## 3 Results and Discussion
### Response in Honeybee Gene Expression after Feeding with a Grape Pomace Supplement
In the Toll pathway case, it is generally associated with fungi and gram-positive bacteria, and, on the other hand, the Imd pathway is associated with gram-negative bacteria [57]. Dorsal (Toll), and Relish (Imd) are both NF-kB family transcription factors that are involved in the production of antimicrobial peptides (AMPs) in fat bodies, a tissue analogous to the mammalian liver. It has been previously suggested that the Toll pathway, particularly the expression of NF-kB factor Dorsal-1A, is a strategic component regulating the immune response of honeybees against DWV [55; 58]. Cactus is an NF-kB inhibitor that is degraded to allow the nuclear translocation of Dorsal [59]. An increased cactus expression in DWV infected adult bees parasitized by _V. destructor_ has been associated with the down-regulation of Dorsal [60]. This is consistent  
Figure 5: Expression level of _Vago_ gene in worker honeybees per collection throughout the assay. Grape pomace powder (GPP) concentrations (0.5, 1, 2.5 and 5%), inoculated control (I-DWV) and non-inoculated control (N-DWV) are shown in different colors. Different letters indicate significant differences according to [PERSON]’s test (\(p<\) 0.005). with the results of the present study, where significant differences were observed for Cactus and Dorsal in both control and GPP treatments (Figure 6), while the expression of Cactus silenced the expression of Dorsal mainly in the non-inoculated control (N-DWV). Figure 6: Expression level of _Cactus_ (**A**) and _Dorsal_ (**B**) genes involved in Toll pathway worker honeybees per collection throughout the assay. Grape pomace powder (GPP) concentrations (\(0.5,1,2.5\) and \(5\%\)), inoculated control (I-DWV), and non-inoculated control (N-DWV). Different letters indicate significant differences according to a [PERSON]’s test (\(p<0.005\)). All of the GPP treatments at different concentrations showed an intermediate expression with respect to N-DWV and I-DWV control treatments, with some exceptions. This clearly indicates that the dietary supplementation based on GPP influences the expression of Toll pathway genes. The immune-deficiency signaling pathway (Imd) in bees and flies is activated by the Relish transcription factor (homologue to NF-xB transcription factor) [56]. In another insect, such as Drosophila, the immune pathways are strongly regulated in order to balance the immune responses. Therefore, the increased expression of pathway inhibitors, such as Cactus, does not necessarily indicate complete or continuous repression of the pathway (e.g., Toll) [22]. Relish is a transcription factor in the Imd pathway, which, upon cleavage, leads to the production of the antimicrobial peptides (AMPs) abaecin and hypmenoptaecin [55]. In the present study, the quantification of the Relish gene (Figure 7) showed significant differences between the treatments with respect to the inoculated control (I-DWV) throughout the assay, showing a higher expression than I-DWV and maintaining a gene expression similar to N-DWV (before 14 days). There is evidence that both the Toll and Imd pathways defend against viral invasion in insects [61; 62]. All of the treatments used with GPP presented differences from the control. However, this was not observed in a dose-dependent manner. The results showed that the GPP dietary supplement inhibited DWV infection in DWV-infected bees. This indicates that the bioactive compounds found in grape pomace strengthen the immune system by upregulating the  
Figure 7: Expression level of Relish gene involved in the Imd pathway in worker honeybees per collection throughout the assay. Grape pomace powder (GPP) concentrations (0.5, 1, 2.5 and 5%), inoculated control (I-DWV), and non-inoculated control (N-DWV). Different letters indicate significant differences according to a Tukey’s test (\(p<0.005\)). expression of genes involved in pathways known to influence immune responses (RNAi, Toll and Imd), protecting honeybees from external stress caused by viral infections. A study on bees infected with DWV conducted by [PERSON], et al.