Stored grain protection, host both primary and secondary endosymbionts, making then suitable models to study the roles of co-existing symbionts and their eventual relevance for pest control. Grain weevils exploit a restrictive food source, cereal grains, and must complete their development within the grain kernel. The association between grain weevils and their primary endosymbiont SPE is hypothesized to be an important requirement allowing survival under such conditions. However, physiological differences do exist among weevil strains, allowing strain variation in how well they are able to cope with cereal amylase inhibitors and insecticide exposure. SPE was initially detected in the rice weevil, where it is referred to as SOPE, and subsequently in the granary and maize weevils, where it is referred to as SGPE and SZPE, respectively. SPE seems to provide vitamins to its weevil hosts, assisting in their amino acid metabolism, in addition to interacting with mitochondrial oxidative phosphorylation, thus enhancing respiration and mitochondrial enzyme activity in the host insect. Such effects of SPE may affect development, immune response and flight activity in their weevil hosts. Curiously, however, the focus of previous SPE studies has remained on the genetics and molecular biology of these endosymbionts, and not on their behavioral or physiological consequences in the weevil hosts. However, the co-occurrence of SPE and Wolbachia in cereal weevils, raises questions ALK5 Inhibitor II regarding their interaction and potential impact on this host species. Here we recognized the presence of both SZPE and Wolbachia in the maize weevil, subjected the colonized weevil hosts to different treatments for endosymbiont inactivation/suppression, assessed the impacts of endosymbiont loads of either one or both symbionts, and analyzed how they affect host reproductive fitness following a structured hierarchical approach. Past studies focused on the simultaneous presence/absence of such endosymbionts, while here presence was quantified and associated with behavioral and physiological traits potentially affecting the insect reproductive output. The insects were maintained for 40 min in the Petri dishes with the desired water-diluted antibiotic and subsequently transferred to maize contained in Petri dishes for 24 h; this procedure was repeated six times for each individual insect. The progeny of the treated insects was also subjected to the same antibiotic treatment. Therefore the antibiotic-treated insects were from the parental generation when the F1 progeny was assessed, and from the P and F1 generations when the F2 progeny was assessed. Only the progenies of the insects treated for one or two generations were used in the endosymbiont quantification and subsequent bioassays in order to eliminate the eventual deleterious effects of the antibiotics themselves on insect performance.