Effects of destruxins on free calcium and hydrogen ions in insect hemocytes

Destruxins, cyclohexadepsipeptidic mycotoxins isolated from the entomopathogenic fungus Metarhizium anisopliae, inhibit innate insect immunity. However, their mechanism of action remains unclear. In this study, the effects of destruxins on changes in free calcium and hydrogen ions in the hemocytes of Exolontha serrulata, Bombyx mori and the Spodoptera litura SL-1 cell line were detected using laser scanning confocal microscopy (LSCM). An instant Ca2+ influx of hemocytes induced by destruxins A and B (DA and DB) was recorded. The DA/DB-dependent Ca2+ influx was not influenced by the Ca2+ channel inhibitors 2-aminoethoxydiphenyl borane (2-APB) and U73122. It also had an apparently different LSCM profile from that of the ionomycin-dependent Ca2+ influx. However, the instant Ca2+ influx was not seen in the SL-1 cells; on the contrary, a slow, moderate enhancement of intracellular Ca2+ was observed. Meanwhile, an instant intracellular free H+ decrease aroused by DA and DB was found. DB at 20 mol/L and DA at 690 mol/L significantly reduced intracellular free H+ levels. Furthermore, the vacuolar H+-ATPase (V-ATPase) inhibitor bafilomycin A1 had obvious effects on the decreases of intracellular free H+ in hemocytes. These results suggest that the mechanism of DA/DB-dependent Ca2+ influx is perhaps not related to Ca2+ channels and ionophores; rather, the intracellular free H+ decrease might be due to V-ATPase inhibition.