Trichobilharzia ocellata infections in its snail host Lymnaea stagnalis: an in vitro study showing direct and indirect effects on the snail internal defence system, via the host central nervous system.

In this in vitro study we investigated whether previously described in vivo plasma-associated effects, that occurred in the period shortly after penetration of Trichobilharzia ocellata into the snail host Lymnaea stagnalis (1.5-72 h post-exposure; p.e.) were direct and/or indirect effects of parasite-derived factor(s). It was investigated whether the effect is mediated by the central nervous system (CNS) of the host. Phagocytic activity of the haemocytes was taken as a parameter for the activity of internal defence of the host. A number of preliminary experiments were performed. When the supernatant of in vitro cultured parasites (33 h; corresponding with their developmental stage in vivo when plasma-associated activation was found) was applied directly to monolayers of haemocytes, it appeared to enhance their phagocytic activity. No direct effect, however, was found with a supernatant of parasites cultured for a longer period of time (72 h; when, in vivo, a plasma-associated suppression was found). In this case, indirect suppression was detected: the parasites appeared to have released a factor that induced the CNS of the host to release material suppressing the activity of the internal defence system of the host. To date the nature of this factor is unknown.

Modulation of the activity of the internal defence system of the pond snail Lymnaea stagnalis by the avian schistosome Trichobilharzia ocellata.

Effects of infection with the avian schistosome Trichobilharzia ocellata on the activity of the internal defence system of the intermediate snail host Lymnaea stagnalis were studied, utilizing an in vitro phagocytosis assay for determining haemocyte activity. A distinction was made between plasma- and cell-associated effects. The period immediately after penetration of the parasite into the snail host (1.5-72 h post-exposure (p.e.)) was extensively studied. In addition, several time-points coinciding with the later-successive-stages of parasite development (2, 4, 6, 8 and 10 weeks p.e.) were investigated. Plasma-associated enhancement of defence activity was found between 1.5 and 6 h p.e., followed by plasma-associated suppression between 12 and 72 h p.e. A cell-associated activation was found between 1.5 and 6 h p.e. and also at 8 and 10 weeks p.e. How these effects on the defence system may be related to phenomena observed in infected snails at these time-points is discussed.

Trichobilharzia ocellata in Lymnaea stagnalis: a flow cytometric approach to study its effects on hemocytes.

Flow cytometric analysis was performed on hemocytes in suspension derived from individual Lymnaea stagnalis. The distribution of cell sizes within the hemocyte population was comparable in all 40 specimens studied. The size distribution of circulating hemocytes is unimodal and continuous, with no discrete subpopulations, and is not affected by age or by infection with Trichobilharzia ocellata. Flow cytometry proved to be a very useful technique in analysis of hemocyte populations in snails and anti-hemocyte monoclonal antibodies can be employed in these studies. The use of individual instead of pooled hemolymph samples in studying hemocyte populations of molluscs is stressed.

Trichobilharzia ocellata: Quantification of effects on haemocytes of the pond snail Lymnaea stagnalis by morphometric means.

To identify functionally different subpopulations, we quantified by morphometric means the spreading activity of circulating haemocytes of the pond snail Lymnaea stagnalis, recognized by monoclonal antibodies (5 surface and 5 cytoplasmic). The influence of snail age and of the different intramolluscan stages of the compatible avian schistosome Trichobilharzia ocellata on this activity were studied. The antibody-recognized cells could be separated into two groups, differing in their spreading activities. The probes detecting cytoplasmic markers recognized the majority of cells (78-95%). These were active (well spreading), differentiated cells. The surface probes recognized a smaller part (12-38%) of the total haemocyte population. These harmocytes were less active (less spreading), and were predominantly immature cells. The relative sizes of the antibody-detected subpopulations were not affected by snail age or infection with T. ocellata. The maximal size (planimetric area) attained after attaching to glass of all cells increased between day 0 (juvenile snails) and about 6 weeks post (sham) exposure and then decreased. Infection had little effect on the spreading activity of the more differentiated cells. The less differentiated cells showed a larger spreading activity when the parasite was present as mother sporocyst and also when the digestive gland area became colonized by growing daughter sporocysts.

Effects of Trichobilharzia ocellata on hemocytes of Lymnaea stagnalis.

We analyzed the effects of infection with Trichobilharzia ocellata on hemocytes of its snail host, Lymnaea stagnalis, and correlated them with successive stages of parasite development. Circulating hemocytes were studied at 0, 2, 4, 6, and 8 weeks post exposure (p.e.) with respect to cell number, distribution of subpopulations (as characterized by morphology, determinants recognized by either of two lectins and a monoclonal antibody) and to proliferative, phagocytic and endogenous peroxidase activity. Infection results in a net elevated level of activity of circulating hemocytes at 2 weeks p.e., when mother sporocysts are present in the head-foot-mantle region, as well as at 4 weeks p.e., when daughter sporocysts are migrating to and growing in the digestive gland region. A lower level of activity was observed at 6 weeks p.e., when cercariae are differentiating within daughter sporocysts. A net activation was again found at 8 weeks p.e., when cercariae are escaping. So, infection with T. ocellata results in a net general activation of the internal defense system of L. stagnalis, during several stages of development of the parasite.