Bacterial community shifts in the olive fly Bactrocera oleae following antimicrobial treatment of infested olives

The olive fly Bactrocera oleae is the most destructive olive pest in the Mediterranean basin, posing a threat to production. Traditionally managed with synthetic insecticides, B. oleae control now requires alternative strategies due to health risks, environmental concerns, and recent European regulations restricting the use of the commonly applied insecticides. The disruption of the strict association between the olive fly and its primary symbiont Candidatus Erwinia dacicola, has been demonstrated through the application of commercial products with putative antimicrobial activity (e.g., Dentamet, a Zn-Cu-citric acid bio-complex) to olive fruit infested with newly laid eggs. In this study, we assessed the impact of Dentamet on the overall composition and structure of the bacterial community associated with B. oleae life stages developed in a laboratory rearing from treated olive fruit infested with newly laid eggs (DT). The bacterial communities from these samples were then compared with those from B. oleae life stages developed from two control groups i.e. untreated olives (NT), and olives collected from untreated olive orchard (wild specimens). Total DNA was extracted from pooled specimens for 16S rRNA gene metabarcoding sequencing of the v3-v4 hypervariable regions using the Illumina MiSeq platform. Moreover, quantitative Real-Time PCR (qPCR) analyses were performed to assess the abundance of core microbiota bacterial genera, including the primary symbiont Ca. Erwinia dacicola, as well as functional genes involved in toxic compound inhibition, nitrate reduction and aromatic compound degradation. 16S metabarcoding results highlighted some differences between DT and NT samples. A reduced bacterial diversity, though not significant, was observed in DT specimens during the larval and pupal stages. However, in the adult stage, the shift in the bacterial community structure was statistically supported by beta diversity analyses (PERMANOVA p<0.01, ANOSIM p<0.01). The core microbiota evaluation highlighted the absence of six bacterial genera in DT specimens, present in both NT and wild specimens. Finally, qPCR analysis showed a significantly lower abundance of the primary symbiont E. dacicola in the larval stage, along with a reduced abundance in the pupal and adult stages of DT. Our results show that Dentamet influences the microbial community of the olive fly especially on its primary symbiont Ca. Erwinia dacicola, potentially disrupting key microbial interactions.