Paramphistomosis, caused by Calicophoron daubneyi, is an emerging disease of ruminant livestock in the UK and Ireland. With only one drug (oxyclozanide) currently proven to be effective against both the adult and juvenile parasites there is a clear need to identify new control strategies to combat rumen fluke infection. C. daubneyi produce thousands of eggs per day, to complete their life-cycle, thus eggshell formation may represent a promising target for therapeutic intervention. Most trematode eggshells (including those of the liver fluke Fasciola hepatica) are formed by tyrosinase-mediated cross-linking of modified tyrosine (L-DOPA) residues present in the vitelline proteins. However, our histochemical analysis and UV fluorescence microscopy suggests that the composition of the eggshell proteins and the chemical nature of their cross-links are distinct in C. daubneyi. In addition, C. daubneyi eggshells were resistant to the de-tanning agent sodium hypochlorite (which readily dissolved F. hepatica eggshells) but were solubilised by the reducing agent DTT. Finally, sequence analysis of C. daubneyi vitelline proteins revealed that the percentage of tyrosine residues present had approximately halved in C. daubneyi compared to sequences from related fluke species, and a similar percentage of cysteine residues had been introduced, whereas no cysteine is present in the vitelline protein sequences from these other trematodes. In some instances the cysteine residues had directly replaced tyrosine where known L-DOPA modifications involved with cross-linking occur. Taken together, our data suggest that C. daubneyi eggshells may be stabilised by disulfide cross-links rather than cross-linking of L-DOPA residues as seen in other trematodes.
