**4. Mechanism of action and selection process of probiotics**

Probiotics' mechanisms of action are not fully understood [45]. These mechanisms have been reviewed for humans through *in vitro* and *in vivo* animal models such as *Drosophila* [46, 47]. The effects of probiotics on medfly were studied, but the mechanisms underlying this were not explored. In general, probiotics affect microorganisms through antimicrobial secretion, competitive adhesion to epithelium and mucosa, intestinal epithelial barrier reinforcement, and immune system regulatory impact [48].

The probiotics used in the initial studies were selected from the prevailing population. The effectiveness of the aforementioned probiotic was then confirmed using the quality control criteria, which can be referred to as *in vivo* analyses, that were used to rate the quality of sterile males [1]. None of the studies adopted the basic selection approaches developed for human or aquaculture. The recent study by Hamden et al., [35] was the first to select strains based on specific criteria established in accordance with probiotics selection criteria and SIT requirements. Stress tolerance (tolerance to irradiation), adhesion ability (hydrophobicity, autoaggregation and coaggregation assays (biofilm formation), and antipathogenic activity (Exopolysaccharides production (EPS)) at specific diet incubation temperatures were the minimum criteria for a probiotic strain prior to integration into medfly food for SIT application. It consists of a series of *in vitro* tests that allowed all of the isolated strains to be screened as a first step before being proven *in vivo*. **Table 1** also shows that *Enterobacter* AA26, isolated from the gut of the Vienna 8D53+ genetic sexing strain (GSS), is a promising probiotic for medfly. When this strain was added to the larval diet, it increased the strain's productivity. Azis et al., [49] thoroughly investigated this strain *in vitro* for its biokinetic properties and nutritional values. Indeed, as demonstrated by this strain, a probiotic can be chosen for its functional molecules'secretory abilities, which could provide amino acids, vitamins, and increased α- and β-glucosidase activities.

From a scientific standpoint, the selection criteria for medfly probiotics could be expanded to include immunostimulatory activity, anti-inflammatory activity, and safety assessment [50]. Combined "omics" approaches including genomics, proteomics, transcriptomics, and metabolomics analyses in a novel scientific discipline called "Probiogenomics" [51] could provide a better comprehension and new insights about the selection of the "best" probiotic strain (see Section 5).
