Elisabeth A. Meuleman

Host-parasite interrelationships between the freshwater pulmonate Biomphalaria pfeifferi and the trematode Schistosoma mansoni.

The influence of infection with S. mansoni on reproduction, growth, food consumption and survival in B. pfeifferi was studied experimentally (Ch. II). The prepatent period in infected snails was shown to last 22-24 days under the conditions prevailing in the relevant experiment. The number of eggs laid by infected snails as compared with controls was significantly reduced from 7-10 days postinfection onwards. After day 13 postinfection it was completely suppressed in the majority of the infected snails. The increase in shell diameter in infected specimens was larger than that of the controls between 7 and 18 days, that in weight between 11 and 21 days postinfection. Gigantism did not occur. There was no difference in mortality between infected and control snails prior to the 11th week after the beginning of the experiment. After that, the mortality among infected specimens showed a sharp rise. Uninfected and infected snails consumed similar amounts of food (dried lettuce) in the first two weeks of the experiment. After that, infected snails consumed less food than controls. The digestive gland of B. pfeifferi was studied with histological, histochemical and ultrastructural methods (Ch. III). The highly folded epithelium of the ducts connecting the digestive gland with the pyloric part of the stomach, consists of cilia and/or microvilli bearing columnar cells with interspersed mucous cells. In the epithelium of the digestive gland two main cell types are distinguished: the digestive cell and the secretory cell. In addition some mucous cells occur. It is concluded that the main functions of the digestive cell are absorption and endocytosis of predigested food material, followed by intracellular digestion. Indigestible residues are accumulated in a large vacuole, which eventually is excreted. The secretory cell produces and secretes proteinaceous substances, presumably digestive enzymes. It does not store calcium. The so-called excretory cells, which are in fact degenerating secretory cells, have vacuoles containing yellow globules. These yellow globules together with cell debris are finally released into the lumen of the digestive gland. The observations indicate that the digestive gland is not important as a storage organ for reserve material. The lobules of the digestive gland are covered by a thin connective tissue sheath, consisting of ground substance, collagen-like fibrils, smooth muscle cells and cells with the appearance of pigment cells. The connective tissue between the lobules of the digestive gland and the acini of the ovotestis, and between these organs and the mantle, consists mainly of vesicular connective tissue cells. These cells are supposed to play an important metabolic role as storage cells for glycogen. The tegument of the daughter sporocyst of S. mansoni (Ch. IV), consisting of an outer syncytial anucleate layer which is joined by cytoplasmic connections to nucleated cell bodies (tegumental cells), has the same basic architecture as that described for various stages of trematodes and cestodes. Well-developed mitochondria, GER cisternae and tubules, free ribosomes and polysomes, and lipid droplets occur throughout the tegument. The ultrastructural features suggest that the outer layer is involved in uptake of nutrients by absorption and endocytosis. The poorly developed musculature is loosely arranged in two layers beneath the outer tegumental layer. The nucleated portions of the muscle fibres are situated between the tegumental cells. The protonephridial system consists of flame cells with efferent tubules. The bundle of cilia, implanted in the flame cell, beats in the cilia chamber or barrel. The proximal part of the barrel consists of alternating rib-like extensions of the flame cell and of the first tubule cell, respectively. Ultrafiltration probably takes place in the ribbed part of the barrel. The development of cercariae from germinal cells is briefly described. The pathological effects of infection with S. mansoni on the digestive gland epithelium of B. pfeifferi (Ch. V) are limited. From about 12 weeks after infection slight changes occur, but they are difficult to distinguish from changes due to ageing of the cells. The effect of starvation on the digestive gland epithelium is much more marked than the adverse influence of infection. Compression of digestive gland lumina by daughter sporocysts was not observed. Changes in the connective tissue of the digestive gland due to the presence of daughter sporocysts were: development of muscle cells into cells with the appearance of pigment cells and/or degeneration of these muscle cells, and an increase in the number of amoebocytes, which become involved in the elimination of cell debris and in the formation of a loose layer over the parasites.

The relationship between miracidial dose, production of cercariae, and reproductive activity of the host in the combination Trichobilharzia ocellata and Lymnaea stagnalis.

The effects of exposure of juvenileLymnaea stagnalis to one, two or, four miracidia ofTrichobilharzia ocellata on the following parameters were studied: infection rate, length of prepatent period, production of cercariae, growth and ovipository activity of the snails, and the weights of their accessory sex organs. An infection rate of 100% was established with all miracidial doses. Mortality of the snails was low in all experimental groups. In cercarial production high-(HP) and low-productive (LP) infections could be distinguished. An increase in miracidial dose at exposure results in an increase in the number of snails with HP-infections and decreases the length of the prepatent period of these infections. Snails with HP-infections grow faster than controls after day 14 post-exposure and show giant growth, while the accessory sex organs remain very small. The snails show hardly any ovipository activity. In all snails with HP-infections these effects are observed independent of the miracidial dose at exposure. Snails with LP-infections demonstrate no signs of gigantism, their accessory sex organs are almost identical to those of controls, and the snails show ovipository activity. It is concluded that the occurrence of HP- and LP-infections indicates differences in reproductive capacity of the parasite. HP-infections can only develop when the parasites can adjust the host to their requirements at an early stage of the infection.

Differences between blood cells of juvenile and adult specimens of the pond snail Lymnaea stagnalis

SummaryBlood cells (amoebocytes) of juvenile and adult specimens of the pond snail Lymnaea stagnalis were compared. Juvenile snails contain fewer circulating amoebocytes per μl haemolymph. However, a higher percentage of these cells shows mitotic activity, as determined by incorporation of 3H-thymidine in vitro. Relatively more amoebocytes of juvenile snails have the characteristics of less differentiated cells: they are small and round with few inclusions, a high nucleus-to-cytoplasm ratio, and a high pyronin stainability. Enzyme cytochemical studies showed that acid phosphatase (AcP), non-specific esterase (NSE), and alkaline phosphatase (AlP) are present in all amoebocytes of juvenile and adult snails. AcP activity is relatively weak. NSE activity is dispersed throughout the cytoplasm and occasionally found in granules, whereas AlP is clearly localized in granules. Differences between the two age groups were found only for the enzyme peroxidase (PO). In juvenile snails a lower percentage of the cells is positive and the granules that contain the activity are less abundant than in amoebocytes of adults. It is suggested that, due to the above-mentioned characteristics of the amoebocytes, the activity of the internal defence system in juvenile L. stagnalis is on a lower level than that in adult snails. This might be an explanation for the fact that juvenile L. stagnalis are highly susceptible to infection by the schistosome Trichobilharzia ocellata, whereas adult snails are less susceptible.

Alterations in the internal defence system of the pond snail Lymnaea stagnalis induced by infection with the schistosome Trichobilharzia ocellata

In order to investigate whether the schistosome Trichobilharzia ocellata interferes with defence activities in its snail intermediate host Lymnaea stagnalis, aspects of the immune system of infected snails and of non-infected controls were compared. The elimination of injected live Staphylococcus saprophyticus bacteria starts at a lower rate in infected snails 1 and 5 weeks after exposure to the parasite, but then proceeds faster than in control snails. During the first 3 weeks of infection, when only mother sporocysts are present, the haemocytes of the infected snails have an increased capacity to phagocytose rabbit red blood cells in vitro. From 5 weeks onwards, when mother and daughter sporocysts are present but cercariae are not yet mature, the phagocytic activity decreases to below control level. The number of circulating haemocytes is also higher in infected snails than in controls at this time. Moreover, the cells are larger, have more inclusions and an increased surface area with many long, branched, spiked pseudopods. The development of the parasite is retarded in a subpopulation of snails in which the haemolymph plasma agglutinates erythrocytes with high titres, compared to a subpopulation with low haemagglutinating activity. The haemagglutinating activity in infected snails of the first decreases significantly from 6 weeks onwards.

The development of daughter sporocysts inside the mother sporocyst of Schistosoma mansoni with special reference to the ultrastructure of the body wall.

The development of the mother sporocyst and the differentiation of the daughter sporocyst ofSchistosoma mansoni inBiomphalaria pfeifferi are described. The tegumental structure of the mother sporocyst, consisting of an outer layer connected to internally situated nucleated cell bodies, forms extensions which enwrap the germinal cells. The parenchyma cells, in which the germinal cells were embedded before, degenerate. When daughter sporocyst embryos develop from germinal cells they are enveloped by a primitive epithelium which is formed by fusion of the extensions of the tegumental structure of the mother sporocyst. Somatic cells located peripherally in the developing daughter sporocyst expand and coalesce beneath the primitive epithelium to form the future outer layer of the tegumental structure of the daughter sporocyst. The primitive epithelium degenerates, the newlyformed layer looses its nuclei, and becomes connected to internally situated nucleated cell bodies. Further developments in the tegumental structure of the daughter sporocyst include the formation of microvillus-like projections, a surface coat, spines, and a basement membrane.

Ultrastructural changes in the body wall ofSchistosoma mansoni during the transformation of the miracidium into the mother sporocyst in the snail hostBiomphalaria pfeifferi

SummaryThe ultrastructure of the body wall of the free miracidium ofSchistosoma mansoni and the changes occurring within 48h after penetration into the intermediate hostBiomphalaria pfeifferi are described. Within 2 h after penetration the ciliated plates are shed into the haemolymph of the snail and phagocytized by amoebocytes. At the same time the narrow ridges between the plates of the free miracidium expand to form the continuous outer layer of the sporocyst. Within 48 h the entire tegumental structure, consisting of a thin outer layer, connected with sunken nucleated areas, develops to its full extent. The observations are compared with those onFasciola hepatica.

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.

The development of the primitive epithelium and true tegument in the cercaria of Schistosoma mansoni

SummaryThe formation of the final cercarial tegument of Schistosoma mansoni is preceded by that of a so-called primitive epithelium. The primitive epithelium is derived from the tegument of the daughter sporocyst. The final cercarial tegument is formed from peripherally located somatic cells of the cercarial embryo, which expand and coalesce beneath the primitive epithelium. The primitive epithelium degenerates and disappears. The ultrastructure of both epithelia in the course of the development of the cercaria is described in detail. Possible functions are discussed.