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

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.

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.