# Impacts of Diverse Natural Products on Honey Bee Viral Loads and Health
## 4 Discussion  
A main priority of this study was to explore additional natural products for their activities against honey bee viruses, namely, DWV and VDV. While we did not find consistent results across all products tested, we did find some that affected viral loads. Intriguingly, raw cacao had significant effects on the level of both DWV and VDV when bees were supplemented with these NPs. The tropical rainforest tree _Theobroma cacao L_ produces cacao beans that must be fermented, dried, and roasted to make chocolate. Cacao beans have a broad range of bioactive chemical compounds, such as polyphenols, flavonoids, and procyanidins [23; 45]. These compounds are found in the nib (used to make chocolate) and the seed coat (a waste product). Many of these chemical compounds are in higher concentrations [46] prior to roasting, which results in losses of up to 71% for total phenolic compounds and 53-77% for antioxidants [47; 48]. For this reason, unroasted and roasted cacao bean shells were used in this study. Interestingly, the DWV levels increased (\(p\) = 0.0307), whereas the VDV levels decreased (\(p\) = 0.0013) with similar 30-fold changes in viral loads, albeit in opposite directions. Hesperidin is a major flavonoid found in citrus fruits such as lemons and sweet oranges, as well as in some other fruits and vegetables [49]. Hesperidin has been linked to a range of health benefits, including antioxidant, analgesic, anti-carcinogenic, anti-hypertensive, anti-viral, and anti-inflammatory effects [50]. Hesperidin also significantly lowered the VDV levels (\(p\) = 0.0208). Generally, of those treatments showing effects, most trends exhibited a lowering of viral loads that were not statistically significant (i.e., chrysin, curcumin, limonene, tocopherol, tyrosine, and vanillin (Table 2)). Others, despite showing promising properties in the literature, showed little or no effect in our experiments. For example, carvacrol is a phenolic monoterenoid found in essential oils of oregano, thyme (with current applications fighting bee pests), pepperwort, and other plants. While carvacrol possesses a wide range of biological activities, including antibacterial, antifungal, antioxidant, and anticancer effects [51] we were unable to achieve differences in disease loads for bees fed carvacrol. Another potential avenue to study is the effect of natural products on eukaryotic parasites afflicting honey bees. Herein, we looked at levels of _Nosema cerane_ (microsporidian parasite) and trypanosomes (parasitic protozoon). During the course of this experiment, we found no _Nosema_ infections in the honey bees tested in either the control or treated groups, and we were therefore unable to test the efficacy. We found no significant differences in the trypanosome levels in the treatments tested (chrysin, curcumin, hesperdin, tocopherol, and vanillin). We also looked at the effects of compounds on the honey bee core microbiota, specifically _Snodgrassella alvi_ and _Gilliamella apicola_. _G. apicola_ showed significant decreases when bees were fed diets supplemented with tocopherol, giving rise to a 28-fold difference in expression (\(p\) = 0.0019). Tocopherol, vitamin E, has been reported in the prevention of oxidative damage or the modulation of signal transduction and gene expression in antioxidant and non-antioxidant manners [52]. Interestingly, almonds have the highest amount oftocopberol of all nuts [53] and are frequented by honey bees in yearly almond pollination services. Likewise, albeit less significant, vanillin (selected for its potential antimicrobial properties [54]), curcumin, hesperidin, and chrysin treatments also led to lower levels of _Gilliamella_. A shift in the honey bee microbiota, in any direction, should be interpreted carefully. For example, _Gilliamella_ is a honey bee gut symbiont that is responsible for the breakdown of potentially harmful/toxic sugars produced by pollen digestion [55]; this argues that more of this bacteria is better for bees.