Virus specific impacts on honey bee flight performance are mediated by the octopamine pathway

Reviewer #1 (Public review):

Summary:

Kaku and Flenniken investigate the mechanistic pathways through which specific viral infections alter the flight capabilities of honey bees. Building on their previous discovery that DWV impairs flight while SBV unexpectedly enhances it, the authors hypothesized that these behavioral shifts are driven by interactions with the insect's octopamine (OA) signaling pathway, which is responsible for the "fight-or-flight" neurohormonal stress response and energy mobilization. To test this, the authors experimentally infected adult honey bees with DWV or SBV and pharmacologically manipulated the OA pathway using either octopamine supplementation or epinastine (EP), an OA-receptor antagonist. They then evaluated the bees' flight performance (distance, duration, and speed) on custom flight mills and profiled their gene expression using qPCR and RNA sequencing.

Strengths:

A major strength of this study is the high prevalence of preexisting background DWV and SBV infections in the honey bee cohorts, which meant there were no completely "virus-free" control groups. However, the authors successfully mitigated this limitation by rigorously quantifying viral RNA copies for every individual bee via qPCR and utilizing these viral abundances as continuous variables in powerful linear mixed-effect models.

Weaknesses:

The primary weakness lies in the methodology used for targeted pharmacological manipulations, as well as the lack of OA quantification across different treatments. Thus, their claims are not sufficiently supported by the current data.

(1) The authors utilize Epinastine to block octopamine signaling, describing it as a highly specific OA receptor antagonist. However, pharmacological inhibitors often lack absolute specificity. Epinastine might bind to other octopamine receptor subtypes present in honey bee neural and flight muscle tissues, or it could potentially cross-react with tyramine and dopamine receptors. Without further genetic validation (e.g., RNA interference targeting specific receptors), it is difficult to definitively conclude that the altered flight performance is solely due to the blockade of the specific Oβ−2R pathway.

(2) As a natural neurotransmitter, insects have evolved highly efficient "cleanup" mechanisms. OA is rapidly cleared from the synaptic cleft via reuptake transporters and quickly inactivated by enzymes such as N-acetyltransferase (NAT) or Monoamine Oxidase (MAO). Consequently, an injection of OA produces only a transient "pulse" of activity. It is often a poor "tool" for inducing prolonged physiological effects compared to synthetic formamidines like Amitraz.

(3) The study relies heavily on transcriptomics and quantitative PCR to measure the mRNA expression of key synthesizing enzymes, namely tyrosine decarboxylase (tdc) and tyramine β-hydroxylase (tβh), to infer the activation or suppression of the octopamine pathway. However, changes in enzyme synthesis at the RNA level are often insufficient to accurately reflect the true physiological levels of biogenic amines. To robustly prove the authors' hypothesis of a "feedback loop that regulates intracellular OA concentrations", direct quantification of actual octopamine and tyramine titers in the bees (e.g., using high-performance liquid chromatography or mass spectrometry) is necessary.

Reviewer #2 (Public review):

Summary:

This highly original and well-designed study provides insight into how honeybee picorna-like viruses, Deformed wing virus ( DWV) and Sacbrood virus (SBV), affect flight performance, and reveals the role of the octopamine (OA) pathway in virus-honeybee interactions. The authors used a flight mill to quantify the flight performance of bees with different levels of DWV and SBV. Bees were treated with OA and/or epinastine (EP) - an OA receptor antagonist; the study also quantified virus loads and expression of two key genes involved in OA biosynthesis.

The results showed that reduced flight performance associated with high DWV levels could be alleviated by OA administration. In contrast, increased levels of SBV had the opposite effect, leading to enhanced flight performance. This suggests distinct physiological responses to DWV and SBV infections. Administration of EP had led to a reduction of flight performance in SBV-infected bees, indicating the involvement of the OA pathway.

The authors also quantified levels of mRNAs of enzymes involved in OA synthesis, tyrosine decarboxylase (TDC) and tyramine beta-hydroxylase (TbH), and concluded that DWV induced expression of TbH, while SBV upregulated expression of TDC. Furthermore, the study identified upregulated and downregulated genes in response to SBV, DWV and DWV in combination with OA.

Strengths:

The study reported opposing effects of infections of related viruses, SBV and DWV, on honeybee flight performance, and identified the central role of the octopamine (OA) signaling pathway in the effect of viruses on honeybee flights.

These findings were achieved by using a combination of approaches, including experimental measurement of flight distance, virus infections, and introduction of OA and EP. Experimental work with honeybees is technically challenging and requires specialized expertise, which makes the results produced in this study more valuable.

DWV and SBV are among the most important honeybee pathogens affecting honeybee health and threatening the pollination service. Therefore, an understanding of the mechanisms underlying DWV and SBV pathogenesis has the potential to develop novel approaches to mitigate the negative impact of these viruses.

Weaknesses:

No weaknesses were identified by this reviewer.

Author response:

Reviewer #1 (Public review):

Summary:

Kaku and Flenniken investigate the mechanistic pathways through which specific viral infections alter the flight capabilities of honey bees. Building on their previous discovery that DWV impairs flight while SBV unexpectedly enhances it, the authors hypothesized that these behavioral shifts are driven by interactions with the insect's octopamine (OA) signaling pathway, which is responsible for the "fight-or-flight" neurohormonal stress response and energy mobilization. To test this, the authors experimentally infected adult honey bees with DWV or SBV and pharmacologically manipulated the OA pathway using either octopamine supplementation or epinastine (EP), an OA-receptor antagonist. They then evaluated the bees' flight performance (distance, duration, and speed) on custom flight mills and profiled their gene expression using qPCR and RNA sequencing.

Strengths:

A major strength of this study is the high prevalence of preexisting background DWV and SBV infections in the honey bee cohorts, which meant there were no completely "virus-free" control groups. However, the authors successfully mitigated this limitation by rigorously quantifying viral RNA copies for every individual bee via qPCR and utilizing these viral abundances as continuous variables in powerful linear mixed-effect models.

Weaknesses:

The primary weakness lies in the methodology used for targeted pharmacological manipulations, as well as the lack of OA quantification across different treatments. Thus, their claims are not sufficiently supported by the current data.

We thank Reviewer #1 for these comments.

(1) The authors utilize Epinastine to block octopamine signaling, describing it as a highly specific OA receptor antagonist. However, pharmacological inhibitors often lack absolute specificity. Epinastine might bind to other octopamine receptor subtypes present in honey bee neural and flight muscle tissues, or it could potentially cross-react with tyramine and dopamine receptors. Without further genetic validation (e.g., RNA interference targeting specific receptors), it is difficult to definitively conclude that the altered flight performance is solely due to the blockade of the specific Oβ−2R pathway.

We thank the reviewer for this thoughtful comment and agree that pharmacological approaches have inherent limitations with respect to receptor specificity. However, among the available octopamine receptor antagonists, epinastine is considered one of the most selective compounds for insect octopamine receptors. Roeder et al. (1998) reported that epinastine exhibits affinities for octopamine receptors that are at least four orders of magnitude greater than those for other insect biogenic amine receptors, including dopamine, tyramine, histamine, and serotonin receptors.

Honeybees encode four β-adrenergic-like receptors AmOARβ1- AmOARβ4) and one αadrenergic-like receptor (AmOARα1). Our transcriptomic analyses indicated that expression of AmOARβ2 was substantially higher than that of other octopamine receptor genes. Specifically, AmOARβ4 transcripts were not detected in our RNA-seq datasets, while AmOARβ1 and AmOARβ3 were expressed at very low levels in most samples (Supplementary Table S9; Figure S5). Although AmOARα1 transcripts were detected in some samples, expression levels were consistently lower than those of AmOARβ2. These observations support the interpretation that the physiological effects observed following epinastine treatment are primarily mediated through disruption of AmOARβ2 signaling. We agree that receptor-specific genetic approaches would provide valuable complementary evidence. RNAi-mediated knockdown of AmOARβ2 is an attractive future direction; however, RNAi efficacy in honey bees is variable and influenced by factors including transcript turnover rates. In addition, dsRNA treatments can induce sequence independent antiviral effects that could confound interpretation in studies involving viral infection (Flenniken and Andino, 2013). We have revised the manuscript to more explicitly acknowledge these limitations and to clarify the basis for our interpretation of the epinastine experiments.

(2) As a natural neurotransmitter, insects have evolved highly efficient "cleanup" mechanisms. OA is rapidly cleared from the synaptic cleft via reuptake transporters and quickly inactivated by enzymes such as N-acetyltransferase (NAT) or Monoamine Oxidase (MAO). Consequently, an injection of OA produces only a transient "pulse" of activity. It is often a poor "tool" for inducing prolonged physiological effects compared to synthetic formamidines like Amitraz.

We thank the reviewer for this important point regarding the pharmacokinetics of octopamine. We agree that octopamine is rapidly metabolized and cleared under physiological conditions and that exogenous administration is unlikely to precisely mimic endogenous signaling dynamics. Our goal was not to induce a prolonged pharmacological activation of octopamine signaling comparable to that produced by synthetic agonists such as amitraz, but rather to determine whether increasing octopaminergic signaling could mitigate the flight impairments associated with DWV infection. Octopamine was administered either by injection or through feeding (Lines 86-89), both of which resulted in significant improvements in flight performance in DWV-infected bees (Figure 2). The observation that two independent delivery methods produced similar outcomes supports the conclusion that enhanced octopaminergic signaling can partially rescue the DWV-associated flight phenotype. We have revised the manuscript to clarify this distinction and to acknowledge that exogenous octopamine administration likely produces transient elevations in signaling rather than sustained receptor activation.

(3) The study relies heavily on transcriptomics and quantitative PCR to measure the mRNA expression of key synthesizing enzymes, namely tyrosine decarboxylase (tdc) and tyramine βhydroxylase (tβh), to infer the activation or suppression of the octopamine pathway. However, changes in enzyme synthesis at the RNA level are often insufficient to accurately reflect the true physiological levels of biogenic amines. To robustly prove the authors' hypothesis of a "feedback loop that regulates intracellular OA concentrations", direct quantification of actual octopamine and tyramine titers in the bees (e.g., using high-performance liquid chromatography or mass spectrometry) is necessary.

We thank the reviewer for this comment and agree that octopamine and tyramine quantification would strengthen the mechanistic interpretation of our findings. Previous studies have successfully quantified OA in honey bees using HPLC-based approaches, including KayaZee et al. (2022, eLife), who measured OA in honey bee muscle tissue (both naturally occurring levels and levels post-treatment with 10 mM OA), and Cook et al. (2017, J. Exp. Bio) who quantified OA in pooled honey bee brain samples.

Prior to submission, we inquired with our institutional mass spectrometry facility regarding the feasibility of measuring OA in individual honey bee samples. The expected concentrations of OA in our samples was below their limit of detection, so we did not pursue these analyses at that time.

We are exploring the possibility of analyzing a subset of samples at external facilities that may have the sensitivity required to quantify OA and tyramine in honey bee tissues. However, our initial discussions indicate that such analyses would require substantial resources, with estimated costs of approximately $5,000–10,000 for 12–15 samples. While we acknowledge that direct measurements of OA and tyramine would provide valuable complementary evidence, the current study relies on multiple independent lines of evidence including gene expression analyses, OA supplementation experiments, and behavioral measurements that collectively support a role for octopaminergic signaling in mediating the observed effects.

Reviewer #2 (Public review):

Summary:

This highly original and well-designed study provides insight into how honeybee picorna-like viruses, Deformed wing virus (DWV) and Sacbrood virus (SBV), affect flight performance, and reveals the role of the octopamine (OA) pathway in virus-honeybee interactions. The authors used a flight mill to quantify the flight performance of bees with different levels of DWV and SBV. Bees were treated with OA and/or epinastine (EP) - an OA receptor antagonist; the study also quantified virus loads and expression of two key genes involved in OA biosynthesis.

The results showed that reduced flight performance associated with high DWV levels could be alleviated by OA administration. In contrast, increased levels of SBV had the opposite effect, leading to enhanced flight performance. This suggests distinct physiological responses to DWV and SBV infections. Administration of EP had led to a reduction of flight performance in SBVinfected bees, indicating the involvement of the OA pathway.

The authors also quantified levels of mRNAs of enzymes involved in OA synthesis, tyrosine decarboxylase (TDC) and tyramine beta-hydroxylase (TbH), and concluded that DWV induced expression of TbH, while SBV upregulated expression of TDC. Furthermore, the study identified upregulated and downregulated genes in response to SBV, DWV and DWV in combination with OA.

Strengths:

The study reported opposing effects of infections of related viruses, SBV and DWV, on honeybee flight performance, and identified the central role of the octopamine (OA) signaling pathway in the effect of viruses on honeybee flights.

These findings were achieved by using a combination of approaches, including experimental measurement of flight distance, virus infections, and introduction of OA and EP. Experimental work with honeybees is technically challenging and requires specialized expertise, which makes the results produced in this study more valuable.

DWV and SBV are among the most important honeybee pathogens affecting honeybee health and threatening the pollination service. Therefore, an understanding of the mechanisms underlying DWV and SBV pathogenesis has the potential to develop novel approaches to mitigate the negative impact of these viruses.

Weaknesses:

No weaknesses were identified by this reviewer.

We thank Reviewer #2 for these comments