Leucine Enhances Stress Resistance in Honeybees (Apis mellifera L.) by Modulating Sestrin-Dependent Antioxidant Responses

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript investigates the protective effects of dietary leucine supplementation against oxidative stress induced by environmental stressors (cadmium and permethrin) in honeybees. The authors propose an interesting mechanism wherein leucine enhances antioxidant defenses primarily through the upregulation of sestrin, rather than the canonical TOR signaling pathway. The study is well-structured, utilizing a combination of in vivo feeding assays, RNA interference, pharmacological inhibition, and molecular docking. The topic is highly relevant to current ecological concerns regarding pollinator decline. However, there are several significant physiological contradictions in the reported oxidative stress markers that require rigorous clarification. Additionally, some methodological validations (such as RNAi efficiency at the time of stress exposure) and computational claims need to be addressed before the manuscript can be considered for publication.

Major Comments:

- In the results section (Page 7, Lines 225-226), the authors state that leucine supplementation increased MDA content while decreasing H₂O₂ and ROS levels. Malondialdehyde (MDA) is a well-established end-product of lipid peroxidation and a primary marker of cellular oxidative damage. It is highly counterintuitive for ROS levels to decrease while lipid peroxidation (MDA) increases. The authors must provide a detailed biological or methodological explanation for this inconsistency. If this is an item of the assay, it must be approved; otherwise, this paradox fundamentally challenges the core conclusion that leucine purely enhances antioxidant capacity.

- The authors report that silencing sestrin led to decreased SOD and CAT activities, as well as reduced ROS and H₂O₂ levels, regardless of whether the bees were exposed to CdCl₂ or permethrin (Page 14, Lines 335-336). Typically, the knockdown of a crucial antioxidant regulator like sestrin should result in the accumulation of ROS. The authors briefly address this anomaly for the non-stressed condition (Page 17, Lines 457-460), attributing it to the absence of exogenous stress. However, they fail to explain why this phenomenon persists even under heavy metal or pesticide stress. If ROS levels were artificially low due to a measurement error, or if the bees underwent severe metabolic depression/apoptosis preventing accurate ROS measurement, this must be explicitly discussed.

- The authors verified the silencing efficiency of dssestrin at 24, 48, and 72 hours post-treatment (Figure S1). However, the stress exposures (CdCl₂ and permethrin) and subsequent samplings occurred on Day 8. Although the bees were administered dsRNA every two days, the authors must confirm and explicitly state that sestrin was still effectively silenced on Day 8 when the stress tests were conducted.

- The authors utilized molecular docking to predict the binding of leucine to sestrin (Figure 5), reporting a binding free energy of -5.2 kcal/mol. While this provides a theoretical basis, computational docking alone is insufficient to claim a direct physical interaction. The authors should moderate their language regarding this binding (e.g., use "predicted to interact" instead of definitive statements) and explicitly acknowledge the lack of in vitro biochemical validation (e.g., SPR, ITC, or mutagenesis of the binding pocket) as a limitation of the current study.

- The study uses a leucine concentration of 1.5 mg/g in a 50% sucrose solution. The authors should discuss how this concentration compares to the natural levels of free amino acids (specifically leucine) found in floral pollen and nectar. Establishing the ecological and physiological relevance of this dose is crucial for interpreting these findings into practical, field-applicable nutritional strategies for beekeeping.

- The histopathological analysis (Figure 7C) provides visual evidence of midgut damage and its partial rescue by leucine. However, the descriptions are purely qualitative. The authors should include a quantitative analysis of the histological sections (e.g., measuring epithelial thickness, assessing the extent of cell vacuolization, or scoring tissue damage) to provide robust statistical support for the morphological claims.

Minor Comments:

- Please clarify the feeding protocol for rapamycin. Was it provided continuously throughout the 9 days? Is the final concentration of 0.02 mg/mL sufficient to fully inhibit the TOR pathway in honeybees? A brief reason or citation supporting this dosage would be helpful.

- There are several grammatical errors that need correction to improve readability. Examples include: Line 140: "The content of determining malondialdehyde" "The content of malondialdehyde"; Line 201: "When P< 0.05 was considered" "A P-value < 0.05 was considered"; Line 287: "Sestrin from Apis mellifera L. closely with this protein" "is closely related to this protein"; Line 491: "TOR does not the key mediator" "TOR is not the key mediator"; Line 496: "functional diversity" "the functional diversity",…..

- In the Simple Summary (Line 20), the sentence "Notably, the protective effect of leucine did not depend on the previously recognized TOR pathway" is a very strong claim. Consider this slightly to "did not primarily depend on..." to align with the nuance shown in the discussion where TOR still plays some regulatory role.

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

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Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have improved the manuscript and implemented the suggested revisions. I think that manuscript can be accepted for publication.