Octopamine signaling pathway (i.e., synthesis, activation, inhibition) and experimental design

(A) Octopamine (OA) is synthesized by conversion of tyrosine to tyramine by tyrosine decarboxylase (TDC) and tyramine conversion to OA by tyramine β-hydroxylase (TβH). (B) OA β-2 receptor (Oβ-2R) is one of the most abundant and specific OA-detecting receptors. Upon activation of Oβ-2R, a G-coupled protein receptor, adenylyl cyclase (AC) is activated and increases intracellular cyclic adenylyl monophosphate (cAMP) levels. High cAMP levels induce protein kinase A activity which phosphorylates metabolic enzymes and transcription factors that enhance metabolic activity. (C) An OA receptor antagonist, epinastine (EP), inhibits OA binding (D) To evaluate relationships between virus infection and the OA response, and prevents activation of the Oβ-2R pathway honey bees were inoculated with either deformed wing virus (DWV) or sacbrood virus (SBV) via injection or mock infected. Bees were exposed to either OA, EP, or both OA and EP via co-injection or feeding, all bees were fed sucrose. (E) Impacts of experimental conditions were quantified in individual bees 72 hours post infection. Panels B-D reproduced from Kaku, 2025.

The flight performance of honey bees infected with DWV and/or SBV is differentially impacted by OA and EP treatment

To evaluate the involvement of the OA pathway on virus infection and/or flight performance, virus- or mock- infected honey bees were exposed to OA, EP, or both OA and EP via co-injection (blue) or feeding (green). Honey bees were experimentally infected with or had preexisting infections of DWV (A-C) or SBV (D-F) in three experiments. Flight distance (log10 m) was evaluated using linear mixed effect models to quantify impacts of DWV and/or SBV infection in the context of OA and/or EP treatment. Each data point represents an individual bee (n = 336); colors indicate treatment (i.e., bees fed only sucrose in pink, bees fed OA and/or EP in green, and bees injected with OA and/or EP in blue). (A) In bees fed only sucrose, DWV infections negatively impacted flight distance, whereas DWV infected bees treated with OA flew further distances. (B) DWV infected bees fed or injected with EP flew shorter distances than DWV infected bees fed only sucrose. (C) DWV infected bees fed or injected with OA and EP flew similar distances to DWV infected bees fed only sucrose. (D) In the control group, SBV infected bees flew greater distances than uninfected bees. SBV infected bees fed OA flew shorter distances than SBV infected bees fed only sucrose. (E) Honey bees with SBV infections fed or injected with EP flew shorter distances than SBV infected bees fed only sucrose. (F) Bees with SBV infections fed or injected with OA and EP resulted in similar flight distances regardless of SBV infection level.

Expression of tyrosine decarboxylase (tdc) and tyramine β-hydroxylase (tβh) across treatments and SBV infection

The relationships between honey bee tyrosine decarboxylase (tdc) and tyramine β-hydroxylase (tβh) expression in the context of virus infection and treatments were evaluated using a linear mixed effect model that included SBV abundance and OA/EP treatments as fixed effects with an interaction. Individual honey bee data plotted by treatment group with either (A) tdc expression or (B) tβh expression. Gene expression was calculated as log2 fold change using the ΔΔCt method relative to housekeeping rpl8 and compared to mock infected bees. SBV abundance was calculated as log10 copies / 2 µg RNA. The background violin plots represent the 95% confidence interval, and the horizontal line in each violin represents the median. SBV abundance is represented by a green color scale and SBV-negative samples with an “X”. When SBV abundance did not impact tdc or tβh expression the dark green points are evenly distributed above and below the control ‘0’ fold change line, whereas unequal distribution indicates an SBV-specific effect; * p<0.05, **p<0.01, and ***p<0.001. (A) Positive association between SBV abundance and tdc expression (p = 0.021). SBV infected bees fed OA or both OA and EP had greater tdc expression levels than uninfected bees fed only sucrose (p = 0.03 and 2×10−8). (B) Expression of tβh was greater in SBV infected bees than uninfected bees (p =0.022). SBV infected bees fed OA had greater tβh expression levels than uninfected bees fed sucrose (p = 2×10−5). SBV infected bees injected with EP or co-injected with OA and EP had lower tβh expression (p = 2×10−5 and 4×10−9).

Relative tdc and tβh expression in honey bees with high DWV or SBV levels

(A) Tyrosine is converted via tyrosine decarboxylase (TDC) to tyramine. Tyramine is a neurohormone that acts as a behavioral antagonist to octopamine (OA), resulting in reduced movement in invertebrates. Tyramine may act as a signaling neurohormone, or tyramine β-hydroxylase (TβH) may convert it to OA. (B) A linear mixed model was used to assess the relative expression of tdc and tβh in honey bees harboring high levels of either deformed wing virus (DWV) or sacbrood virus (SBV) (i.e., 108 virus copies / 2μg RNA) in the context of OA and/or epinastine (EP) injection. Estimates of relative tdc and tβh expression were generated based on data from 336 honey bees. The single bold dash indicates tdc or tβh expression was similar in virus infected bees and mock infected bees and arrows indicate estimates that were higher or lower than controls.

Transcriptome level comparison of honey bees that were SBV infected, DWV infected, or DWV infected and OA-treated

Gene expression was evaluated in honey bees that were mock infected, SBV infected, DWV infected, or DWV infected bees that were co-injected with OA (DWV+OA) after flight. Differentially expressed genes (DEGs) for each treatment group were identified relative to mock infected bees and compared using a Venn diagram. Full list of DEGs, fold change, and Benjamini-Hochberg corrected significance values are included in SI Appendix Table S6.