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

  1. Zhi Li
  2. Yuedi Wang
  3. Qiqian Qin
  4. Lanchun Chen
  5. Xiaoqun Dang
  6. Zhengang Ma
  7. Zeyang Zhou

  • Curated by eLife

    eLife logo

    eLife assessment

    This investigation of the changes in gene expression and some of the physiological consequences of sublethal exposures to the neonicotinoid insecticide imidacloprid in honeybee larvae is useful, although numerous experiments were not considered based on technical issues. The methodological design leads to concerns and it is therefore not obvious that all conclusions are justified. The study adds to our understanding of how this insecticide impacts development and growth of honeybees, but the evidence supporting the major claims is incomplete.

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

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 on 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.

Article activity feed

  1. Version published to 10.7554/elife.88772.4 on eLife
  2. Version published to 10.7554/elife.88772 on eLife
  3. Version published to 10.7554/elife.88772.3 on eLife
  4. eLife

    Author Response

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

    On behalf of all the authors, I'd like to thank you for your insightful comments and valuable suggestions, which fully reflect your high level of scientific thinking and point the direction of our research and help us and other future researchers in the field to more comprehensively study and interpret the toxic effects of imidacloprid on honey bee larvae and its potential mechanisms, as well as the mechanisms of larval resistance and adaptations to imidacloprid. We have addressed each of the questions and revised the manuscript point-by-point in response to your comments. Below are detailed point-by-point responses to each question.

    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.

    Recommendations for the authors:

    This study presents a useful investigation on changes in gene expression by real time PCR and some of the physiological consequences of sublethal exposures to the neonicotinoid insecticide imidacloprid in honeybee larvae. It offers preliminary evidence of imidacloprid impacts on the development of bee larvae by interfering with molting and metabolism. Whereas the study provides evidence that small doses of imidacloprid affect larval growth rate, there is no investigation on whether that could affect the overall colony health, and some of the results open the possibility that the larvae may overcome some of the impacts of the exposure. As the authors state, the doses tested show no impact on larvae survival, food consumption or time to pupation. The investigation and methodological design lack in depth to explain the findings and provide incomplete evidence to support the authors conclusions. The study would benefit from a more thorough mechanistic characterization to better sustain the findings and demonstrate their biological relevance.

    Response: I would like to express, on behalf of all the authors, our sincere appreciation for your insightful and insightful comments and suggestions, which significantly enhanced the quality of the manuscript. Your incisive insights point the way for future research in the field of bee biology on the mechanisms underlying imidacloprid-induced delays in larval development.

    In this study, we investigated the effects of imidacloprid on honey bee larval development, including macro and micro changes and possible causes. This is the first of its kind in the field of honeybee biology research. However, we found that the underlying mechanism is extremely complex. The effects of toxic substances on animals and their interactions with larval development are complex and far-reaching. They include oxidative stress and damage; disruption of nutrient metabolic homeostasis; inhibition of detoxification and immunity; adverse effects on the nervous, circulatory, and digestive systems; inflammation, disease, and even organ failure; and subsequent effects on physiological activities such as development, reproduction, and behavior, and even death. These toxic effects interact in complex ways with the development of young animals, with some effects directly or indirectly affecting development while others do not.

    Addressing this complex mechanistic issue based solely on the results of this study is a formidable challenge, which leads to some limitations of our study as pointed out by the reviewers. Although our study is not comprehensive enough in terms of mechanistic analysis and does not fully elucidate the mechanism, we believe it is an important and valuable first step in this area.

    In the future, we will follow the reviewers' suggestions and deliberately redesign the experiments to focus on further research on the issues they raised. These include examining the effects of larval developmental delay on adult and colony health, investigating the post-pupal situation, identifying the source of ROS, and determining whether the larval gut damage observed after pupalization recovers.

    In accordance with the reviewers' comments and suggestions, we have revised the manuscript to improve its rigor and scientific quality. We sincerely ask the reviewers to understand and accept this modification from us!

    Next is our response to each of the questions and valuable suggestions provided by reviewers:

    Recommendations For The Authors:

    1. The authors found a reduction in growth index and body mass, but document no impact on survival, food consumption or time to pupariation. How much exactly is the reduction in growth index? It seems to be from ~0.96 to ~0.93. Is this biologically relevant? Would that be enough to impact the colony health?

    Response: Thank you for your comments. In this study, we observed a gradual decrease in larval growth index from day 4, which stabilized by day 6. At the 4th, 5th and 6th instars, the growth index of the imidacloprid-treated groups were significantly lower than those of the control group by an average of 1.35%, 4.49% and 2.76%, respectively (Figure 1, source data 8). Statistical analysis confirmed the significance of the difference in these results. We have incorporated the above description into the red text on lines 148-152 of the Results section. Regarding the reviewer's inquiry on colony health, including imidacloprid-induced delayed larval development and some reduction in growth index and body weight with no effect on survival, food consumption, or time develop to pupation, because we do not currently have the technical capabilities to culture larvae to adulthood in laboratory incubators, this has resulted in a failure to further investigate the effects of imidacloprid-induced delayed larval development on adult colony health. However, this is a very important scientific question for future colony health. We will design experiments to address this issue in a follow-up study.

    1. The authors find that P450s can help in detoxifying mechanisms to mitigate imidacloprid impacts. That however is a well-known fact. What is new about this claim?

    Response: The point at which the ability to detoxify toxic substances is acquired during early development varies widely among animals. Although many studies have reported that the detoxification function of P450s helps mitigate the effects of imidacloprid in adult honey bees, there is no conclusive evidence as to whether or not honey bee larvae have acquired this ability at early stages of development. This ability is critical to the defense and health of honey bee larvae. Therefore, it is incumbent upon this study to clarify this issue, which is important in explaining the effects of imidacloprid on honey bee larvae.

    1. Some references are cited incorrectly. The first and last name are swapped, for instance Charles et al.

    Response: Thank you very much for pointing out this error, which we have corrected. Please see lines 92 and 889 in our revised version.

    1. I still encounter important methodological flaws. The authors acknowledge my previous suggestions but only address a small fraction of them. The most relevant points regarding the understanding of the mechanisms behind the delayed growth rate remain unexplored. The expression levels of other nAChRs target of imidacloprid in honeybees were not investigated. The expression analyses are still based on a single time point and using whole larvae, which only superficially explore the problem and may lead to misinterpretations. I do not understand the authors claim that a technological breakthrough is required to address these issues, when performing more PCRs and doing dissections should cover the matter.

    Response: Thank you very much for your important comment. You point out several unexplored issues related to understanding the mechanisms behind delayed growth rates. For example, The most relevant points regarding the understanding of the mechanisms behind the delayed growth rate remain unexplored. The expression levels of other nAChRs target of imidacloprid in honeybees were not investigated. The expression analyses are still based on a single time point and using whole larvae. Please allow me to explain. Honeybees (Apis mellifera) have nine different α-subunits, Amelα1-9, and two β-subunits, Amelβ1-2. Amelα5, Amelα7, and Amelα8 are expressed in MB Kenyon cells and AL neurons, and the Amelβ2 subunit is present in Kenyon cells. Amelα2, Amelα3, and Amelα7-2 are expressed in the optic lobes. The aim of this study was to investigate whether imidacloprid induces larval neurotoxicity. Based on the above information, we selected the two most representative nAChRs (Alph1 and Alph2) for analysis. The results showed that exposure to imidacloprid increased the expression of the Alph2 gene and inhibited AChE activity, indicating that imidacloprid is neurotoxic to larvae. This result answered our question of whether imidacloprid induces neurotoxicity in larvae. Therefore, we did not further analyze the expression levels of other nAChRs. We believe that this does not affect the understanding of the mechanism behind the delayed growth rate and that it is not necessarily necessary to analyze all 11 nAChRs to find an answer. We sincerely hope that the reviewers will understand and agree with this.

    Furthermore, regarding the expression analysis based on a single time point and whole larvae. In this study, 72 h after imidacloprid exposure Fig. 1J, 5 days of age) was chosen for sampling because this is when imidacloprid has the greatest and most representative effect on larval development. Therefore, analyzing samples at this time point did not interfere with our exploration of the mechanisms by which imidacloprid causes larval developmental retardation. We used whole larvae rather than individual tissues for sample selection, which is a shortcoming for us. This was mainly due to technical challenges where we were unable to obtain pure single tissues through dissection. Nevertheless, we will make technical breakthroughs in the future so that we can sample and compare different tissues and developmental stages to obtain more comprehensive and accurate data. Thank you again for raising this important issue and for your valuable suggestions.

    1. The authors could in many different ways explore what are the origin of ROS is. That is important to further develop their hypothesis on reduced energy levels.

    Response: Thank you very much for your insightful comment and suggestion, it gives us great insight. Mitochondria are the main producers of ATP for cellular metabolism, accounting for approximately 90% of the total. However, mitochondria are also involved in the generation of reactive oxygen species (ROS). Excessive accumulation of ROS in mitochondria leads to oxidative stress, which in turn damages mitochondria and further increases ROS levels, creating a vicious cycle (Boovarahan and Kurian, 2018). In the present study, it was found that imidacloprid exposure led to increased ROS and MDA levels in larvae (Figure 5A and Figure 5-source data 14), indicating that imidacloprid induced severe oxidative stress and lipid damage, which may damage mitochondria and in turn affect mitochondrial ATP production, resulting in insufficient energy supply for larval development. This factor may also be an important explanation for the larval developmental delay caused by imidacloprid. We have included the above text in our revised manuscript. Please see the lines 432-442 in the revised manuscript.

    1. If there is gut damage, is it restored in the adults? It is not clear from the methods which precise stage of larval development was used for gut preparations. That information is important because prior to pupation larvae defecate for the first time and undergo shedding of the gut lining. That could profoundly affect some of the results in case gut preparations for microscopy were made close to this stage. If no food residues are found in the gut of control larvae, does it mean that they are close to pupation? Could the apoptosis found in gut of exposed larvae be the natural shedding of gut lining prior to pupation? All these possibilities have to be discussed and authors should clarify the precise larval stage used in every assay.

    Response: Thank you for your important comments. In this study, all samples used for the assay were larvae that had developed to 5-day-old after oral administration imidacloprid at 2-day-old. This is described in detail in the Materials and Methods. See lines 507, 517-521 in the revised manuscript. In general, 6-day-old bee larvae cease feeding and begin their first defecation at approximately 7-day-old. However, in our study, intestinal sections were prepared from 5-day-old larvae that had not fasted or defecated, when the intestinal mucosa was normal and not undergoing shedding. In this case, we found that imidacloprid caused damage to intestinal structures, apoptosis of intestinal cells, incomplete formation of the peritrophic membrane, and undigested food residues in the intestine. We believe that these results are objective and reliable.

    1. Honeybee have 5 larval instars, not 7 (Figure 1). That creates confusion about which larval stage the authors used.

    Response: Thank you very much for pointing out this editorial error, which we have corrected, please see Figure 1.

    1. The Results section does not state the numbers by which parameters measures have changed, neither the values of significance. How much is the impact in growth index, body mass, gene fold change, etc?

    Response: Thank you very much for pointing out this important problem. We have revised the Results section according to your suggestions. Please see the revised manuscript.

    1. Mention figures in order (5c comes before 5b in the text)

    Response: Thank you very much for the comment. We have revised according to your suggestions. Please see the lines 208-212 in the revised manuscript.

    1. Paraquat is a herbicide not a pesticide

    Response: Thank you for pointing out the loose wording. We have revised according to your suggestions. Please see the lines 316-319 in the revised manuscript.

    1. What is the evidence that imidacloprid reduces growth index by inhibiting 20E? The authors provide real time data and discuss the data in terms of correlation. But correlation does not mean causation. Reduction in growth index could come from multitude of factors such as ROS affecting mitochondrial energy metabolism.

    Response: We deeply appreciate your insightful comments and valuable suggestions. In this study, although we conducted an in-depth analysis of ecdysone regulation, which is crucial for insect larval development, and found some clues, as you pointed out, this is not the sole reason for larval developmental delay. In fact, animal growth and development are collectively regulated by numerous physiological, biochemical, and genetic factors. The the decline in the growth index may be due to other factors as you mentioned, such as oxidative stress impairing mitochondria, dysregulated neuro-endocrine axis caused by imidacloprid targeting neurons, poor nutrient absorption, impaired movement, etc, as animal growth and development are collectively regulated by numerous physiological, biochemical, and genetic factors. We have incorporated this understanding into the revised manuscript. Please see the lines 389-394 in the revised manuscript.

    1. The authors state that "digestion and breakdown of nutrients is impaired by imidacloprid", the evidence discussed in the paragraph however supports only that imidacloprid impairs some of the genes involved in these processes.

    Response: Thank you for your comments and valuable insights. In this paragraph, a lack of clarity and completeness in our writing may have led to the misconception that the evidence discussed only demonstrates the effects of imidacloprid on specific genes in these processes. In fact, our intent in this paragraph was to analyze and discuss the effects of imidacloprid on nutrient digestion and breakdown in larvae and to explore the causes of larval developmental delay. We demonstrated this using tissue sections, qRT-PCR and correlation analysis, which showed that the intestinal structure was disrupted and the expression of genes involved in nutrient digestion and catabolism was suppressed, resulting in defects in the catabolic utilization of food and consequently the presence of many food residues. In addition, there was a positive correlation between these genes and larval developmental delay. All this may be another important factor contributing to imidacloprid-induced larval developmental delay. We have revised and incorporate the above logic into the revised manuscript. Please see the lines 407-431 in the revised manuscript.

    1. There is no evidence for the claim that overexpressing P450s and antioxidant enzymes cause a reduction in growth index. No transcriptome analysis was performed so it is unknown under the circumstances presented here how all the other P450s, antioxidant genes and overall gene profiles are responding. Surely, some genes will be repressed. Reduction in growth index could stem from, oxidative stress impairing mitochondria, dysregulated neuro-endocrine axis caused by imidacloprid targeting neurons, poor nutrient absorption, impaired movement, etc.

    Response: Thank you for your comments and valuable insights. Indeed, as you have pointed out, drawing the conclusion that antioxidants and detoxification are significant contributors to larval developmental retardation solely based on correlation analysis is inherently flawed and lacks critical support, especially in the absence of P450 and antioxidant enzyme overexpression and comprehensive transcriptome analysis of other P450s, antioxidant genes, and the entire gene map. We have revised and included in the revised manuscript. Please see lines 461-467 in the red text in the revised manuscript. We have revised and incorporate the above logic into the revised manuscript. Please see the lines 407-431 in the revised manuscript.

    1. How come the decreased ATP and glycogen levels have no effect on time to pupation? Extra time points for gene expression, measurements of gut damage, ATP levels, ROS, etc, are vital to answer how the exposed larvae eventually catch up with the unexposed group. Also, it is vital to understand whether these larval impacts translate to impacts on adults.

    Response: We sincerely thank you for your insightful comments and suggestions! These important scientific issues you've raised are a good example of your high-level scientific thinking, and they will help us and other future researchers in the field to more comprehensively study and interpret the toxic effects of imidacloprid on honey bee larvae and their potential mechanisms, as well as the mechanisms of larval resistance and adaptation to imidacloprid. According to your comments, we will adapt our experiments and conduct more thorough research in the future to address the above issues.

    1. I am confused about the author's definition of developmental rate; rate gives the notion of speed to achieve something. But the authors use developmental rate as a measure of viability (number of larvae that successfully pupated). There seems to be a significant decrease in their developmental rate plot (Fig 1i), but at the same time the authors show in Figure 1c (and mention throughout the manuscript) that there is no difference in probability of survival. This is quite confusing and the method section regarding these data is too concise and does little to help explain what the authors were trying to measure. The whole section on developmental traits would benefit of more details on how experiments were conducted and equipment used.

    Response: Thank you so much for your valuable comments. Yes, as you can see, there appears to be a significant decrease in developmental rate but no difference in survival probability, which is an intriguing finding of this study. This finding suggests that the 377 ppb imidacloprid dose is not as harmful to the larvae as previously thought. Imidacloprid appeared to limit the larval ability to molt and develop only to a certain extent, but had no effect on the developmental process, let alone survival. It's worth investigating the underlying mechanism. As a result, we have included this question in the design of future studies. In addition, following your suggestion, we have revised the description of the material and methods in this section, including the experimental method in more detail. For more information, please see the revised manuscript, lines 530-541.

    1. The authors should try to make it clear what percentage of exposed larvae become adults? I am confused because the plot called developmental rate might be trying to convey this message, but developmental rate and viability are very distinct traits. What is the difference, if any, in the time it takes for exposed larvae to become adults in comparison to non-exposed ones? Is there a difference in adult body weight? The answers to these last two questions are important to start understanding if the impacts of imidacloprid on larvae alimentation would still impact these same individuals once they become adults, i.e., would there be impacts for the colony and workers activity?

    Response: Thank you very much for your insightful comments. Unfortunately, this is where the research falls short. Culturing larvae to adulthood in 24-well cell culture plates is a significant technical challenge that we have yet to overcome. As a result, the important questions you raise, such as what percentage of exposed larvae become adults? How does the time to adulthood differ (if at all) for exposed larvae versus non-exposed larvae? Is there a difference in adult weight? Do the effects of imidacloprid on larval feeding persist after these individuals reach adulthood? Does imidacloprid damage to larvae affect colony and adult activity? We do not have answers at this time. We are aware that answers to the above questions will help people better understand how serious the effects of imidacloprid environmental residues on honey bee larvae and adults, as well as bee colonies as a whole, are, and will draw sufficient attention to them. We intend to break through this technological bottleneck of culture larvae to adulthood in future studies and incorporate the above scientific questions into our next research design. Thank you again for your insightful comments! This gives us new research ideas.

  5. eLife

    eLife assessment

    This investigation of the changes in gene expression and some of the physiological consequences of sublethal exposures to the neonicotinoid insecticide imidacloprid in honeybee larvae is useful, although numerous experiments were not considered based on technical issues. The methodological design leads to concerns and it is therefore not obvious that all conclusions are justified. The study adds to our understanding of how this insecticide impacts development and growth of honeybees, but the evidence supporting the major claims is incomplete.

  6. eLife

    Joint Public Review:

    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 employing these different approaches to investigate the impacts of imidacloprid exposure. The study weaknesses come from the lack of a in depth investigation and drawing many conclusions solely from punctual gene expression, that are not representative of complete biological processes. Though relevant to understand the impacts on neonicotinoid contamination on insect pollinators, the study conclusions should be carefully weighted as they are often not fully substantiated. Follow up studies using in-depth investigation and more robust methodological design testing whether the impacts observed lead to post-metamorphosis effects and impacts in the colony would have a significant impact.

  7. Version published to 10.1101/2023.06.05.543803v3 on bioRxiv
  8. Version published to 10.7554/elife.88772.2 on eLife
  9. eLife

    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.

  10. eLife

    eLife assessment

    This is a useful investigation of the changes in gene expression and some of the physiological consequences of sublethal exposures to the neonicotinoid insecticide imidacloprid in honeybee larvae. While the study adds to our understanding of how this insecticide impacts development and growth of honeybees, the evidence supporting the major claims is incomplete. The study would benefit from a more thorough mechanistic characterization of the phenotypes to substantiate the conclusions.

  11. eLife

    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.

  12. Version published to 10.1101/2023.06.05.543803v2 on bioRxiv
  13. Version published to 10.7554/elife.88772.1 on eLife
  14. eLife

    eLife assessment

    This is a useful investigation of the changes in gene expression and some of the physiological consequences of sublethal exposures to the neonicotinoid insecticide imidacloprid in honeybee larvae. While the study adds to our understanding of how this insecticide impacts development and growth of honeybees, the evidence supporting the major claims is incomplete. The study would benefit from a more thorough mechanistic characterization of the phenotypes to substantiate the conclusions.

  15. eLife

    Reviewer #1 (Public Review):

    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.

  16. eLife

    Reviewer #2 (Public Review):

    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.

  17. Version published to 10.1101/2023.06.05.543803v1 on bioRxiv