Effects of insulin on E. multilocularis larval development. A) Morphology of primary cell aggregates. Primary cells were isolated from axenic metacestode vesicles and cultivated in 2% FCS/(D)MEM supplemented with or without 10 nM human insulin for one week. The aggregates were fixed and embedded in Technovit 8100. Sections (4 μm) were stained with haematoxylin/eosin. B) Formation of metacestode vesicles. Primary cells were cultivated in conditioned medium supplemented with human insulin for three weeks and mature metacestode vesicles were counted. Control was set to 1 and results were normalised against the control. (*) P values below 0.05, (**) very significant for P between 0.001 and 0.01, (***) extremely significant for P <0.001. C) BrdU uptake by parasite cell cultures. Primary cells were isolated and incubated for 24 hours with insulin. BrdU was added for four hours and BrdU uptake was measured with the colorimetric BrdU ELISA kit (Roche, Mannheim, Germany). Asterisks mark significant values. D) BrdU uptake by mature metacestode vesicles. Metacestode vesicles were incubated for two days in the presence or absence of insulin and BrdU. BrdU uptake was measured after chromosomal DNA isolation with the colorimetric BrdU ELISA kit (Roche). E) Re-differentiation and microcyst formation of E. multilocularis protoscoleces. Examples of microcysts forming in in vitro protoscolex cultures. F) Microcyst formation of in vitro cultivated protoscoleces incubated with insulin for three weeks. Control was set to 1 and results were normalised against the control. (*) P values below 0.05, (**) very significant for P between 0.001 and 0.01, (***) extremely significant for P <0.001. BrdU, bromodeoxyuridine.