To evaluate the mRNA expression level in different developmental stages, quantitative real time PCR (Q-rt.-PCR) was performed. Primers were designed using Oligo-primer analysis software v. 6.71 (Molecular Biology Insights, Cascade, WA, USA) (Additional file 1: Table S1). Calcyphosine (CAP) and phosphoglycerate kinase (PGK) were employed as the reference genes . Q-rt.-PCR reaction mixtures were prepared in triplicate using LightCycler FastStart DNA Master SYBR Green I Kit (Roche, Mannheim, Germany), with each reaction containing 50 ng of total cDNA of the adults or metacercariae. Q-rt.-PCR was performed on LightCycler 2.0 (Roche, Penzberg, Germany) with the following thermal cycle parameters: pre-heating (95 °C for 15 min), 40 cycles of 95 °C for 10 s, 48 °C for 10 s, and 72 °C for 30 s. The relative transcription ratio was calculated according to the 2-ΔΔCt method .