Enterobacter supplement and ambient conditions determine the performance of sterile male Mediterranean fruit flies

Sterile Insect Technique (SIT) efficacy depends on field survival, dispersion capacity and critically, mating competitiveness of the released sterile males. These parameters are influenced by mass‐rearing, handling, sterilization and environmental conditions. In true fruit flies (Diptera: Tephritidae), symbionts influence host physiology and behaviour; however, the potential of inactivated bacterial supplements to improve sterile male mating performance under field conditions remains insufficiently explored. We investigated whether partial replacement of dietary protein with inactivated Enterobacter sp. AA26 biomass during larval development improves mating performance of sterile Ceratitis capitata (Wiedemann) males, and whether performance varies with ambient conditions. VIENNA 8 Genetic Sexing Strain (GSS) males were reared on either an Enterobacter ‐enhanced or a non‐enhanced larval diet (typical) and evaluated in outdoor field‐cage mating competitiveness assays during summer, autumn and spring. Enterobacter ‐enhanced larval diet increased mating competitiveness, quantified by the Relative Sterility Index (RSI), across all seasons. Season‐associated ambient conditions significantly affected mating competitiveness, with males demonstrating higher RSI during spring and the lowest in summer under high ambient temperatures. Mating latency and duration were influenced by ambient conditions but not by larval diet. Matings initiated sooner and lasted less during summer than during autumn or spring. No significant larval diet × season interaction was observed for mating competitiveness, latency or duration. Overall, larval supplementation with autoclaved Enterobacter biomass increased sterile male mating competitiveness under outdoor field‐cage conditions, while seasonal ambient conditions remained a major driver of mating performance, indicating that combining nutritional interventions with seasonally optimized release strategies may maximize SIT efficacy.