Effect of piperazine (diethylenediamine) on the moulting, proteome expression and pyrophosphatase activity of Ascaris suum lung-stage larvae

Piperazine (diethylenediamine) is an anthelmintic widely used against animal and bird ascariasis. In this study, we show that treatment with piperazine blocks Ascaris suum larval moulting and development processes and affects larval proteome expression profiles. A. suum lung-stage L3 (LL3) obtained from an infected rabbitʹs lungs were cultured in RPMI medium in the presence of increasing concentrations of piperazine sulfate (Pzes). Our results showed that Pzes potently inhibited moulting of A. suum LL3 in a dose-dependent manner and that moulting was completely blocked (100%) at 50 mM concentrations. We then examined the changes in A. suum LL3 proteome expression patterns following Pzes exposure using two-dimensional (2D) electrophoresis. Pzes exposure inhibited expression of at least 16 major protein spots in unmoulted LL3 out of more than 200 visible protein spots resolved on 2D gels prepared from moulted larvae (i.e., lung-stage L4). Pzes exposure also inhibited expression of 13 immunogenic protein spots in unmoulted LL3. More importantly, Pzes exposure inhibited activity of a moulting-specific enzyme, inorganic pyrophosphatase of A. suum (AsPPase), by 26%. Expression of native AsPPase was also reduced following Pzes exposure as detected by immunoblotting and immunofluorescent staining. Transmission electron microscopy showed that Pzes interfered with growth and ecdysis of the cuticle and caused damage to gut tissues of the larvae. Our results suggest that A. suum LL3 may become a suitable model to screening new-class anthelmintics with antimoulting functions and that A. suum LL3-Pzes may serve as a useful tool for identification of moulting-specific potential proteins in Ascaris roundworms.