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

Abstract

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 Ca(2+) influx of hemocytes induced by destruxins A and B (DA and DB) was recorded. The DA/DB-dependent Ca(2+) influx was not influenced by the Ca(2+) channel inhibitors 2-aminoethoxydiphenyl borane (2-APB) and U73122. It also had an apparently different LSCM profile from that of the ionomycin-dependent Ca(2+) influx. However, the instant Ca(2+) influx was not seen in the SL-1 cells; on the contrary, a slow, moderate enhancement of intracellular Ca(2+) was observed. Meanwhile, an instant intracellular free H(+) decrease aroused by DA and DB was found. DB at 20 μmol/L and DA at 690 μmol/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 Ca(2+) influx is perhaps not related to Ca(2+) channels and ionophores; rather, the intracellular free H(+) decrease might be due to V-ATPase inhibition.

Interactions of Destruxin A with Silkworms’ Arginine tRNA Synthetase and Lamin-C Proteins

“…In BLI assays, the results indicated that the responses of BmArgRS and BmLamin-C proteins were positively correlated with DA concentrations. There were affinity constant (K D ) values of 5.53 × 10 −5 and 8.64 × 10 −5 M respectively for DA with BmArgRS and BmLamin-C (Table 1, Figure 1B). However, the results showed that BmLamin-C is not a DA-binding protein, because a significant correlation between DA concentration and response of BmPRP1 was not detected.…”

“…For this, it is necessary to elucidate the targets of DA acting on insects. In the past decade, several studies indicated that DA changes the morphology of hemocytes and brings on equilibrium chaos of intra-and extra-cellular hydrogen and calcium ion in Bombyx mori [4,5], and regulates immune related gene expression [6,7]. Recently, a few DA binding-proteins in silkworm were found [8][9][10].…”

COMPARATIVE PROTEOMIC ANALYSIS OF Bombyx mori HEMOCYTES TREATED WITH DESTRUXIN A

“…The expression of antimicrobial peptides was also reduced after Drosophila melanogaster were treated with DA (Pal et al., ). Destruxin also acts as a kind of calcium ionophore and an inhibitor of V‐H + ‐ATPase (Bandani et al., ; Chen et al., ). Other studies have reported that destruxins damage the organs and tissues of insects, including the midgut, Malpighian tubules, salivary glands, and muscle (Kershaw et al., ; Sowjanya Sree et al., ).…”

The Effects of Destruxin A on Relish and Rel Gene Regulation to the Suspected Immune-Related Genes of Silkworm

“…Destruxin A (DA), the common analogue of entomopathogenic fungus, Metarhizium anisopliae , has substantial insecticidal activity [ 1 , 2 , 3 ]. Research has shown that DA can destroy an insect’s innate immune system, which includes breaking the balance between calcium ion and hydrogen ion in hemocytes [ 4 ], affecting the function of phagocytosis and encapsulation in hemocytes [ 5 ] and inhibiting the biosynthesis of the antibacterial peptides in drosophila [ 6 ]. Our previous research suggests that, although relatively large doses of DA is required to kill the silkworm ( Bombyx mori ) Bm12 cell, smaller dosages are sufficient to cause morphological changes [ 7 ].…”