Imidacloprid causes larval developmental retardation in Apis mellifera. Larvae were orally administered imidacloprid (IMI) or artificial food (CK) beginning at three-day-old, followed by developmental monitoring for 96 hours. (A) Larval developmental phenotypes at 0.7, 1.2, 3.1, and 377 ppb environmental concentrations of imidacloprid exposure; (B) Larval developmental phenotype at 377 ppb imidacloprid exposure; (C) Effect of 0.7, 1.2, 3.1, and 377 ppb imidacloprid on larval survival; (D) and (E) body width and weight of larvae exposed to 0.7, 1.2, 3.1, and 377 ppb imidacloprid for 96 hours; (F) and (G) statistics for daily (F) and three-day cumulative feeding (G) for larvae exposed to 377 ppb imidacloprid; (H) larval development time from three-day-old to pupal stage; (I) The number of larvae that successfully reached the pupal stage as a percentage of the total initial sample size; (J) The growth index of larvae. Statistical significance was set at *P < 0.05 and **P < 0.01.

Imidacloprid neurotoxicity in Apis mellifera larvae. (A) AChE activity and (B) nerve conduction-related gene expression analysis in larvae exposed to 377 ppb imidacloprid for 72 hours or a control group. IMI is the larvae exposed to imidacloprid for 72 hours. CK is the artificial food control group. Statistical significance was set at *P < 0.05 and **P < 0.01.

Effects of imidacloprid on the homeostasis of developmental regulation in Apis mellifera larvae. (A) Relative gene expression and (B) hormone levels of developmental regulatory-related genes in larvae exposed to 377 ppb imidacloprid for 72 h or a control group. Statistical significance was set at *P < 0.05 and **P < 0.01.

Imidacloprid induced a detoxification response in Apis mellifera larvae. Relative expression levels of cytochrome P450 monooxygenase family genes in larvae exposed to imidacloprid for 72 hours. IMI is the larvae exposed to imidacloprid for 72 hours. CK is the artificial food control group. Statistical significance was set at *P < 0.05, **P < 0.01, and ***P < 0.001.

Imidacloprid exposure causes oxidative stress, gut apoptosis, and tissue structural damage while inducing antioxidant defenses in Apis mellifera larvae. (a) Analysis of oxidative stress and damage in larvae exposed to imidacloprid for 72 hours. ROS levels indicate the degree of oxidative stress. MDA is a marker for lipid peroxidation. PCO levels reflect the extent of protein carbonylation damage; (b) Activity of antioxidants in larvae exposed to imidacloprid for 72 hours; (c) The relative expression levels of antioxidant genes in larvae exposed to imidacloprid for 72 hours; (d) Histological sections of the larval gut stained with H&E. The circled inserts in the figure show magnified views of the muscle layer. The blue arrow indicates the cell nucleus of the muscle layer. The boxed inset in the figure is a magnified view of the basal layer (black arrow), peritrophic membrane (red arrow), and food residues (green arrow). IMI is the larvae exposed to imidacloprid for 72 hours. CK is the artificial food control group. Statistical significance was set at *P < 0.05, **P < 0.01, and ***P < 0.001.

Imidacloprid toxicity inhibited the expression of genes involved in nutrient catabolism, protein synthesis, and energy metabolism and reserves. (A) Carbohydrate catabolism; (B) Proteolysis; (C) Amino acid transport; (D) Mitochondrial oxidative phosphorylation; (E) Glycolysis; (F) Protein synthesis. (G) The total contents of ATP, glycogen (H), and protein (I). IMI is the larvae exposed to imidacloprid for 72 hours. CK is the artificial food control group. Statistical significance was set at *P < 0.05, **P < 0.01, and ***P < 0.001.

Correlation between phenotypes and molecular characteristics in imidacloprid-exposed larvae with developmental retardation. Data were normalized using GraphPad software, followed by a Pearson correlation coefficient calculation using SPSS, and a heatmap was generated.

A model interpreting the ecotoxicological effects of imidacloprid on Apis mellifera larvae and the mechanisms of imidacloprid-induced developmental retardation. Imidacloprid toxicity caused growth and developmental delay in A. mellifera larvae. It disrupts the regulatory balance of molt development, resulting in restricted development. In addition, the damage caused by imidacloprid restricts the metabolism and utilization of food nutrients and energy in larvae. The additional energy consumed by larval P450 detoxification and antioxidant defenses further reduces the supply of nutrients and energy supply for growth and development. The arrows indicate induction or promotion, the straight lines indicate inhibition, and the green dotted lines indicate speculative inhibition.