Abstract
In continuation of the search for new anthelmintic natural products, the study at hand investigated the nematicidal effects of the two naturally occurring quassinoids ailanthone and bruceine A against the reproductive system of the model nematode Caenorhabditis elegans to pinpoint their anthelmintic mode of action by the application of various microscopic techniques. Differential Interference Contrast (DIC) and the epifluorescence microscopy experiments used in the presented study indicated the genotoxic effects of the tested quassinoids (c ailanthone = 50 µM, c bruceine A = 100 µM) against the nuclei of the investigated gonadal and spermathecal tissues, leaving other morphological key features such as enterocytes or body wall muscle cells unimpaired. In order to gain nanoscopic insight into the morphology of the gonads as well as the considerably smaller spermathecae of C. elegans, an innovative protocol of polyethylene glycol embedding, ultra-sectioning, acridine orange staining, tissue identification by epifluorescence, and subsequent AFM-based ultrastructural data acquisition was applied. This sequence allowed the facile and fast assessment of the impact of quassinoid treatment not only on the gonadal but also on the considerably smaller spermathecal tissues of C. elegans. These first-time ultrastructural investigations on C. elegans gonads and spermathecae by AFM led to the identification of specific quassinoid-induced alterations to the nuclei of the reproductive tissues (e.g., highly condensed chromatin, impaired nuclear membrane morphology, as well as altered nucleolus morphology), altogether implying an apoptosis-like effect of ailanthone and bruceine A on the reproductive tissues of C. elegans.
Highlights
Infectious diseases caused by pathogenic helminths are responsible for a striking global health burden, especially in socioeconomically less developed countries all over the world [1,2]
The presented study aimed at the detailed investigation of morphological defects of reproductive tissues in L4 C. elegans hermaphrodites after treatment with two quassinoids in order to potentially pinpoint their anthelmintic mode of action
Initial Differential Interference Contrast (DIC) investigations carried out during this study already proved significant structural alterations induced in germ line tissues of L4 C. elegans hermaphrodites after quassinoid treatment compared to untreated individuals
Summary
Infectious diseases caused by pathogenic helminths are responsible for a striking global health burden, especially in socioeconomically less developed countries all over the world [1,2]. On C. elegans ultra-sections indicated morphological impairments in the gonadal tissues Based on these preliminary investigations, the present study aimed at the conformation of the mentioned results on ailanthone against C. elegans as well as on the identification of a potential anthelmintic mode of action of both nematicidal quassinoids by means of advanced light microscopic assessments as well as by Atomic Force Microscopy (AFM)-based ultrastructural evaluations of quassinoid-treated compared to untreated C. We used within the following study a newly developed, facile, and economic technique [20], allowing the rapid identification of specific tissues in immobilized C. elegans ultra-sections by epifluorescence microscopy and subsequent ultrastructural data acquisition on those tissues by AFM This new protocol enabled us to identify and to nanoscopically characterize specific tissues of the reproductive system of C. elegans such as the gonads or the rather small spermathecae by AFM with high efficiency. This subsequently led to the identification of localized ultrastructural defects induced in the reproductive tissues of C. elegans by quassinoid treatment and allowed pinpointing an apoptosis-like mechanism of action concerning the infertility effect of the tested quassinoids against C. elegans
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