A Comparative Study Of Digestive Poisons Periplocoside P And CelangulinⅤ On Midgut Cell Transmembrane Potential Ofmythimna Separata Larvae

Pesticides which target on the insect digestive system are mainly destroyed the gut structures or influenced the digestive enzymes. Preliminary studies suggest that periplocoside P(PSP) and celangulin V(CV) act on the midgut tissues of lepidopteran larvae, and there are some binding sites of compounds on the midgut cell membranes of Mythimna separata larvae.It is well known that the physiology of the larval midgut epithelium of lepidopteran insects is characterized by a strong active transport of K+ from haemolymph to lumen. This activity,which is generally thought to be mediated by a vacuolar-type H+-ATPase coupled with an electrogenic K+/H+ exchanger, both located in the apical membrane of goblet cells, maintains a large potential difference across the epithelium. In the present study, we established a standard microelectrode technique to measure the membrane potential in freshly isolated midguts from lepidopteran larvae. Then we measured the membrane potential in freshly isolated midguts from M. separata larvae to analyze PSP and CV how to influence transmembrane potentials, compared with Bacillus thuringiensis toxin Cry1 Ab, inactive periplocoside E(PSE) and celangulin V-MIA(CV-MIA). The main results are as follows:1. Establishing of stable experimental system. In vivo measurement showed midgut cell transmembrane potential was completely abolished at 4 h after ingestion of 1 μg activated Cry1 Ab. In vitro, activated Cry1 Ab caused a distinct concentration-dependent depolarization of the apical membrane. The Vam values were deplorized by 50% after 14.7 ± 0.2, 9.8 ± 0.4and 7.6 ± 0.6 min at 1, 5 and 10 μg/mL, respectively. The concentrations of Cry1 Ab had no significant effect on the basolateral side of the epithelium. Our results were consistent with the published values of lepidopteran larvae, which indicated that the experimental system was established.2. The Vam values of M. separata larvae treated with 50 μg PSP were rapidly deplorization with time, the Vam was deplorized to-25.1±7.2 mV after 6 h, and the midgut cell transmembrane potential was completely abolished. The response tendency of PSP wassimilar to that of Cry1 Ab acting on Vam. However, PSE had not any effects on Vam and Vbm of M. separata larvae. In vitro, PSP caused a distinct concentration-dependent depolarization of the apical membrane, but the tendency was slowing compared with 5 μg/mL Cry1 Ab.Alternatively, the Vbm of larvae had no any significant changes between treated and untreated with PSP and PSE.3. M. separata larvae were treated with 25 μg CV, the Vam of control was stable at different period, but Vam of treatment showed a time-dependent depolarization to-35.5±10.2mV after 8 h and had no effects on Vbm. However, there has no effect on Vam and Vbm after treated with CV-MIA. In vitro, 0.333 mg/mL inactive CV-MIA had no effect on Vam, but CV caused a concentration-dependent depolarization. Both compounds had no effects on Vbm.Based on PSP and CV have significant effects on the larval midgut apical membrane of M. separata, we may speculate V-type H+ ATPase of the midgut goblet cells in lepidopteran insects is the most important target of insecticidal PSP and CV. PSP and CV are act on V-type H+ ATP enzyme, leading to K+ and H+ transport failure, dysfunction. It may cause the electrochemical gradient to collapse, thus abolishing the capacity of the cells to transport solutes and allowing disequilibrium of the pH between the cytoplasm and the highly alkaline content of the lumen, finally leading to disruption of midgut epithelium and larvae death.