Excretory–secretory products of Trichobilharzia ocellata and their modulating effects on the internal defence system of Lymnaea stagnalis

Excretory–secretory (E–S) products, released by Trichobilharzia ocellata cultured in vitro, were assessed for their modulatory effects on the internal defence system of the freshwater snail, Lymnaea stagnalis. E–S products were concentrated and fractionated using high-pressure liquid chromatography (HPLC) and bacterial clearance activity of haemocytes was taken as a parameter for the internal defence system. Two main fractions of E–S products were produced by T. ocellata, a low molecular fraction (low MW) and a high molecular fraction (high MW), weighing approximately 2 kDa and 40 kDa, respectively. Stage-dependent release of E–S products by transforming miracidia and mother sporocysts was indicated by the difference in proportions of these two fractions, which varied over time. After a culture time of 0–33 h, the smaller fraction was prominent whilst E–S products released between 33 and 72 h consisted of a higher proportion of the high MW fraction. After a culture of 72–96 h, lower and comparable quantities of both fractions were produced by the same batch of cultured parasites. Assaying the 2 fractions released during 0–33 h revealed that the high MW fraction had a suppressing effect on the bacterial clearance activity of haemocytes from juvenile snails and the low MW fraction activated haemocyte activity. Combined fractions obtained during 0–33 h had an activating effect on haemocyte activity but fractions separated from media collected at 33–72 h culture had a suppressing effect on the bacterial clearance activity of the haemocytes. The combined E–S fractions from the 72–96 h incubation had no effect on haemocyte activity. The present study shows that T. ocellata modulates the internal defence system of L. stagnalis by actively interfering with haemocyte function. MALDI-MS analysis of the low MW fraction revealed several components ranging from 450 to 1600 Da. Although it is as yet not clear which factor is responsible for the activating effect it is supposedly a peptide as it is pronase-sensitive and resistant to TFA and acetonitrile.