Imidacloprid disrupts larval molting regulation and nutrient energy metabolism, causing developmental delay in honey bee Apis mellifera

Joint Public Review:

This study provides evidence of the ability of sublethal imidacloprid doses to affect growth and development of honeybee larva. While checking the effect of doses that do not impact survival or food intake, the authors found changes in the expression of genes related to energy metabolism, antioxidant response, and metabolism of xenobiotics. The authors also identified cell death in the alimentary canal, and disturbances in levels of ROS markers, molting hormones, weight and growth ratio. The study strengths come from exploring different aspects and impacts of imidacloprid exposure on honeybee juvenile stages and for that it demonstrates potential for assessing the risks posed by pesticides. The study weaknesses come from the lack of in depth investigation and an incomplete methodological design. For instance, many of the study conclusions are based on RT-qPCR, which show only a partial snapshot of gene expression, which was performed at a single time point and using whole larvae. There is no understanding of how different organs/tissues might respond to exposure and how they change over time. That creates a problem to understand the mechanisms of damage caused by the pesticide in the situation studied here. There is no investigation of what happens after pupation. The authors show that the doses tested have no impact on survival, food consumption and time to pupation, and the growth index drops from ~0.96 to ~0.92 in exposed larvae, raising the question of its biological significance. The origin of ROS are not investigated, nor do the authors investigate if the larvae recover from the damage observed in the gut after pupation. That is important as it could affect the adult workers' health. One of the study's central claims is that the reduced growth index is due to the extra energy used to overexpress P450s and antioxidant enzymes, but that is based on RT-qPCR only. Other options are not well explored and whether the gut damage could be causing nutrient absorption problems, or the oxidative stress could be impairing mitochondrial energy production is not investigated. These alternatives may also affect the growth index. The authors also state that the honeybee larvae has 7 instars, which is an incorrect as Apis mellifera have 5 larval instars. It is not clear from methods which precise stage of larval development was used for gut preparations. That information is important because prior to pupation larvae defecate and undergo shedding of gut lining. That could profoundly affect some of the results in case gut preparations for microscopy were made close to this stage. A more in-depth investigation and more complete methodological design that investigates the mechanisms of damage and whether the exposures tested could affect adult bees may demonstrate the damage of low insecticide doses to a vital pollinator insect species.

Author Response

The following is the authors’ response to the original reviews.

We sincerely appreciate the opportunity to revise the manuscript and the reviewers' critical comments and valuable suggestions. After carefully revising the manuscript, we strongly believe that the reviewers' comments are invaluable and will significantly enhance the quality of the manuscript and contribute to our future research. Following the reviewers' comments, we conducted a comprehensive and meticulous review, addressing each point individually and making extensive modifications and corrections. The responses to each question are provided in a point-by-point manner as follows:

Reviewer 1:

This study delves into the impact of imidacloprid, an insecticide documented for its toxicity towards honeybees, on the development of bee larvae. The investigation involved exposing bee larvae to various concentrations of imidacloprid, and observing the resultant effects.

The findings of this study revealed that imidacloprid exerted a dose-dependent delay in the development of bee larvae, marked by reductions in body mass, width, and an overall decline in the growth index. Moreover, at elevated concentrations, imidacloprid was observed to impair neural transmission, induce oxidative stress, inflict damage to the gut, and inhibit hormones and genes essential for development. The larvae were found to engage antioxidant defense systems and deploy detoxification mechanisms to mitigate these effects.

However, the manuscript could be significantly enhanced through several improvements. Firstly, the structure of the manuscript warrants refinement to foster coherence and clarity. Additionally, there is a need for careful reevaluation of the concentrations of imidacloprid employed in the study, to ensure their relevance and applicability. In terms of references, greater attention to accuracy in citation is imperative.

Furthermore, while the authors have provided an overview of the general effects of imidacloprid on both vertebrates and invertebrates, the inclusion of a more exhaustive literature review with a specific focus on honey bees and other insects would bolster the context and significance of this research. This would be particularly beneficial in the introduction section, which should be subjected to a major revision.

In summary, this study offers preliminary evidence of the detrimental effects of imidacloprid on the development of bee larvae by interfering with molting and metabolism. This research holds potential as a valuable resource for assessing the risks posed by pesticides to juvenile stages of various animal species.

On behalf of all the authors, I express our most sincere gratitude for your critical comments and suggestions. Following your suggestions, we have thoroughly reviewed and revised the entire manuscript, including the issues of imidacloprid concentration and citation accuracy you raised. More importantly, we have significantly revised the structure and content of the introductory section of the manuscript to include many more detailed reviews of critical literature, with particular attention to the overview of relevant research on honey bees, Drosophila, and other insects, to promote coherence and clarity of the introduction and to enhance the context and importance of this research. We hope that these changes meet with your approval. Overall, your valuable comments have greatly improved the quality of the manuscript and will facilitate our future research.

Q1: Line 48, "Adults exposed to high doses of imidacloprid experience", please provide a more precise value for the high doses.

Thank you very much for your comments. Following your suggestion, we have provided precise values for high doses of imidacloprid for adult exposure based on the study by Dr. Wu et al. 2001.

Q2: Line 82, There are several larvae effect reports using next generation sequencing approach. The authors should include those related references in this section.

Thank you for your comments. We have included relevant references in our revised manuscript.

Q3: Line 394, for the concentration design, the maximum concentration of imidacloprid used in this study is 377 ppb, which is from the imidacloprid residue level in beeswax. Bees don't consume beeswax, and the reference is wrong.

Thank you for raising this critical issue. As you point out, bees do not eat beeswax, but it is important to stress that this may well mean that the bee larvae themselves are exposed to higher doses. Therefore, in this study, we ultimately designed for the worst-case scenario of 377 ppb of imidacloprid residues in beeswax. We would like your agreement on this point. In addition, we have corrected the citation errors in the references here and included them in the revised manuscript.

Reviewer 2:

This study provides evidence on the ability of sublethal imidacloprid doses to affect growth and development of honeybee larva. While checking the effect of doses that do not impact survival or food intake, the authors found changes in the expression of genes related to energy metabolism, antioxidant response, and P450 metabolism. The authors also identified cell death in the alimentary canal, and disturbances in levels of ROS markers, molting hormones, weight and growth ratio. The study strengths come from applying these different approaches to investigate the impacts of imidacloprid exposure. The study weaknesses are not providing an in-depth investigation of the mechanisms behind the impacts observed and not bringing the results in light of the current literature. For instance, the authors' hypothesis is based on two main points, the generation of ROS that leads to gut cell death and energy dysfunction, and the increased P450 expression. They propose this increases P450 expression which in turn increases energy consumption and could contribute to developmental retardation. There is however no investigation on the mechanisms of ROS generation (it could be through mitochondrial damage, Nox/ Duox activity, NOS activity, P450s activity, etc). A link between higher P450 expression and increased energy consumption leading to energy deprivation is also missing. It would also be important for the authors to provide a more complete literature review as previous works have investigated imidacloprid sublethal dose impacts in larval stages for bees and other insect models.

I greatly appreciate your insightful comments and valuable suggestions on behalf of all the authors. Thank you for identifying the limitations of this study and providing valuable comments and suggestions. These comments and suggestions have significantly improved the quality of the paper and will facilitate our future research. Following your comments, we have revised and corrected the manuscript point by point. We hope that these corrections meet with your approval.

Q1: Abstract: It would be important to rephrase the abstract to make it clear when authors are talking about gene expression results or functional assays.

Thank you for your comment. Following your suggestion, we have revised the abstract to make it clearer, especially the description of the gene expression results. Please see lines 15-34 in our revised manuscript.

“Abstract Imidacloprid is a global health threat that severely poisons the economically and ecologically important honeybee pollinator, Apis mellifera. However, its effects on developing bee larvae remain largely unexplored. Our pilot study showed that imidacloprid causes developmental delay in bee larvae, but the underlying toxicological mechanisms remain incompletely understood. In this study, we exposed bee larvae to imidacloprid at environmentally relevant concentrations of 0.7, 1.2, 3.1, and 377 ppb. There was a marked dose-dependent delay in larval development, characterized by reductions in body mass, width, and growth index. However, imidacloprid did not affect larval survival and food consumption. The primary toxicological effects induced by elevated concentrations of imidacloprid (377 ppb) included inhibition of neural transmission gene expression, induction of oxidative stress, gut structural damage, and apoptosis, inhibition of developmental regulatory hormones and genes, suppression of gene expression levels involved in proteolysis, amino acid transport, protein synthesis, carbohydrate catabolism, oxidative phosphorylation, and glycolysis energy production. In addition, we found that the larvae may use antioxidant defenses and P450 detoxification mechanisms to mitigate the effects of imidacloprid. Ultimately, this study provides the first evidence that environmentally exposed imidacloprid can affect the growth and development of bee larvae by disrupting molting regulation and limiting the metabolism and utilization of dietary nutrients and energy. These findings have broader implications for studies assessing pesticide hazards in other juvenile animals”

Q2: Line 55-58: rephrase the sentences to make it clear that imidacloprid was not created in 1925, but only in the 90's.

Thank you for pointing out this error. We have corrected the citation. Please see the line 58 in our revised version.

Q3: Line 88: typo: " remain to be systematically investigated"

Thank you for pointing out this error. We have rewritten the sentence. Please see lines 121-122 in our revised manuscript.

Q4: Introduction is lacking important citations, a few of the important ones are: Farooqui 2013 (doi: 10.1016/j.neuint.2012.09.020.) - hypothesis linking neonic exposure, nAChRs receptors, and ROS in honeybees; Ihara et al 2020 (https://doi.org/10.1073/pnas.2003667117) - the targets of imidacloprid in honeybees; Martelli et al 2020 (https://doi.org/10.1073/pnas.2011828117) - mechanistic investigation of imidacloprid sublethal damage in Drosophila; Whitehorn et al 2018 (doi: 10.7717/peerj.4772) - investigation of imidacloprid sublethal dose impact on growth and development of butterflies; Chen et al 2021 (doi: 10.3390/ijms222111835) - sublethal effects of imidacloprid exposure on gene expression in honeybees at different life stages. It is important that the authors perform a more complete literature search to compare their work to previous ones, drawing conclusions and highlighting their novelties.

We greatly appreciate your insightful comments and valuable suggestions. Following your suggestions, we have made significant revisions to the structure and content of the Introduction section. We have incorporated the critical literature you provided and other relevant literature reviews, with a particular emphasis on studies of bees, fruit flies, and other insects. These revisions aim to improve the coherence, clarity, background, and significance of the Introduction. We hope that these modifications meet with your approval. Please see the red text in the Introduction section in our revised version.

Q5: Line 104: Explanation on the doses used should be included here, not later in the methods. Also, important to highlight that whereas the doses tested were found in bee products, they likely mean that the bees themselves were being exposed to even higher doses.

Thank you for your comment. Following your suggestions, we have moved the explanation of the imidacloprid doses used in this study to the Results section, as you mentioned. Please see lines 138-142 in our revised manuscript.

Q6: Line 112: It is important to identify the neuronal targets of imidacloprid in honeybees. Many are known. Some of the nAChRs targets were not investigated in this study (such as subunit alpha8 and beta1). Plus, is alpha2 an imidacloprid target? How does the expression of other nAChRs subunits compares? Importantly, these genes are expressed mostly in the nervous system, so a more correct approach would be a tissue specific analysis. The lack of tissue specific analysis is a consistent flaw throughout the methodological design.

Thank you very much for your important comment. Bees have more than ten nAChR subunit members. Imidacloprid inhibits acetylcholinesterase activity by competitively binding to acetylcholinesterase receptors. As you noted, this study did not investigate the expression of all nAChR subunits, including the alpha8 and beta1 subunits, in different tissues, which is a shortcoming of our study. We have always failed to make a technological breakthrough and cannot dissect to obtain important tissues from developing larvae alone. We have therefore had to abandon this design and use the whole larva as a sample for measurement. We are aware that this is a shortcoming of this research. In the future, we will make a breakthrough in technology and conduct a comparative analysis of all nAChR subunit genes in different organizations and developmental stages to obtain more comprehensive and accurate data. Thank you again for raising this important issue and for your valuable suggestions.

Q7 ~ Q9: Line 125: P450s expression may have opposite behavior when exposed to insecticides depending on tissue (such as brain and fat body). When checking whole larva gene expression, the tissue specific profiles become diluted and thus less reliable (for reference, check: https://doi.org/10.1073/pnas.2011828117); Line 131: Again, for the analysis of oxidative stress it would be important to investigate a tissue specific expression pattern and measurement of ROS markers. Investigating different time points during the exposure also adds to the mechanistic understanding. Do all tissues respond in the same way? In which tissue does an increase in ROS generation start? How? Does it spread to other tissues? By which mechanisms is it generated; Results in general: Tissue specific analyses and more time points can provide a better understanding of how sublethal imidacloprid doses impact growth and survival. Thinking about the doses of choice in light of what bees might be exposed is also important. The mechanistic understanding is missing in the paper, and without it the study does not add much in comparison to previous ones.

Thank you very much for your valuable comments. As you pointed out, the intensity of P450 detoxification and oxidative stress varies considerably between tissues. When checking whole larva gene expression, the tissue-specific profiles become diluted, which is detrimental to elucidating mechanisms. In this study, we encountered technical barriers in obtaining independent samples of specific tissues for anatomical sampling. As a result, we had to forego analysis of some specific tissues, including the tissue-differentiated analyses of P450 gene expression patterns and ROS markers that you mentioned. We only examined larval overall detoxification and antioxidant responses to imidacloprid toxicity. While we do not believe that data from specific tissues are fully representative of the complex overall picture of larvae, there is no doubt that the decision to study larvae as a whole does not contribute to our complete understanding of the mechanisms by which imidacloprid causes larval developmental retardation and larval responses to imidacloprid toxicity. In addition, the fact that this study only analyzed one-time points during imidacloprid exposure and did not design and comparatively analyze different time points limits our complete understanding of the above mechanisms. In summary, as you have pointed out, tissue-specific analyses and more time points could better understand how sublethal doses of imidacloprid affect growth and survival. In future studies, we will overcome the technical challenges and refer to your suggestions for further systematic and in-depth mechanistic studies specifically targeting imidacloprid toxicity in different tissues at different exposure times and incorporate your suggestions, such as whether the response is consistent across all tissues, the origin of the increase in ROS production, how it increases, whether it spreads to other tissues, and the underlying mechanisms into the next experimental design. Again, Thank you for your constructive and valuable comments, which have provided valuable insight for our study on mechanisms. Undoubtedly, these comments will enhance the innovativeness of our study and greatly facilitate our future research.

Q10: Line 236: The conclusion that mitochondrial dysfunction is taking place is not well corroborated. Are there changes in mitochondrial aconitase activity to suggest the mitochondrial origin of ROS? How do mitochondria look like under electron microscopy? Evidence for mitochondrial damage from functional assays? Could the ATP reduced levels be caused by increased consumption by other systems, instead of reduced production? Without functional assays to demonstrate mitochondrial dysfunction the indirect measurements of gene expression at most suggest expression perturbations in mitochondria for the point in time when gene profiles were examined.

Thank you for the comments. Based on the data of the present study, i.e., suppression of mitochondrial oxidative phosphorylation (COX17, NDUFB7) and expression of genes of its alternative glycolytic pathways (Gapdh, Oscillin), as well as a decrease in the ATP content, suggests that imidacloprid exposure leads to impaired energy metabolism in larvae and not to mitochondrial dysfunction. We have corrected this uncritical language presentation error. Please see the lines 267 and 275 red text in our revised version. We hope that this correction will meet with your approval.

Q11: Though not the aim of the study, an important step forward would be to investigate whether these doses that do not impact survival but cause growth retardation could affect the many stereotypical behaviors displayed by the worker bees when they reach the adult life. Without this sort of analysis, it is difficult to stablish whether the doses tested will impact the colony health.

Thank you very much for your valuable suggestions, which give us broader ideas for our subsequent, more in-depth work on the mechanism of toxicity. Inspired by your suggestion, we plan to conduct further studies to investigate the effects of different levels of imidacloprid exposure on the developmental process of bee larvae and the underlying mechanism of toxicity. We will also investigate the intrinsic link between this juvenile toxicity and behavioral and physiological defects in adult individuals.

Q12: Line 376: the authors do not provide a link to their hypothesis that increased P450, and antioxidant response is reducing larvae nutrient supply.

Thank you for your comment. I apologize for not fully understanding your point. If you mean that the hypothesis proposed in this study that increased P450 and antioxidant responses reduce larval nutrient energy supply is not well-founded, we have already addressed this in the previous paragraph. See Figure 7 and lines 395-399 for more details in our revised manuscript.

Q13: Line 393: Were the colonies single-cohort? Were the frames from different hives mixed together to create the experimental groups? Or each experimental group comes from a different frame/colony? This information is important to establish how much genetic variation might exist between the different experimental groups.

Thank you for your comment. In this study, the selected colonies were healthy and not exposed to pathogens or pesticides. Two-day-old larvae from the same frames of the same hive were individually transferred to sterile 24-well cell culture plates. The plates contained a standard diet containing royal jelly, glucose, fructose, water, and yeast extract. We have included the above text in our revised manuscript. Please see the lines 430-432 red text in the revised manuscript.