Electrophysiological and pharmacological properties of the slowpoke channel in the diamondback moth, Plutella xylostella.

The slowpoke channel responds to the intracellular calcium concentration and the depolarization of the cell membrane. It plays an important role in maintaining the resting potential and regulating the homeostasis of neurons, but it can also regulate circadian rhythm, sperm capacitation, ethanol tolerance, and other physiological processes in insects. This renders it a potentially useful target for the development of pest control strategies. There are relatively few studies on the slowpoke channels in lepidopteran pests, and their pharmacological properties are still unclear. So, in this study, the slowpoke gene of Plutella xylostella (Pxslo) was heterologous expressed in HEK293T cells, and the I-V curve of the slowpoke channel was measured by whole cell patch clamp recordings. Results showed that the slowpoke channel could be activated at -20 mV with 150 µM Ca2+. The subsequent comparison of the electrophysiological characteristics of the alternative splicing site E and G deletions showed that the deletion of the E site enhances the response of the slowpoke channel to depolarization, while the deletion of the G site weakens the response of the slowpoke channel to depolarization. Meanwhile, the nonspecific inhibitors TEA and 4-AP of the Kv channels, and four pesticides were tested and all showed an inhibition effect on the PxSlo channel at 10 or 100 µM, suggesting that these pesticides also target the slowpoke channel. This study enriches our understanding of the slowpoke channel in Lepidopteran insects and can aid in the development of relevant pest management strategies.

Identification and pharmacological characterization of the voltage-gated potassium channel Shab in diamondback moth, Plutella xylostella.

BACKGROUND: Voltage-gated potassium channel Kv2 is the primarily delayed rectifier in insect nerves and muscles involved in several crucial biological processes, including action potential regulation, photoreceptor performance, and larval locomotor. It is a potential molecular target for developing a novel pesticide for mosquitos. However, there are few studies on the Kv2 channel in agricultural pests. RESULTS: The only α-subunit gene of the Kv2 channel in Plutella xylostella (L.), PxShab, was cloned, and its expression profile was analyzed. The relative expression level of PxShab was highest in the pupal stage of both sexes and male adults but lowest in female adults. Meanwhile, PxShab had the highest expression in the head in both larvae and adults. Then, PxShab was stably expressed in the HEK-293 T cell line. Whole cell patch clamp recordings showed an outward current whose current-voltage relationship conformed to a typical delayed-rectifier potassium channel. 20 µM quinidine could effectively inhibit the potassium current, while the channel was insensitive to 4-AP even at 10 mM. Several potential compounds and botanical pesticides were assessed, and carvedilol (IC50  = 0.53 µM) and veratrine (IC50  = 2.22 µM) had a good inhibitory effect on the channel. CONCLUSION: This study revealed the pharmacological properties of PxShab and screened out several high potency inhibitors, which laid the foundation for further functional research of PxShab and provides new insight into designing novel insecticides. © 2022 Society of Chemical Industry.

Isolation and yield optimization of lipopeptides from Bacillus subtilis Z-14 active against wheat take-all caused by Gaeumannomyces graminis var. tritici.

Wheat take-all, caused by the soil-borne fungus Gaeumannomyces graminis var. tritici, is one of the major constraints on wheat production worldwide. Bacillus subtilis Z-14 exerts significant biocontrol activity against wheat take-all, and lipopeptide antibiotics are the main antifungal substances. Herein, lipopeptide antibiotics C14-C15 iturin A, C14-C16 fengycin A, and C15-C17 fengycin B from B. subtilis Z-14 culture filtrates were separated and identified by high-performance liquid chromatography, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and mass spectrometry/mass spectrometry, respectively. The optimal medium components for Z-14 lipopeptide antibiotic production were 3.85 g/L corn flour, 1.57 g/L soybean meal, 0.03 g/L FeSO4 ·7H2 O, 0.2 g/L NaH2 PO4 ·2H2 O, and 0.4 g/L Na2 HPO4 ·2H2 O. Quantification analysis by high-performance liquid chromatography showed that fengycins played a main role in antifungal activity against Gaeumannomyces graminis var. tritici. Quantitative reverse transcription polymerase chain reaction showed that lipopeptide synthesis genes fenD and ituC reached maximum expression levels after 48 h of fermentation. The strongest control of wheat take-all by Z-14 was achieved by adding 30 mL of culture filtrate per 350 g of soil in pot experiments, during which disease reduction reached 88.15%. This study provides theoretical support and a material basis for the prevention and treatment of wheat take-all disease.