Culture for genetic manipulation of developmental stages of Schistosoma mansoni

Abstract

Genomes of the major human helminth parasites, and indeed many others of agricultural significance, are now the research focus of intensive genome sequencing and annotation. A draft genome sequence of the filarial parasite Brugia malayi was reported in 2007 and draft genomes of two of the human schistosomes, Schistosoma japonicum and S. mansoni reported in 2009. These genome data provide the basis for a comprehensive understanding of the molecular mechanisms involved in schistosome nutrition and metabolism, host-dependent development and maturation, immune evasion and invertebrate evolution. In addition, new potential vaccine candidates and drug targets will likely be predicted. However, testing these predictions is often not straightforward with schistosomes because of the difficulty and expense in maintenance of the developmental cycle. To facilitate this goal, several developmental stages can be maintained in vitro for shorter or longer intervals of time, and these are amenable to manipulation. Our research interests focus on experimental studies of schistosome gene functions, and more recently have focused on development of transgenesis and RNA interference with the longer term aim of heritable gene manipulation. Here we review methods to isolate and culture developmental stages of Schistosoma mansoni, including eggs, sporocysts, schistosomules and adults, in particular as these procedures relate to approaches for gene manipulation. We also discuss recent advances in genetic manipulation of schistosomes including the deployment of square wave electroporation to introduce reporter genes into cultured schistosomes.

Fig. 1

Cultured eggs of Schistosoma mansoni. These eggs were isolated from livers of experimentally infected mice. Panel A and B: micrographs illustrating a mixed population, mature and immature eggs, three days after isolation from mouse liver. Scales bars=50 μm. Panel C: two immature eggs (black arrow-heads) and a mature egg containing the miracidium (black arrow); scale bar=20 μm. Panel D: a mature egg containing miracidium (black arrow) and an empty egg shell after hatching has taken place (black arrow-head); Scale bar=20 μm. The micrographs were taken using a digital camera (Zeiss AxioCam ICc3) fitted to an inverted microscope (Zeiss Axio Observer A1).

Fig. 2

Miracidia of Schistosoma mansoni hatching from eggs and transforming into mother sporocysts in vitro. Panel A: mature egg ready to hatch (arrow-head) and two immature eggs. Panel B: two miracidia after hatching. Panel C: miracidium losing its ciliated plates (arrow-head). Panel D: in vitro cultured mother sporocyst (black arrow). Scale bars: 20 μm. The micrographs were taken using a digital camera (Zeiss AxioCam ICc3) fitted to a Zeiss Axio Observer A1 microscope.

Fig. 3

Schistosomules of Schistosoma mansoni. The schistosomules were mechanically transformed from cercariae and cultured for 14 days. Black pigmented guts are evident in many of the larvae. Erythrocytes have been added to the cultures. Panels A to D: schistosomules viewed at 5, 20, 20 and 40× magnification, respectively. Scale bars are shown in each panel. The micrographs were taken using a digital camera (Zeiss AxioCam ICc3) fitted to a Zeiss Axio Observer A1 inverted microscope.