MicroRNA-281-X Modulates Self-Grooming Behavior in Honeybees by Targeting Tyrosine Decarboxylase 2 in the Octopaminergic Pathway

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Journal: Insects

Title: MicroRNA-281-X Regulates Self-Grooming Behavior in Honey-2 bees by Targeting Tyrosine Decarboxylase 2 in the Octopamin-3 ergic Pathway 4

Authors: Yang Lü, Wenyao Ouyang, Jiali Liao, Liuchang Miao, Zhiguo L, Songkun Su

 

Thesis (central claim)

Animals adjust their behavior in response to internal and environmental conditions. This flexibility is necessary for survival and fitness. A key question in neuroscience is how neural circuits modify fixed motor patterns using molecular mechanisms. Self-grooming is a useful model behavior and has been conserved across species in a predictable sequence.

 

This study identifies such a link. The authors show that miR-281-x regulates grooming behavior by targeting tdc2 and altering octopamine synthesis. Increasing miR-281-x reduces grooming. Decreasing miR-281-x increases grooming. These results reveal a molecular pathway that explains variation in grooming behavior.

 

Reviewer Comments

Methods are comprehensive and support a potentially high-impact study, spanning behavior, neurochemistry, transcriptomics, and functional validation. The experimental logic is strong (correlative > manipulative > rescue). However, this section suffers from incomplete reporting, inconsistencies in design, and insufficient detail for replication, particularly in behavioral assays, sample sizes, and statistical structure. Addressing these would substantially strengthen the manuscript.

Recommendations: 1. Justify why different age groups were included in groups. 2. In the behavioral assay, provide quantitative scoring metrics for weak and strong groomers. 3. Report full sample sizes for every experiment and every group. 4. Redo statistical analysis with GLMMs using bee colony as a random effect; apply FDR correction for the RNA-seq. 5. Clarify experimental flow by adding a schematic linking behavior > sampling > molecular assays > manipulations.  

 

Results are logically organized and potentially strong, but authors repeatedly make causal claims that exceed the data.

Recommendations: 1. The manuscript states that OA and miR-281-x “regulate” grooming behavior, but should be written as “are associated or correlated with grooming.” 2. The MS and MW categories are defined by grooming performance (e.g., latency and grooming intensity) and are then reused as outcome variables (e.g., proportion of MS bees after treatment). This creates circular reasoning because the analysis tests for changes in a category that is already constructed from the behavior being measured. As a result, treatment effects may appear stronger than they are, because the classification and the outcome are not independent. 3. The Results report medians, p-values, and proportions but do not provide n for behavioral assays, molecular assays, or manipulations, preventing evaluation of statistical power and robustness. 4. The Results rely on p-values without reporting effect sizes, confidence intervals, or model structure, and RNA-seq initially uses p < 0.05 before later invoking FDR, indicating inconsistent statistical thresholds. 5. The claim that miRNA profiles are “globally different” is based on PCA separation without reporting explained variance, clustering robustness, or batch effects, and the biological interpretation of 61/64 miRNAs being upregulated in MW bees is not explored. 6. The transition from 64 miRNAs to 7 candidates and then to miR-281-x relies on prediction and enrichment without demonstrating that this miRNA has the strongest or most biologically relevant effect size. 7. The use of “proportion of MS bees” discards quantitative behavioral data (latency, bout number) and reduces statistical sensitivity while reinforcing the circular classification issue (#2 issue). 8. The OA rescue and tdc2 knockdown rescue are presented as mechanistic confirmation, but alternative explanations (e.g., general arousal effects, off-target effects) are not excluded. 9. The inverse relationship between miR-281-x and tdc2 across age is reported but not linked quantitatively to grooming behavior, weakening its relevance to the central claim. 10. The luciferase assay confirms binding but does not demonstrate functional regulation in honeybee neurons, yet the Results present it as strong evidence of in vivo mechanism. 11. OA levels differ between groups and treatments, but the manuscript does not quantitatively link OA variation to behavioral metrics, leaving a gap between neurochemistry and phenotype. 12. The final mechanistic model is presented as complete despite missing quantitative linkage.
The miR-281-x → tdc2 → OA → grooming pathway is supported directionally, but the Results do not demonstrate effect sizes across each step or test the full pathway in a single integrated model (GLMM).

 

Discussion: In summary, the Discussion is well written and logically organized, but it should be revised to tighten causal language, reduce speculative extensions, and align claims more closely with the experimental evidence. The study has the potential to make a meaningful contribution, but its impact will depend on presenting the miR-281-x–tdc2–OA pathway as a supported component of behavioral modulation, rather than a fully resolved mechanistic framework.

Recommendations: 1. This section presents a clear and compelling narrative that a miR-281-x–tdc2–octopamine axis regulates variation in honeybee grooming behavior. However, the interpretation consistently extends beyond what the data directly support. The manuscript frames this pathway as a definitive mechanistic system that “controls” grooming intensity, yet much of the evidence remains correlative or partially validated through indirect assays. 2. The Discussion also overinterprets molecular and transcriptomic findings. The asymmetry in miRNA expression is presented as evidence that miRNAs broadly inhibit grooming-related neural pathways, but this conclusion is not directly tested and remains speculative. Similarly, the identification of miR-281-x as a key regulator is framed as a central discovery, although the selection process from many candidate miRNAs is not sufficiently justified by effect size, specificity, or functional dominance. 3. The integration of endocrine pathways and developmental history into the interpretation is conceptually interesting, but these mechanisms are not empirically evaluated in this study and should be presented as hypotheses rather than conclusions. 4. The proposed neurobiological mechanism linking brain miRNA activity to thoracic motor output is another area where inference exceeds evidence. The Discussion invokes descending octopaminergic pathways to explain how brain-level regulation influences grooming movements, but this connection is not experimentally demonstrated. 5. Likewise, the claim that miR-281-x acts as a “molecular switch” controlling octopamine production simplifies what is likely a multi-step, distributed regulatory system. The data support an association between miR-281-x, tdc2 expression, and OA levels, but they do not establish that this axis alone quantitatively determines behavioral output across individuals. 6. The treatment of neurochemistry is directionally consistent with existing literature, but the Discussion assumes a tight coupling between OA levels and grooming intensity without presenting quantitative linkage across individuals or treatments. This limits the strength of the claim that OA functions as a “key signal” driving grooming behavior in this context. In addition, alternative explanations—such as generalized arousal, stress responses, or broader neuromodulatory effects—are not sufficiently considered.

7. To its credit, the Discussion does acknowledge several limitations, including the lack of field validation and the focus on a single miRNA. However, these limitations are understated relative to the strength of the claims made throughout the section. A more balanced interpretation would clearly distinguish between (1) supported findings (e.g., miR-281-x affects tdc2 expression and OA levels under experimental manipulation) and (2) inferred mechanisms (e.g., system-level control of grooming behavior, integration with endocrine pathways, and ecological relevance).

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, the authors investigate the molecular and neural regulation of self-grooming behavior in Apis mellifera, focusing on the differences between bees with strong (MS) and weak (MW) grooming phenotypes. They identify a microRNA-mediated neuromodulatory pathway that influences grooming intensity. Their findings show that octopamine (OA) levels are significantly higher in MS bees. Through small RNA sequencing, they discovered that miR-281-x is differentially expressed in the brains of MS and MW bees and plays a crucial role in OA regulation. Furthermore, overexpressing miR-281-x decreases grooming behavior, while inhibiting it increases grooming. They suggest that miR-281-x targets tdc2, which is involved in OA biosynthesis; thus, the miR-281-x–tdc2-OA regulatory axis provides a post-transcriptional mechanism linking a microRNA to behavioral adaptation in honeybees. Overall, it is an interesting study. Although all experiments appear to have been performed accurately and the subject is placed in the context of similar studies, I have only a few suggestions to further improve the manuscript, as detailed below, that came to my mind.

Comment #1: Lines 9-15. Although Varroa mites are part of this study, the simple summary omits the word Varroa, which should probably be added to the keywords (lines 36-37).

Comment #2: Please make sure that you explain all abbreviations when they are first mentioned in the main text. Currently, this is not done for OA line 65 and DMF at line 170. Also, in the thought manuscript, it is better to use a single definition with 5-Hydroxytryptamine (5-HT), serotonin.

Comment #3: It is not clear whether the experiments were carried out side by side or how the series of experiments was done. Please include a clearer description of biological/technical replicates for each set of experiments, given that it is mentioned for MS (n=108) and MW (n=103). It is a bit confusing; which set of bees was used for which experiments? For example, (Lines 205-207), the text mentions pooling 8 brains per sample. Please clarify how many independent biological replicates (pools) were analyzed to generate the statistical significance.

Comment #4: Lines 119-128: Regarding behavioral categorization, on what basis did you decide to define strong groomers and weak groomers? Did you evaluate/test other conditions of bees that exhibited intermediate responses? Was the behavior characterization performed manually, visually, or by tracking software? Furthermore, (Lines 129-131) a second trial for the MW group to confirm their phenotype. How much time elapsed between the first and second trials? Please, indicate how many bees out of the total sampled population fell into the MS vs. MW categories.

Comment #5: Lines 167-169: The 5 μg OA dose is justified by citing a study on waggle dancing. However, how can one be sure that a dose that modulates waggle dancing might be the exact physiological threshold for grooming? Did you perform a preliminary dose-response curve for grooming? How was the dose injected, i.e., with a microinjector using a glass capillary or a needle?

Comment #6: (Lines 199-200: In the initial pharmacological assay, bees were observed at 30 minutes post-treatment. In the rescue assay, they observed 30-60 minutes post-application. What was the rationale for this broadened observation window?

Comment #7: If available, please state the tool or software used to construct the fluorescence in situ hybridization (FISH) probes.

Comment #8: Lines 228-230: I am wondering why HEK293T cells were used for the luciferase assay rather than other cells, for example, Drosophila S2 cells or Chinese hamster ovary (CHO) cells.

Comment #9: Figure 1 related: In Fig. 1A and 1C, the y-axis is labeled (Value). While at the headers' first grooming time(s) and total grooming bouts. I think it is better to have descriptive y-axis labels for each graph (e.g., Time (seconds) for the left plot and Number of Bouts for the right plot). For Fig. 1B, I am a bit confused regarding violin plots for tyramine and 5-hydroxytryptamine; the y-axis extends below zero. Again, pick one term for serotonin and use it consistently across the manuscript and figures.

Comment #10: Figure 2 related: Why is a confidence ellipse only drawn for the MW group?

Comment #11: Figure 3 related: I am wondering on what basis the behavioral phenotypic data in Fig. 3 B-D were analyzed. The legend states that the group differences in these categorical proportions were tested using a Mann-Whitney U test. I think, if I am not mistaken, if you are comparing the percentage of bees falling into two distinct categories (MS vs. MW), a non-parametric continuous test is mathematically inappropriate. You must use a chi-square test of independence or Fisher's exact test for categorical proportional shifts. Please clarify.

Comment #12: In my opinion, lines 436-445, the discussion regarding juvenile hormone and 20-hydroxyecdysone is highly speculative. While interesting, I recommend softening this claim or explicitly stating that it is a hypothetical link requiring future testing.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for providing a revised version of the manuscript. I appreciate that all of my comments have been addressed and reflected in the revised version. I have no further comments.