In vitro manipulation of gene expression in larval Schistosoma: a model for postgenomic approaches in Trematoda

Abstract

With rapid developments in DNA and protein sequencing technologies, combined with powerful bioinformatics tools, a continued acceleration of gene identification in parasitic helminths is predicted, potentially leading to discovery of new drug and vaccine targets, enhanced diagnostics and insights into the complex biology underlying host-parasite interactions. For the schistosome blood flukes, with the recent completion of genome sequencing and comprehensive transcriptomic datasets, there has accumulated massive amounts of gene sequence data, for which, in the vast majority of cases, little is known about actual functions within the intact organism. In this review we attempt to bring together traditional in vitro cultivation approaches and recent emergent technologies of molecular genomics, transcriptomics and genetic manipulation to illustrate the considerable progress made in our understanding of trematode gene expression and function during development of the intramolluscan larval stages. Using several prominent trematode families (Schistosomatidae, Fasciolidae, Echinostomatidae), we have focused on the current status of in vitro larval isolation/cultivation as a source of valuable raw material supporting gene discovery efforts in model digeneans that include whole genome sequencing, transcript and protein expression profiling during larval development, and progress made in the in vitro manipulation of genes and their expression in larval trematodes using transgenic and RNA interference (RNAi) approaches.

Fig. 1

Schistosoma mansoni miracidia (t=0 h) as they undergo morphological transformation to the primary sporocyst stage (t=24 h). Miracidia, cultured in isotonic snail saline (Chernin’s balanced salt solution, CBSS; Chernin, 1963), first cease swimming, assume a rounded shape (t=2 h) as expansion of intercellular ridges force ciliated epidermal plates to round up and detach from the larval surface (t=6 h). ‘t’ indicate the approximate time elapsed from initial introduction of miracidia into culture.

Fig. 2

In vitro cultivation of Fascioloides magna (Fasiciolidae) miracidia in untreated CBSS (Chernin’s balanced salt solution; Chernin, 1963). Within 24 h, most miracidia cease swimming activity but the majority will not fully transform (i.e. will retain their ciliated epidermal plates (A). However, miracidia cultured in CBSS, pre-conditioned by cultivation with snail Biomphalaria glabrata embryonic (Bge) cells, are induced to transform (rounding and shedding of ciliated epidermal plates (B) to become fully-transformed primary sporocysts (C).

Fig. 3

Schematic representation of Schistosoma intramolluscan larval stages; miracidia, primary (mother) sporocysts, secondary (daughter) sporocysts and cercariae. For each larval stage, specific biological functions corresponding to predominant up-regulated transcripts (black lettering) or proteins (red lettering) are described. Functional categories of differentially-expressed excretory-secretory proteins/products (ESPs) and larval transformation proteins (LTPs) released inside the molluscan intermediate host during miracidium-to-sporocysts transformation, and cercarial ESPs release during and following emergence from the snail host are also listed (red lettering).

Fig. 4

Example of Schistosoma mansoni primary sporocysts subjected to square-wave electroporation in the presence of rhodamine-labeled double-stranded (ds)RNA. Localization of electroporated dsRNA is mainly tegumental in discrete ‘patches’ (right panel; epifluorescence). Intact sporocysts are shown in the left panel.