increased as the exposure time lengthened, while a decrease in adult survivorship was observed after exposure. Complete suppression of the survived larval instar percentage and pupation percentage was noted after 7 days of exposure to 40 and 20 min at 400 and 800 ppmv of ozone, respectively, compared to the control group, which had a 96.67% survivorship. Pupal exposure to 800 ppmv for 20 min was sufficient to completely inhibit adult emergence. Larvae exhibited greater tolerance to ozone gas generated by cold plasma than eggs and pupae 24 h after exposure. Additionally, ozone treatment at 400 and 800 ppmv significantly increased total hemolymph protein content after 24 h, reaching 17.55 g/L and 18.50 g/L, respectively, compared to 16.52 g/L for the control. Cold plasma ozone treatment altered the stored beeswax matrix by modifying essential hydrocarbons and enhancing fatty acid diversity, while preserving the fundamental ester structure, thus converting it into a functionally changed biomaterial. Exposure to ozone gas at 800ppmv caused remarkable abnormalities in appearance in wax moths throughout their life stages, resulting in shrunken, twisted, and crippled insects unable to complete normal development.

The online version contains supplementary material available at 10.1038/s41598-026-36802-w.