Semiochemicals Produced by Microbes in Mealybug Honeydew Attract Fire Ants

Semiochemicals produced by microbes in hemipteran honeydew play an important role in mediating the ant-hemipteran interaction. However, the specific volatile compounds and their origins remain unclear. Here, we found that honeydew produced by Phenacoccus solenopsis exhibited strong attractiveness to fire ant workers, but sterilized honeydew was much less attractive. Four compounds were identified from the honeydew volatiles, including 1-octen-3-ol, limonene, phenylethyl alcohol, and 2,4-ditert-butylphenol. All the identified compounds triggered strong electroantennography response of fire ants and showed repellence at relatively high concentrations while attractiveness at low concentrations to fire ant workers. Furthermore, six bacterial isolates were identified based on 16S rRNA sequencing, namely, Bacillus, Brachybacterium, Kocuria, Microbacterium, Pseudomonas, and Staphylococcus. Fire ants exhibited a strong preference for Brachybacterium, Kocuria, and Microbacterium, suggesting that these bacterial isolates are involved in the attracting effect of honeydew. Both limonene and phenylethyl alcohol were detected from Brachybacterium, while limonene was only detected from Kocuria, and phenylethyl alcohol was exclusively detected from Microbacterium. Reinoculation of these bacteria restored the attractiveness of honeydew to fire ants, and the active compounds, limonene and phenylethyl alcohol, were detectable in bacteria-reinoculated honeydew. Collectively, our results reveal the active compounds in hemipteran honeydew and their association with honeydew bacteria. The findings will contribute to the development of novel attractants for efficient monitoring of fire ants.