Effects of the cyclopeptide mycotoxin destruxin A on the Malpighian tubules of Rhodnius prolixus (Stål)

Abstract

The production of peptide toxins by entomopathogenic fungi during the infection process plays critical roles in pathogenesis. To gain insight into the mechanism of action of these mycotoxins on insect internal organs, we have evaluated the effects of destruxin A, a cyclic peptide produced by Metarhizium anispliae, on Rhodnius prolixus Malpighian tubules measuring fluid secretion rate, transepithelial electrical potential (TEP), pH and ion composition of secreted fluid, and ATP content. Destruxin A dramatically inhibited fluid secretion rate on tubules stimulated by 5-Hydroxytryptamine (5-HT) or cAMP. The calculated IC(50) for destruxin A on 5-HT-stimulated tubules was 3 x 10(-7) M. Fluid secretion rate by Malpighian tubules exposed for 20 min to 10(-6) M destruxin A recovered completely when tubules were washed with saline; however, when tubules were exposed to 5 x 10(-6) M destruxin A the fluid secretion rate only partially recovered upon wash off. The use of Ca(2+)-free saline or addition of the calcium channel blocker CoCl(2) to the bathing saline did not interfere with the effects of destruxin A, and neither did the modification of intracellular calcium by TMB-8. Measurement of TEP of tubules challenged with 5-HT after preincubation for 10 min in saline containing 10(-6) M destruxin A showed that the second and third phases of the typical triphasic response to 5-HT were disrupted. Likewise, the positive shift in TEP in response to 5-HT in chloride-free bathing saline was significantly reduced when tubules were preincubated for 10 min in 10(-6) M destruxin A. The pH of the secreted fluid, but not the Na(+) or K(+) concentration, increased significantly when 5-HT-stimulated tubules were exposed to 10(-6) M destruxin A. The ATP content was not significantly different when tubules stimulated with 5-HT were exposed to destruxin A. Taken together, these results show that destruxin A, without interfering with the intracellular ATP production, strongly inhibits fluid secretion rate by the Malpighian tubules of R. prolixus. Changes in properties of the TEP suggest that one of the target sites for this peptide toxin might be associated with inhibition of the apical V-type H(+) ATPase of tubule cells.