## 3 Results and Discussion
### Response in Honeybee Gene Expression after Feeding with a Grape Pomace Supplement  
Studies on the transcriptional level in honeybees involve uncharacterized genes/pathways in antiviral responses [22]. In this sense, the roles of genes in the Toll, Imd, JakSTAT, JNK, and RNAi pathways are the best characterized. In the present study, colonies with very low levels of DWV were used in the absence of _Varroa_ or other pathogens according to the strategy described in other comparable studies [51; 52; 33]. RNAi is the main antiviral mechanism in insects [53]; therefore, honeybees were expected to mount an RNAi response when inoculated with DWV. In fact, the RNAi-pathway is initiated by the Dm Dicer-2 cleavage of viral dsRNA into 21-22 bp siRNAs. It is important to point out that Am Dicer-like share 30% aa identity with Dm Dicer-2. In addition, the siRNAs are loaded into AGO2 (Argonaute-2), the catalytic component of the RISC (RNA Induced Silencing Complex) [54]. In this situation, a single strand of the siRNA is retained in the RISC and used to specifically target cognate the viral genome sequences  
Figure 3: DWV loads in worker honeybees recorded in days post-inoculation throughout the assay. Grape pomace powder (GPP) concentrations (0.5, 1, 2.5 and 5%), and inoculated control (I-DWV) and non-inoculated control (N-DWV) are shown in different colors. Different letters indicate significant differences according to a Tukey’s test (\(p<0.005\)). for cleavage. In addition, Dm Dicer-2 works as a dsRNA sensor that mediates a signal transduction cascade resulting in increased expression of Dm Vago and suppression of viral replication. Am Dicer-like may serve as a dsRNA sensor, and honeybees have a Vago orthologique, which is upregulated in DWV-infected honeybees [22]. Even though no significant differences in Dicer and Argo-2 expression were found between the treatments, peaks of expression were observed on collection days 1 and 8 for low and medium doses (0.5, 1, 2.5%) which showed higher gene expression (Figure 4). The relationship between the _Varroa_ parasite and DWV was studied, showing that the expression of both Dicer and Argonaute-2 increased in parallel with DWV viral loads in pupae over time. However, in this study it was observed that Dicer was the only gene  
Figure 4: Expression level of Dicer (**A**) and Argo-2 (**B**) genes involved in the RNAi-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) are shown in different colors. Different letters indicate significant differences according to [PERSON]’s test (\(p<0.005\)). that was up-regulated in DWV-injected pupae compared to the two control groups at all time points [55]. In our research, Dicer showed jumps in expression throughout the assay (Figure 4A). Although the quantification of the transcript expression of the Vago gene revealed some significant differences, it is important to highlight the low expression observed for the lowest concentration (0.5%) throughout the assay compared to the controls and the other GPP treatments (Figure 5). A direct relationship between the expression of these two genes and the expression of Vago was also found. In _B. terrestris_, Vago limits viral infection in fat bodies in a Dicer-dependent manner [54]. The principal insect innate immune response for the detecting and inhibiting virus replication in insects is through RNA interference (RNAi) via double stranded RNA (dsRNA) that is a sequence specific post-transcriptional gene-regulator [53]. Hence, the activation of this pathway in bees increases the expression of the Vago gene, an orthologue also found in _Drosophila_, and a suppressor viral replication [56]. The latest data suggest that the Toll and Imd pathways similarly contribute to defense against viral pathogens [50].