Purification, characterization, and sensitivity to pesticides of carboxylesterase from Dendrolimus superans (Lepidoptera: Lasiocampidae).

Through a combination of steps including centrifugation, ammonium sulfate gradient precipitation, sephadex G-25 gel chromatography, diethylaminoethyl cellulose 52 ion-exchange chromatography and hydroxyapatite affinity chromatography, carboxylesterase (CarE, EC3.1.1.1) from sixth instar larch caterpillar moth, Dendrolimus superans (Lepidoptera: Lasiocampidae) larvae was purified and its biochemical properties were compared between crude homogenate and purified CarE. The final purified CarE after hydroxyapatite chromatography had a specific activity of 52.019 µmol/(min·mg protein), 138.348-fold of crude homogenate, and the yield of 2.782%. The molecular weight of the purified CarE was approximately 84.78 kDa by SDS-PAGE. Three pesticides (dichlorvos, lambda-cyhalothrin, and avermectins) showed different inhibition to crude CarE and purified CarE, respectively. In vitro median inhibitory concentration indicated that the sensitivity of CarE (both crude homogenate and final purified CarE) to pesticides was in decreasing order of dichlorvos > avermectins > lambda-cyhalothrin. By the kinetic analysis, the substrates alpha-naphthyl acetate (α-NA) and beta-naphthyl acetate (ß-NA) showed lesser affinity to crude extract than purified CarE. The results also indicated that both crude homogenate and purified CarE had more affinity to α-NA than to ß-NA, and the Kcat and Vmax values of crude extract were lower than purified CarE using α-NA or ß-NA as substrate.

Steroid hormone 20-hydroxyecdysone disturbs fat body lipid metabolism and negatively regulates gluconeogenesis in Hyphantria cunea larvae.

The steroid hormone 20-hydroxyecdysone (20E) has been described to regulate fat body lipid metabolism in insects, but its accurate regulatory mechanism, especially the crosstalk between 20E-induced lipid metabolism and gluconeogenesis remains largely unclear. Here, we specially investigated the effect of 20E on lipid metabolism and gluconeogenesis in the fat body of Hyphantria cunea larvae, a notorious pest in forestry. Lipidomics analysis showed that a total of 1 907 lipid species were identified in the fat body of H. cunea larvae assigned to 6 groups and 48 lipid classes. The differentially abundant lipids analysis showed a significant difference between 20E-treated and control samples, indicating that 20E caused a remarkable alteration of lipidomics profiles in the fat body of H. cunea larvae. Further studies demonstrated that 20E accelerated fatty acid ß-oxidation, inhibited lipid synthesis, and promoted lipolysis. Meanwhile, the activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase were dramatically suppressed by 20E in the fat body of H. cunea larvae. As well, the transcriptions of genes encoding these 4 rate-limiting gluconeogenic enzymes were significantly downregulated in the fat body of H. cunea larvae after treatment with 20E. Taken together, our results revealed that 20E disturbed fat body lipid homeostasis, accelerated fatty acid ß-oxidation and promoted lipolysis, but negatively regulated gluconeogenesis in H. cunea larvae. The findings might provide a new insight into hormonal regulation of glucose and lipid metabolism in insect fat body.