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Background GC16 is a novel pesticide with acaricidal properties against the spider mite Tetranychus pueraricola (Ehara & Gotoh). Its physiological mechanisms have been described previously, but its molecular mechanisms of action remain unclear. Thus, we aimed to explore the acaricidal mechanisms of GC16 through transcriptomic and proteomic analyses. The results were verified using transmission electron microscopy (TEM), immunofluorescence assay, and western blotting. Results Transcriptomic and proteomic analyses revealed 2717 differentially expressed genes (DEGs) and 374 differentially expressed proteins (DEPs) between the GC16-treated and control mites. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the DEGs and DEPs were enriched in the autophagy pathway. TEM showed that the number of autophagosomes and autolysosomes was higher in the GC16-treated mites than in the control mites. Immunofluorescence assay and western blot results consistently indicated that GC16 treatment significantly enhanced the relative expression of the autophagy protein LC3 in insect Sf9 cells. The intracellular calcium concentration in the GC16-treated Sf9 cells was 2.30 times higher than that in the control cells, suggesting that GC16 disrupted calcium homeostasis and potentially acted as a calcium-driven nerve agent. Conclusions Autophagy is involved in the toxicity of GC16 against T. pueraricola and may be activated by elevated Ca 2+ levels. This study reveals the molecular insecticidal mechanisms of GC16 and provides rationale for the field application of GC16 to control pest mites.

The spider mite, Tetranychus pueraricola (Ehara & Gotoh; Acari: Tetranychidae), is a serious pest in agriculture and horticulture. Application of chemical pesticides is the main mode of this pest control. Due to pesticide residues and resistance-induced resurgence of pests, there is a need to discover alternatives for spider mite management. GC16 comprises a mixture of calcium chloride (CaCl2, 45%) and lecithin (55%), which was recently found to have acaricidal properties. We evaluated the sublethal effects of GC16 on T. pueraricola using life table and enzyme [catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), carboxylesterase (CarE), glutathione S-transferases (GST), and Ca2+-ATPase (Ca2+-ATP)] activity assays. The results showed that fecundity of T. pueraricola increased at LC30 but decreased at LC50 of GC16. The intrinsic rate of increase (r) of T. pueraricola decreased under the LC30 and LC50 of GC16. GC16 concentration and exposure time significantly influenced the activities of CAT, POD, CarE, GST, and Ca2+-ATP in adult mites. Twelve hours later after the treatment, GST and Ca2+-ATP activities were significantly inhibited by LC30 but enhanced by LC50. Moreover, the demographic parameter r and enzyme activities were negatively correlated. In sum, sublethal amounts of GC16 had an adverse effect on mites, and there was a trade-off between developmental performance and physiological enzyme activity of mites under GC16 stress, and GC16 showed an acaricidal potential for T. pueraricola. This work provides guidance for the application of GC16 to control T. pueraricola. Graphical Abstract

Chemical control plays a crucial role in pest management but has to face challenges due to insect resistance. It is important to discover alternatives to traditional pesticides. The spider mite Tetranychus pueraricola (Ehara & Gotoh) (Acari: Tetranychidae) is a major agricultural pest that causes severe damage to many crops. GC16 is a new agent that consists of a mixture of Calcium chloride (CaCl2) and lecithin. To explore the acaricidal effects and mode of action of GC16 against T. pueraricola, bioassays, cryogenic scanning electron microscopy (cryo-SEM) and transmission electron microscopy (TEM) were performed. GC16 had lethal effects on the eggs, larvae, nymphs, and adults of T. pueraricola, caused the mites to dehydrate and inactivate, and inhibited the development of eggs. GC16 displayed contact toxicity rather than stomach toxicity through the synergistic effects of CaCl2 with lecithin. Cryo-SEM analysis revealed that GC16 damaged T. pueraricola by disordering the array of the cuticle layer crest. Mitochondrial abnormalities were detected by TEM in mites treated by GC16. Overall, GC16 had the controlling efficacy on T. pueraricola by cuticle penetration and mitochondria dysfunction and had no effects on Picromerus lewisi and Harmonia axyridis, indicating that GC16 is likely a new eco-friendly acaricide.