**3.2 Ameliorative effects on medfly colonies productivity and biological quality of sterile males**

The initial interest in probiotics for medfly was focused on their use to improve colony productivity and the biological quality of released sterile males, such as longevity, flight ability, and mating competitiveness; however, new areas have been found, such as their effect on stress tolerance, although this requires more scientific development. The following section discusses some functional properties of gut bacteria supplemented as probiotics in medfly feeding. **Table 2** provides an overview of the main results obtained in several studies. There have been several studies in which potential bacterial strains such as *K. oxytoca* and *Enterobacter* sp. have been used to improve the egg to the adult recovery of medfly colonies [29, 32, 35] as well as the biological quality of released sterile males in the laboratory and/or field cages [16, 17, 28, 31, 32, 34, 35]. These studies revealed that the incorporation of gut bacteria in larval or adult artificial diets can positively affect pupal weight [17, 31, 35], adult size [17], survival ability [2, 17, 28, 32, 35], flight ability [17, 30, 35], mating competitiveness [17, 28, 34, 35], and sperm transfer [17].

However, **Table 2** also demonstrates that inconsistencies between results for the same bacterial strain can be found for some parameters, including pupal weight and sexual competitiveness [28, 29, 35]. This might be explained by the methodological setup used in each study. Since experiments are conducted with different medfly strains, isolated bacterial taxa, feeding stages, and lab or field-based applications, the different effects of the bacteria additives on medfly fitness may be explained. Probiotic bacteria have the potential to establish themselves, modify the existing gut microbial community, and play a more discrete role in nutrition and development. Follow-up experiments regarding the localization/quantification of these bacteria after incorporation in larval or adult artificial diets in the medfly's gut during development can provide more insight into how probiotic diets work. More research could enhance mass-rearing even further by upscaling the experimental design, using more replicates and generations, and potentially combining these beneficial isolates (consortium) or testing new bacteria isolated either from the medfly or other insect species. In general, increased pupal and adult productivity, decreased developmental time of the immature stages, and improved fly longevity would result in increased production of insects in shorter periods. This would facilitate mass-rearing of this insect pest species for SIT applications as well as small-scale laboratory rearing required for research.

*Probiotics as a Beneficial Modulator of Gut Microbiota and Environmental Stress… DOI: http://dx.doi.org/10.5772/intechopen.110126*

### **3.3 Colonization of the probiotics and host origin importance**

An effective probiotic should be able to adhere to and colonize the mucus layer of the insect gut [36]. According to **Table 1**, some studies chose to supplement the probiotic daily [17], whereas others only did so once. The initial step in establishing a symbiotic relationship between a microorganism and its host is colonization. Since the ingested food moves from the oral to the anal opening, the digestive tract is exposed to the environment. The term "colonization" can therefore be used for a wide range of associations, ranging from the simple transition of environmental bacteria to the replication, proliferation, and persistence of specific symbionts in the insect gut [37, 38]. The research on *Drosophila* revealed that each strain had a different capacity to reside in the gut following initial colonization [39]. The first day after consuming probiotics, the gut's probiotic levels grew quickly. After ceasing the probiotics, their number in the *Drosophila* intestine dropped and remained at a low level [39]. On the contrary, Lee et al., [40] did not find any differences in the extent of colonization and proliferation in the *Drosophila* gut among the tested bacteria. Successful colonization of the probiotics was demonstrated for medfly by [16, 28, 34]. However, to confirm the presence of *E. agglomerans* and *K. pneumoniae* in the guts of the probiotically treated insects, Niyazi et al., [28] only stated that the later strains were retrieved from the treated males, whereas control flies were found to be largely free of these bacteria (90% of the cases) (**Table 1**). There was no information provided about the isolates' identification procedure. Similarly, Gavriel et al., [34] confirmed that they recovered probiotics (*K. oxytoca* N8-S stereptomycine-resistant strain) from enriched sterile flies even after more than 7 days with no bacteria replacement by comparing bacterial counts on an antibiotic (Sm) treated LB agar and LB agar without antibiotics. However, Ben Ami et al., [16] went further in their explanation of the colonization by comparing the total bacterial count (SmKo strain) from adult guts on chromogenic medium and LB medium containing antibiotics for five consecutive days for the enriched diet and two additional days with a diet devoid of bacteria. Colonization is a fairly complex phenomenon that would also depend on stochastic factors and preexisting populations. The latter reduces the chances of subsequent colonization as was suggested for irradiated males of *B. dorsalis* fed with *K. oxytoca* BD177 [3], thus increasing the stability of the highly-diverse guts [41]. The direct and indirect colonization resistance from the commensal gut microbiota will limit the long-term effect of the probiotic. Indeed, Akami et al., [42], working on *Bactrocera dorsalis*, discovered that axenic flies preferred probiotic diets over symbiotic flies, confirming colonization resistance due to resident microbiota. They hypothesize that the native probiotic isolates were able to recolonize their natural habitat in the axenic flies' guts and revive appetitive behaviors that had been slowed due to bacterial suppression.

The provenance of the strain studied, however, is something we want to highlight here since it is crucial. All of the aforementioned studies used the *Drosophila* model to examine the probiotic human strains. Isolating putative probiotics from the host or environment where the bacteria are intended to exert their beneficial effect, on the other hand, makes more sense. The origin of the host should be considered even if for human purposes this requirement was negated since some strains showed to be effective even if they were of not human origin [43]. Recently, a study used a mixture of non-native and native bacteria for honey bees [44], however, without any proof of persistence in bee guts.
