Investigating the antimicrobial and immunomodulatory effects of honeybee venom peptide apamin in the Drosophila genetic platform

Article activity feed

1. 

   Review Commons

   Dec 26, 2024

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   Reviewer #1

   General Comment. *Using ubiquitous and targeted heterologous expression of the honeybee venom peptide Apamin in Drosophila, the authors find that apamin has antimicrobial activity that is enhanced by membrane-tethering and dependent on the Drosophila pattern-recognition receptors PGRP-LA and PGRP-SC2. Expression of apamin in the Drosophila gut or ingestion of Apamin by honeybees has positive effects on gut health as shown by a number of metrics. *

   __ Answer: __We thank the reviewer for their insightful comments. We agree that the findings of this study are significant and have broad implications for understanding the antimicrobial …

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   Reviewer #1

   General Comment. *Using ubiquitous and targeted heterologous expression of the honeybee venom peptide Apamin in Drosophila, the authors find that apamin has antimicrobial activity that is enhanced by membrane-tethering and dependent on the Drosophila pattern-recognition receptors PGRP-LA and PGRP-SC2. Expression of apamin in the Drosophila gut or ingestion of Apamin by honeybees has positive effects on gut health as shown by a number of metrics. *

   __ Answer: __We thank the reviewer for their insightful comments. We agree that the findings of this study are significant and have broad implications for understanding the antimicrobial properties of apamin. As suggested, we have further delved into the molecular mechanisms underlying apamin's antimicrobial activity, providing additional details on its interactions with target bacteria. We have also expanded our discussion on the role of membrane-tethering in enhancing apamin's activity and its potential impact on its localization. We believe that these additional illustrations strengthen our conclusions and provide a more comprehensive understanding of apamin's biological functions.

   *Major comments: *

   Comment 1. *The key conclusions are convincing and largely supported by the data as shown. The data is presented clearly, save for some areas in the results where the authors should be more explicit about the methods that were used as they affect the reader's interpretation of the results (see minor comments). *

   __ Answer: __We would like to express our gratitude to the reviewer for their constructive feedback and positive remarks regarding our manuscript. We are pleased to note that the reviewer found our key conclusions convincing and largely supported by the data presented. This affirmation encourages us as we strive to contribute meaningful insights to the field. We acknowledge the reviewer's suggestion to enhance clarity in certain areas of the Results section, particularly concerning the methods employed. We appreciate this guidance and have taken it into account. In our revised manuscript, we have made explicit revisions to ensure that the methodology is clearly articulated, thereby improving the reader's interpretation of our results. Thank you once again for your valuable feedback, which has undoubtedly strengthened our work. We look forward to your continued guidance as we finalize our manuscript.

   Comment 2. *If the authors wish to conclude that PGRP-LE and PGRP-LC are not required for the demonstrated functions of Apamin, the authors should do a double knock-down of PGRP-LC and LE together, as these pattern recognition receptors function partially redundantly in activation of the Imd pathway (e.g. doi: 10.1038/ni1356). *

   __ Answer:__ We appreciate reviewer's interesting suggestion to know PGRP-LE and LC's functions are redundant to activate Imd pathway or Apamin is totally independent of Imd pathway. As reviewer suggested, we have conducted double knockdown of PGRP-LE and PGRP-LC and showed that apamin still suppress bacterial infection regardless of these double knockdowns of these genes. This data suggests that apamin's antimicrobial function is totally not dependent on PGRP-LE or LC and open new questions about apamin's unique function as AMP. We added new data in Fig. 5d and described in main text as below:

   "Knockdown of PGRP-LC or LE, as well as their combined knockdown, did not affect the antimicrobial efficacy of apamin (Fig. 5b-d), suggesting that the antimicrobial properties of apamin are independent of PGRP-LC and LE functions (Fig. 5a)."

   *Comment 3. **The Introduction and Discussion would benefit from providing more context that helps the reader understand the significance of the research. Where is apamin expressed in the honeybee? Is it likely to be ingested and have effects on gut health in natural conditions? Do honeybees have homologs of PGRP-LA and PGRP-SC2? Do these findings translate to the honeybee system in any way or are they restricted to heterologous expression in Drosophila? *

   __Answer: __We thank the reviewer for valuable suggestions. We agree that providing additional context on the natural role of apamin in honeybees and the relevance of our findings to the honeybee system is crucial.

   Natural expression and function of apamin: While apamin is primarily known for its neurotoxic effects, studies have suggested that it may also play a role in antimicrobial defense. While its specific expression pattern in honeybees is not fully understood, it is conceivable that it is mainly expressed in venom sacs according to research on biochemistry and pharmacology of apamin (Habermann, 1972; Schumacher et al, 1994; INOUE et al, 1987) . We have outlined this information in the Introduction section as follows:

   "Apamin, an 18 amino acid peptide neurotoxin, is one of the bioactive components of bee venom, making up 2%-3% of its total dry weight, naturally expressed in bee venom sacs (RIETSCHOTEN et al, 1975; E.H., 1976; Son et al, 2007; Zhou et al, 2010; Habermann, 1972)."

   Potential for ingestion and gut effects: Although direct evidence for apamin ingestion and its impact on gut health in natural conditions is limited, it is plausible that honeybees could be exposed to apamin through various means, including foraging and social interactions. However, artificial interference is the potential application method that we are more focusing on. We have included additional details regarding the function of apamin in the Introduction section as follows:

   "It is the smallest known neurotoxic polypeptide and exhibits elevated basicity and sulfur content, demonstrating prolonged action relative to other pharmacological agents influencing the central or peripheral nervous systems(Habermann, 1972)."

   Honeybee homologs of PGRPs: Concerning the honeybee PGRPs and their homologs in Drosophila, we have provided an explanation as follows:

   "While honeybees possess homologs of PGRP family, including PGRP-LC and PGRP-S2, their specific roles in response to apamin and other antimicrobial peptides remain to be elucidated(Larsen et al, 2019a)."

   Relevance to the honeybee system: While our study primarily utilized Drosophila as a model system, the conserved nature of innate immune pathways suggests that the findings may have broader implications for honeybee health. Future studies aimed at directly investigating the effects of apamin in honeybees will be essential to fully understand its role in their physiology and behavior. We have incorporated these points into the Discussion sections to provide a more comprehensive and informative overview of our research as below:

   "In conclusion, it is important to note that much of our understanding of the honeybee immune system is derived from studies conducted on the Drosophila model, owing to the evolutionary proximity of these two species (Larsen et al, 2019b). This close relationship allows for valuable insights into immune mechanisms that are conserved across species (Evans et al, 2006; Morfin et al, 2021). Research has demonstrated that the fruit fly Drosophila melanogaster serves as an effective model for studying the effects of insecticides on honeybees, particularly in understanding the sub-lethal impacts of neonicotinoids, which are known to affect pollinators significantly (Tasman et al, 2021).

   By investigating the function of honeybee AMPs within the Drosophila platform, we can further enhance our knowledge of immune responses and their implications. Just as research on Drosophila has significantly advanced our understanding of human genetic diseases (Bellen et al, 2010; Casci & Pandey, 2015; Bier, 2005; Perrimon et al, 2016; Rieder & Larschan, 2014; Bilder et al, 2021), studying honeybee AMPs in this context holds the potential to uncover novel therapeutic avenues and deepen our comprehension of immune function across taxa."

   *Comment 4. **It is surprising that there is no speculation or hypothesis provided about why PGRP-LA and -SC2 may enhance apamin activity whereas other components are nonessential. It was a significant part of the paper but receives almost no discussion. *

   Answer: We thank the reviewer for highlighting this important point. The specific mechanism by which PGRP-LA and PGRP-SC2 enhance apamin's activity is an intriguing question that warrants further investigation. Our findings indicate that both PGRP-LA and PGRP-SC2 are crucial for the antimicrobial action of apamin, as their knockdown abolishes this effect, suggesting a specific functional relationship between these peptidoglycan recognition proteins and apamin's mechanism of action in the gut environment.

   PGRP-LA is known to play a significant regulatory role as positive regulator of immune responses, while PGRP-SC2 has been shown to promote gut immune homeostasis and prevent dysbiosis, which is essential for maintaining a balanced microbiome in Drosophila (Guo et al, 2014). The enhancement of apamin activity by these proteins could be attributed to their ability to modulate the immune response and facilitate a more effective antimicrobial environment, thereby allowing apamin to exert its effects more efficiently.

   Furthermore, our study aligns with previous research indicating that PGRP-SC2 can limit commensal dysbiosis and promote tissue homeostasis, which may enhance the overall efficacy of antimicrobial peptides like apamin in combating pathogenic bacteria (Guo et al, 2014). By leveraging the evolutionary insights gained from Drosophila, we can better understand how these mechanisms operate in honeybees, ultimately contributing to our knowledge of immune function across species. We have provided a detailed explanation of the potential roles of PGRP-LA and PGRP-SC2 in the action of apamin, as outlined below:

   "The PGRP-LA gene is located in a cluster with PGRP-LC and PGRP-LF, which encode a receptor and a negative regulator of the Imd pathway, respectively; structural predictions suggest that PGRP-LA may not directly bind to peptidoglycan, indicating a potential regulatory role for this PGRP in modulating immune responses (Gendrin et al, 2013). PGRP-SC2 possesses amidase activity, which means it can cleave the peptidoglycan layer of bacterial cell walls, rendering them susceptible to further degradation and ultimately leading to bacterial cell death. This amidase activity contributes to the insect's innate immune response by directly targeting and neutralizing bacterial threats (Takehana et al, 2002; Park et al, 2007; Paredes et al, 2011)."

   *Comment 5. **Line 264: The fact that Rel knockdown did not impair antimicrobial activity of Apamin is a bit odd since upregulation of PGRP-SC2 upon infection is at least partially dependent on Rel (de Gregorio 2002, EMBO J), and the authors find that PGRP-SC2 is required for apamin activity. This is somewhat incongruous. *

   __Answer: __We thank the reviewer for highlighting this important point. The observation that Rel knockdown did not impair apamin's antimicrobial activity, despite its role in upregulating PGRP-SC2, is indeed intriguing.

   Several factors may contribute to this discrepancy:

   Redundancy in PGRP-SC2 regulation: It is possible that other transcription factors, in addition to Rel, may regulate PGRP-SC2 expression. Therefore, even in the absence of Rel, sufficient levels of PGRP-SC2 may be maintained to support apamin's activity(Bischoff et al, 2006) . Direct effects of apamin: Apamin may directly interact with bacterial cells or host immune cells and contribute to its antimicrobial activity, even in the absence of optimal PGRP-SC2 levels.

   We cited (de Gregorio 2002, EMBO J) paper and added explanation for this result as below:

   "It is known that the upregulation of PGRP-SC during infection is partially reliant on the Rel pathway (Gregorio et al, 2002). Our findings indicate that apamin can exert its antimicrobial activity independently of Rel's transcriptional activation function. This observation can be attributed to two key factors. First, there may be redundancy in the regulation of PGRP-SC2 expression, as other transcription factors could compensate for the absence of Rel, allowing sufficient levels of PGRP-SC2 to be maintained to support apamin's activity. Second, apamin may have direct interactions with bacterial cells or host immune cells, contributing to its antimicrobial effects even when optimal levels of PGRP-SC2 are not present. These mechanisms suggest that apamin can function effectively in the immune response, highlighting its potential as a versatile antimicrobial agent."

   *Comment 6. **I cannot comment on the adequacy of the statistical analyses. Some recommendations to improve the methods: *

   *- Be specific about the kind of medium used to rear flies (provide or cite recipe). Different cornmeal-yeast media have very different compositions and can affect fly physiology and microbiome characteristics. *

   *- Specify flipping schedule (every 2-3 days?) - this also affects microbiome. *

   __Answer: __We thank the reviewer for their valuable comments. We agree that precise experimental details are crucial for reproducibility and accurate interpretation of results.

   To address the reviewer's specific concerns:

   Culture medium: We used a standard cornmeal-molasses-agar medium. The specific recipe for this medium is as follows: water add up to 5 L，agar 47g, inactive yeast 65.5g, corn flour 232.5g, soy flour 30g, molasses 350 ml, tegosept sol. 35g, propionic acid 12.5ml, phosphoric acid 2.5ml. Flipping schedule: Flies were flipped every 2-3 days to prevent overcrowding and maintain optimal culture conditions.

   We have included these details in the Methods section to enhance the clarity and reproducibility of our experiments.

   *Minor comments: *

   *- Line 90: Be specific about how the constructs differ from endogenous Melittin and Apamin. Do the endogenous versions have signal peptides? *

   Answer: The endogenous versions do not have signal peptides we have used, we have specified this in the manuscript for readers to have a better understanding as below:

   "To assess the functionality of genetically encoded honeybee VPs in the Drosophila model, we developed UAS-Melittin, and UAS-Apamin constructs that incorporate a previously characterized signal peptide at their N-termini (Choi et al, 2009), which original AMP and VP sequences do not have (Fig. 1a)."

   - Line 92: What is 'broad expression'? Ubiquitous? Specify driver or extent of expression.

   Answer: We have added "by tub-GAL4 driver"

   *- Line 93: Was this oral or septic P. aeruginosa infection? *

   Answer: We have added "oral"

   *- Lines 97-98: Melittin expressed genetically did not show activity against the one pathogen that was tested; making a broad statement without qualification about activity seems excessive. *

   Answer: We have added "against P. aeruginosa"

   *- Line 105: Various Gal4 drivers that express in different tissues or a similar subset of tissues? *

   Answer: We utilized tub-GAL4 and da-GAL4 in this part of screening, they both drive expression in ubiquitous tissues. Daughterless (da) involves in the transcriptional regulation of various processes, including oogenesis, neurogenesis, myogenesis, and cell proliferation. While tub-GAL4 is ubiquitous expression throughout most tissues and cell types in the Drosophila body. We have added "various ubiquitously expressing"

   *- Line 134: Present as a commensal? Pathobiont? Pathogen? *

   Answer: Apibacter raozihei is generally considered a commensal bacterium in the honeybee gut. We have added to manuscript "which is present as a commensal bacterium in the guts of".

   *- Line 149: Are Cyanobacteria naturally present in gut microbiota? What are photosynthetic bacteria doing as part of a gut microbiome? *

   Answer: While cyanobacteria are not typically found in the gut, cyanobacterial 16S rRNA-like sequences have been previously detected in human gut samples, bovine rumen, termite gut, and other animal intestines, suggesting the presence of a non-photosynthetic cyanobacterial lineage in these aphotic environments(Hu & Rzymski, 2022; Hongoh et al, 2003).

   *- Line 171: Where is apamin endogenously expressed in the honeybee? Only in the venom gland? Or in gut cells as done here in Drosophila? *

   Answer: Natural expression and function of apamin: While apamin is primarily known for its neurotoxic effects, studies have suggested that it may also play a role in antimicrobial defense. While its specific expression pattern in honeybees is not fully understood, it is conceivable that it is mainly expressed in venom sacs according to research on biochemistry and pharmacology of apamin (Habermann, 1972; Schumacher et al, 1994; INOUE et al, 1987) .

   *- Line 252: -LC and -LE work in a complementary/semi-redundant fashion, so single knockdown is not an effective method of indicating that they are not required for antimicrobial function. *

   Answer: We appreciate reviewer's interesting suggestion to know PGRP-LE and LC's functions are redundant to activate Imd pathway or Apamin is totally independent of Imd pathway. As reviewer suggested, we have conducted double knockdown of PGRP-LE and PGRP-LC and showed that apamin still suppress bacterial infection regardless of these double knockdowns of these genes. This data suggests that apamin's antimicrobial function is totally not dependent on PGRP-LE or LC and open new questions about apamin's unique function as AMP. We added new data in Fig. 5d and described in main text as below:

   "Knockdown of PGRP-LC or LE, as well as their combined knockdown, did not affect the antimicrobial efficacy of apamin (Fig. 5b-d), suggesting that the antimicrobial properties of apamin are independent of PGRP-LC and LE functions (Fig. 5a)."

   *- Lines 279-283: The bacterial infections that expression of these AMPs were tested against should be mentioned in the text, as all bacteria are not equivalent. *

   Answer: Added with "P. aeruginosa"

   *- Line 296: Challenged with which bacteria? *

   Answer: Added with "P. aeruginosa"

   *- Line 328: Provide brief explanation of what Ttk depletion is for reader context. *

   Answer: Added with short explanation as below:

   "which refers to the reduction or elimination of a protein called TTK (Monopolar Spindle 1 Kinase) that plays a crucial role in cell division, specifically in ensuring accurate chromosome segregation during mitosis (Mason et al, 2017)."

   *- Line 719: This should say, '5 days after eclosion'. *

   Answer: Corrected

   *- General comment on figures: The little icons used to denote what the figure is depicting (gut health, climbing, aging, etc.) are very effective. *

   Answer: We thank the reviewer for their appreciation on figures.

   *- General comment on figure titles: Use of the term 'infectious dose' throughout does not make sense. I think what the authors mean is 'pathogen load' as they are testing using CFUs. 'Infectious dose' should only be used to refer to the amount/OD of pathogen that was initially administered to establish an infection. Also, 'oral feeding' should be used throughout instead of 'orally feeding'. *

   Answer: We thank the reviewer for their insightful comment. We agree that the use of the term 'infectious dose' was inaccurate in certain contexts. We have revised the manuscript to use 'pathogen load' to refer to the number of CFUs administered or recovered, as this more accurately reflects the bacterial burden.

   We have also replaced 'orally feeding' with 'oral feeding' throughout the manuscript to improve clarity and consistency.

   We appreciate the reviewer's attention to detail and believe that these changes have significantly enhanced the clarity and accuracy of the manuscript.

   *- Figure 1O: Abrupt die-offs at 1000hrs and 2800hrs in the UAS-Melittin line suggest that lifespan experiment was only performed once and that die-offs may have been exacerbated due to infrequent flipping. This is perhaps not an issue as the lifespans appear to be quite different between the active line and control regardless. *

   Answer: We thank the reviewer for their careful observation. The abrupt die-offs in the UAS-Melittin line at 1000 hours and 2800 hours were unexpected. While we cannot definitively rule out the possibility that infrequent flipping might have contributed to these events, we believe that the overall lifespan difference between the experimental and control groups is substantial and likely reflects a genuine biological effect of Melittin overexpression.

   *- Figure 2F would be improved by putting the legend in the same descending order that the genotypes are displayed on the graph (tApamin infected, GFP infected, tApamin, GFP) *

   Answer: We have corrected error.

   *- Figure 3I: Unclear what small image inserted in the graph depicts. *

   Answer: This is an image of fly stem cells that is available for free licensing.

   *- Figures 3N and 3O are verry low resolution and difficult to identify the differences that the authors **intend to show. *

   Answer: We have utilized a higher resolution image and revised the figure accordingly.

   - Figure 4 title is confusing. Do the authors mean, "Locomotion of flies expressing neuronal Apamin, sleep in flies with ubiquitous expression of Apamin, and Smurf results induced by different types of stress."?

   Answer: Corrected as below:

   "Locomotion of flies expressing neuronal tApaminDC, sleep in flies with ubiquitous expression of tApaminDC, and Smurf results induced by different types of stress."

   *- Figure 5: Some of these graphs are very cluttered and difficult to parse (particularly 5H). Suggest putting peptide sequences in figure title rather than underneath graphs to simplify and increase visual effectiveness. *

   Answer: We have improved by removing the sequences to figure legend part.

   *-Throughout: Methods section in particular could use a solid edit for grammar. Homogenize capitalization of "Gram-negative/-positive" and "gram-negative/-positive" *

   Answer: We have corrected error.

   *- Line 98: "an AMPs" should be "an AMP" *

   Answer: We have corrected error.

   *- Line 119: Incorrect grammar. Suggest, "which did not affect the lifespan of female flies and had only a slight effect on male flies" *

   Answer: We have corrected error.

   *Reviewer #1 (Significance (Required)): *

   *The paper reveals that apamin has antimicrobial properties. The intended significance seems to be an exploration of apamin for therapeutic potential in gut health, but this is not explicitly stated by the authors. The contribution mainly appears to be conceptual in nature. *

   *The findings appear to be in line with other recent in vitro results suggesting that apamin has antimicrobial properties (DOI: 10.9775/kvfd.2024.32125). *

   *Researchers interested in developing therapeutic applications for bee venom constituents or promoting gut health and microbiome balance will likely find this research of interest. *

   *My expertise is primarily in Drosophila molecular genetics and immunity. I have a broad understanding of Drosophila immune pathways, epithelial immunity, and infection dynamics. I do not feel qualified to comment on the statistics or data analysis aspects of this paper. *

   Answer: We sincerely appreciate the reviewer's positive feedback regarding our findings on the antimicrobial properties of apamin. We are grateful for the acknowledgment that our results align with recent in vitro studies, such as the one referenced (DOI: 10.9775/kvfd.2024.32125), which further supports the significance of our work. We have cited this paper in the Discussion section as below.

   "Our findings are consistent with recent in vitro studies demonstrating the antimicrobial and antibiofilm effects of apamin (AYDIN et al, 2024)."

   We recognize the reviewer's observation that our intended significance-specifically, the exploration of apamin's therapeutic potential for gut health-was not explicitly stated in the original manuscript. To address this, we have revised the Introduction and Discussion sections to clearly articulate our aim of investigating apamin as a candidate for promoting gut health and microbiome balance. We believe this clarification will enhance the conceptual contribution of our study and its relevance to researchers interested in therapeutic applications of bee venom constituents.

   "Apamin shows promising therapeutic potential for enhancing bee gut health by exhibiting antimicrobial properties that can help maintain a balanced microbiome. Its ability to modulate immune responses and promote gut integrity, particularly in the presence of harmful bacteria, positions apamin as a valuable candidate for developing strategies aimed at improving gut health in honeybees."

   Additionally, we appreciate the reviewer's expertise in Drosophila molecular genetics and immunity, and we are grateful for their insights regarding the broader implications of our research. We will ensure that our manuscript reflects these considerations more explicitly.

   Thank you once again for your valuable feedback, which has helped us improve the clarity and impact of our work.

   Reviewer #2

   *Reviewer #2 (Evidence, reproducibility and clarity (Required)): *

   *General Comment. **The reviewer would like to thank the authors for their contributions to the research of animal venoms and their therapeutic value. The manuscript is very well and clearly written. Additionally, the choice of using a model organism such as D. melanogaster in the context of venoms research strengthens the manuscript by providing evidence that is both robust and broadly applicable, thus enhancing the manuscript's scientific merit and relevance to the field. *

   Answer: We would like to express our sincere gratitude to the reviewer for the positive feedback and thoughtful comments regarding our manuscript. We are pleased to hear that the reviewer appreciates our contributions to the research on animal venoms and their therapeutic potential. The reviewer's acknowledgment of the clarity and quality of our writing is particularly encouraging, as we strive to communicate our findings effectively. Additionally, we are glad that the choice of Drosophila melanogaster as a model organism was recognized for its ability to strengthen our research by providing robust and broadly applicable evidence. This endorsement enhances the scientific merit and relevance of our work within the field. Thank you once again for the constructive feedback, which has been invaluable in refining our manuscript.

   *Comment 1. **What is the significance of that the biological property of apamin is independent of its disulfide bonds? Does it suggest that the core functional parts of apamin might not entirely depend on its stabilized structure? Could it mean that modifications to the molecule that disrupt disulfide bonds wouldn't necessarily eliminate all of its activity, which could be important in designing analogs or derivatives of apamin for research or therapeutic purposes etc.? This sentence is written in the abstract which means that it should be a key finding, and it should be clear and a given to the reader. However, it is not the case, and it should be stated more clearly. *

   Answer: We greatly appreciate the reviewer for the perceptive notation. The fact that the biological functioning of apamin needs no disulfide bonds should bring forth the attention of the scientists because it has further implications. This hints that apamin's major functional units are most likely to compose from its polypeptide instead of being rooted in the disulfide-stabilized tertiary structure(Habermann, 1972). The strategy can then lead to the optimization of apamin-based drugs with altered disulfide bridges granting them either higher activity or reduced toxicity. These changes can give apamin additional properties like stability, bioavailability, or selectivity, which make it suitable for research and applied use. We have included an explanation for this in both the Results and Discussion sections.

   "This finding suggests that the core functional components of apamin may not be entirely reliant on its stabilized structure."

   "We discovered that apamin lacking the C-terminus retains its function as an antimicrobial agent, despite missing one of its two disulfide bridges. This finding suggests that the core functional components of apamin may not be entirely dependent on its stabilized structure, indicating that modifications to the molecule that disrupt these disulfide bonds could still maintain some level of activity. These insights are vital for designing analogs or derivatives of apamin, as they pave the way for developing new compounds that could retain therapeutic potential even without the native disulfide bond configuration (Habermann, 1972)."

   *Comment 2. **The authors well explained the evolutionary proximity between apamin producing Honeybees and D. melanogaster in order to justify the choice of the model organism which we can all agree on for genetics and developmental biology studies. However, when addressing the behavior of the insects (sleeping, locomotion, social etc.) which are driven by their ecological roles, evolutionary strategies, and social complexity. How much can you really tell about the role of apamin in the behavior of Honeybees (highly social and form colonies) by studying it on an insect (D. melanogaster) which has a completely different and divergent behavior (solitary and exhibit only few basic forms of social interaction)? *

   Answer: We appreciate the reviewer's insightful comment. While Drosophila melanogaster is an excellent model organism for investigating fundamental biological processes, we recognize the limitations of using it to fully comprehend the complex behavioral effects of apamin in honeybees. Nevertheless, our study establishes a foundational understanding of apamin's potential impact on behavior, including its effects on sleep and locomotion-core behavioral processes that are conserved across many organisms, including insects (Zimmerman et al, 2008).

   By employing Drosophila as a model, we were able to identify potential mechanisms of action for apamin, particularly regarding its effects on intestinal systems. Although honeybees and fruit flies exhibit ecological differences, there is substantial consensus and experimental evidence that many molecular pathways involved in immune responses are conserved between these species. Thus, while the interpretation of behavioral changes induced by apamin may be limited by the ecological and evolutionary divergence between honeybees and fruit flies, the molecular pathways governing the immune response in honeybees can be effectively studied using the Drosophila platform. This approach has previously revealed functions of genes related to human genetic diseases. We have clearly articulated this limitation and the advantages of using the fly model to study the honeybee immune system in the Discussion section as follows:

   "In conclusion, it is important to note that much of our understanding of the honeybee immune system is derived from studies conducted on the Drosophila model, owing to the evolutionary proximity of these two species (Larsen et al, 2019b). This close relationship allows for valuable insights into immune mechanisms that are conserved across species (Evans et al, 2006; Morfin et al, 2021). Research has demonstrated that the fruit fly Drosophila melanogaster serves as an effective model for studying the effects of insecticides on honeybees, particularly in understanding the sub-lethal impacts of neonicotinoids, which are known to affect pollinators significantly (Tasman et al, 2021).

   By investigating the function of honeybee AMPs within the Drosophila platform, we can further enhance our knowledge of immune responses and their implications. Just as research on Drosophila has significantly advanced our understanding of human genetic diseases (Bellen et al, 2010; Casci & Pandey, 2015; Bier, 2005; Perrimon et al, 2016; Rieder & Larschan, 2014; Bilder et al, 2021), studying honeybee AMPs in this context holds the potential to uncover novel therapeutic avenues and deepen our comprehension of immune function across taxa."

   *Comment 3. **Please include the following references: *

   • Wehbe R, Frangieh J, Rima M, El Obeid D, Sabatier JM, Fajloun Z. Bee Venom: Overview of Main Compounds and Bioactivities for Therapeutic Interests. Molecules. 2019 Aug 19;24(16):2997. *

   • Nader RA, Mackieh R, Wehbe R, El Obeid D, Sabatier JM, Fajloun Z. Beehive Products as Antibacterial Agents: A Review. Antibiotics. 2021; 10(6):717. *

   Answer: We have incorporated the references mentioned above in appropriate sections of the manuscript. We appreciate the reviewer's suggestions.

   *Reviewer #2 (Significance (Required)): *

   The manuscript is very well and clearly written. Additionally, the choice of using a model organism such as D. melanogaster in the context of venoms research strengthens the manuscript by providing evidence that is both robust and broadly applicable, thus enhancing the manuscript's scientific merit and relevance to the field.

   Answer: We would like to express our sincere gratitude to the reviewer for their positive feedback regarding our manuscript. We are thrilled to hear that the clarity and quality of our writing were appreciated. Additionally, we are glad that the choice of Drosophila melanogaster as a model organism in our venoms research was recognized for its ability to provide robust and broadly applicable evidence. This endorsement underscores the scientific merit and relevance of our work within the field, and we appreciate the reviewer's acknowledgment of this important aspect. Thank you for your encouraging comments, which motivate us to continue exploring this vital area of research.

   Read the original source
2. 

   Review Commons

   Dec 26, 2024

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   The reviewer would like to thank the authors for their contributions to the research of animal venoms and their therapeutic value. The manuscript is very well and clearly written. Additionally, the choice of using a model organism such as D. melanogaster in the context of venoms research strengthens the manuscript by providing evidence that is both robust and broadly applicable, thus enhancing the manuscript's scientific merit and relevance to the field.

   Some comments to add:

   • What is the significance of that the biological property of apamin is independent of its disulfide bonds? Does it suggest that the core functional parts of …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   The reviewer would like to thank the authors for their contributions to the research of animal venoms and their therapeutic value. The manuscript is very well and clearly written. Additionally, the choice of using a model organism such as D. melanogaster in the context of venoms research strengthens the manuscript by providing evidence that is both robust and broadly applicable, thus enhancing the manuscript's scientific merit and relevance to the field.

   Some comments to add:

   • What is the significance of that the biological property of apamin is independent of its disulfide bonds? Does it suggest that the core functional parts of apamin might not entirely depend on its stabilized structure? Could it mean that modifications to the molecule that disrupt disulfide bonds wouldn't necessarily eliminate all of its activity, which could be important in designing analogs or derivatives of apamin for research or therapeutic purposes etc.? This sentence is written in the abstract which means that it should be a key finding, and it should be clear and a given to the reader. However, it is not the case, and it should be stated more clearly.
   • The authors well explained the evolutionary proximity between apamin producing Honeybees and D. melanogaster in order to justify the choice of the model organism which we can all agree on for genetics and developmental biology studies. However, when addressing the behavior of the insects (sleeping, locomotion, social etc.) which are driven by their ecological roles, evolutionary strategies, and social complexity. How much can you really tell about the role of apamin in the behavior of Honeybees (highly social and form colonies) by studying it on an insect (D. melanogaster) which has a completely different and divergent behavior (solitary and exhibit only few basic forms of social interaction)?

   Please include the following references:

   1. Wehbe R, Frangieh J, Rima M, El Obeid D, Sabatier JM, Fajloun Z. Bee Venom: Overview of Main Compounds and Bioactivities for Therapeutic Interests. Molecules. 2019 Aug 19;24(16):2997.
   2. Nader RA, Mackieh R, Wehbe R, El Obeid D, Sabatier JM, Fajloun Z. Beehive Products as Antibacterial Agents: A Review. Antibiotics. 2021; 10(6):717.

   Significance

   The manuscript is very well and clearly written. Additionally, the choice of using a model organism such as D. melanogaster in the context of venoms research strengthens the manuscript by providing evidence that is both robust and broadly applicable, thus enhancing the manuscript's scientific merit and relevance to the field.

   Read the original source
3. 

   Review Commons

   Dec 26, 2024

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   Using ubiquitous and targeted heterologous expression of the honeybee venom peptide Apamin in Drosophila, the authors find that apamin has antimicrobial activity that is enhanced by membrane-tethering and dependent on the Drosophila pattern-recognition receptors PGRP-LA and PGRP-SC2. Expression of apamin in the Drosophila gut or ingestion of Apamin by honeybees has positive effects on gut health as shown by a number of metrics.

   Major comments:

   The key conclusions are convincing and largely supported by the data as shown. The data is presented clearly, save for some areas in the results where the authors should be more …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   Using ubiquitous and targeted heterologous expression of the honeybee venom peptide Apamin in Drosophila, the authors find that apamin has antimicrobial activity that is enhanced by membrane-tethering and dependent on the Drosophila pattern-recognition receptors PGRP-LA and PGRP-SC2. Expression of apamin in the Drosophila gut or ingestion of Apamin by honeybees has positive effects on gut health as shown by a number of metrics.

   Major comments:

   The key conclusions are convincing and largely supported by the data as shown. The data is presented clearly, save for some areas in the results where the authors should be more explicit about the methods that were used as they affect the reader's interpretation of the results (see minor comments). If the authors wish to conclude that PGRP-LE and PGRP-LC are not required for the demonstrated functions of Apamin, the authors should do a double knock-down of PGRP-LC and LE together, as these pattern recognition receptors function partially redundantly in activation of the Imd pathway (e.g. doi: 10.1038/ni1356 ). The Introduction and Discussion would benefit from providing more context that helps the reader understand the significance of the research. Where is apamin expressed in the honeybee? Is it likely to be ingested and have effects on gut health in natural conditions? Do honeybees have homologs of PGRP-LA and PGRP-SC2? Do these findings translate to the honeybee system in any way or are they restricted to heterologous expression in Drosophila? It is surprising that there is no speculation or hypothesis provided about why PGRP-LA and -SC2 may enhance apamin activity whereas other components are nonessential. It was a significant part of the paper but receives almost no discussion. Line 264: The fact that Rel knockdown did not impair antimicrobial activity of Apamin is a bit odd since upregulation of PGRP-SC2 upon infection is at least partially dependent on Rel (de Gregorio 2002, EMBO J), and the authors find that PGRP-SC2 is required for apamin activity. This is somewhat incongruous.

   I cannot comment on the adequacy of the statistical analyses. Some recommendations to improve the methods:

   • Be specific about the kind of medium used to rear flies (provide or cite recipe). Different cornmeal-yeast media have very different compositions and can affect fly physiology and microbiome characteristics.
   • Specify flipping schedule (every 2-3 days?) - this also affects microbiome.

   Minor comments:

   Line 90: Be specific about how the constructs differ from endogenous Melittin and Apamin. Do the endogenous versions have signal peptides?

   Line 92: What is 'broad expression'? Ubiquitous? Specify driver or extent of expression.

   Line 93: Was this oral or septic P. aeruginosa infection?

   Lines 97-98: Melittin expressed genetically did not show activity against the one pathogen that was tested; making a broad statement without qualification about activity seems excessive.

   Line 105: Various Gal4 drivers that express in different tissues or a similar subset of tissues?

   Line 134: Present as a commensal? Pathobiont? Pathogen?

   Line 149: Are Cyanobacteria naturally present in gut microbiota? What are photosynthetic bacteria doing as part of a gut microbiome?

   Line 171: Where is apamin endogenously expressed in the honeybee? Only in the venom gland? Or in gut cells as done here in Drosophila?

   Line 252: -LC and -LE work in a complementary/semi-redundant fashion, so single knockdown is not an effective method of indicating that they are not required for antimicrobial function.

   Lines 279-283: The bacterial infections that expression of these AMPs were tested against should be mentioned in the text, as all bacteria are not equivalent.

   Line 296: Challenged with which bacteria?

   Line 328: Provide brief explanation of what Ttk depletion is for reader context.

   Line 719: This should say, '5 days after eclosion'.

   General comment on figures: The little icons used to denote what the figure is depicting (gut health, climbing, aging, etc.) are very effective.

   General comment on figure titles: Use of the term 'infectious dose' throughout does not make sense. I think what the authors mean is 'pathogen load' as they are testing using CFUs. 'Infectious dose' should only be used to refer to the amount/OD of pathogen that was initially administered to establish an infection. Also, 'oral feeding' should be used throughout instead of 'orally feeding'.

   Figure 1O: Abrupt die-offs at 1000hrs and 2800 hrs in the UAS-Melittin line suggest that lifespan experiment was only performed once and that die-offs may have been exacerbated due to infrequent flipping. This is perhaps not an issue as the lifespans appear to be quite different between the active line and control regardless.

   Figure 2F would be improved by putting the legend in the same descending order that the genotypes are displayed on the graph (tApamin infected, GFP infected, tApamin, GFP)

   Figure 3I: Unclear what small image inserted in the graph depicts.

   Figures 3N and 3O are verry low resolution and difficult to identify the differences that the authors intend to show.

   Figure 4 title is confusing. Do the authors mean, "Locomotion of flies expressing neuronal Apamin, sleep in flies with ubiquitous expression of Apamin, and Smurf results induced by different types of stress."?

   Figure 5: Some of these graphs are very cluttered and difficult to parse (particularly 5H). Suggest putting peptide sequences in figure title rather than underneath graphs to simplify and increase visual effectiveness.

   Throughout: Methods section in particular could use a solid edit for grammar. Homogenize capitalization of "Gram-negative/-positive" and "gram-negative/-positive"

   Line 98: "an AMPs" should be "an AMP"

   Line 119: Incorrect grammar. Suggest, "which did not affect the lifespan of female flies and had only a slight effect on male flies"

   Significance

   The paper reveals that apamin has antimicrobial properties. The intended significance seems to be an exploration of apamin for therapeutic potential in gut health, but this is not explicitly stated by the authors. The contribution mainly appears to be conceptual in nature.

   The findings appear to be in line with other recent in vitro results suggesting that apamin has antimicrobial properties (DOI: 10.9775/kvfd.2024.32125).

   Researchers interested in developing therapeutic applications for bee venom constituents or promoting gut health and microbiome balance will likely find this research of interest.

   My expertise is primarily in Drosophila molecular genetics and immunity. I have a broad understanding of Drosophila immune pathways, epithelial immunity, and infection dynamics. I do not feel qualified to comment on the statistics or data analysis aspects of this paper.

   Read the original source
4. 

   Version published to 10.1101/2024.07.25.605111v1 on bioRxiv

   Jul 25, 2024