U examination was applied to study the effect of short' (4 days) and long (10 days) exposures to sublethal concentration (1 ppm ) of ethanol extract of Solarium elaeagnifolium seeds (S. etaeag. ) (Family: Solanecae) on the digestive gland and hermaphrodite gland of infected and uninfected the pulmonate snail Biomphalaria alexandrina. Ultrastructural studies of the above two glands of infected snails illustrated remarkable cytologicai changes due to the infection with marked increase in the cytoplasmic vacuolation that almost occupied the whole cell of the digestive gland, The ultrastructural changes in the digestive and hermaphrodite gland cells due to exposure of snails to ethanol extract of the candidate plant showed degeneration in the nucleus, nucleolus and mitochondria with abnormal Golgi complex as well as in glycogen digestion. INTRODUCTION Schistosomiasis is one of the serious parasitic diseases which attack. many people in tropical and subtropical regions of the world causing serious losses in health and economy. In Egypt, the disease stands on the top of the public health problems and its impact on the health and economy is tremendous. Thus a considerable attention has been focused on its control by various means of which the control of the intermediate host snails of the parasite seems to be most possible if properly handled. 2 Abdalla M Ibrahim et ah Biomphalaria alexandrina ( Ehrenberg) is known to be the pulmonate species that acts as the intermediate host of Schistosoma mansoni in Egypt. The high cost and toxicity of synthetic molluscicides, used for the control of intermediate snail hosts of parasitic diseases, resulted in renewing the interest in plant molluscicides (Massoud and Habib,2003). The screening of local plants for their molluscicidal activity has received increasing attention by many authors (Tantawy et aL9 2000; Bakiy et aL, 2002). Extracts of plants in the genus Solarium (Solanaceae) showed significant mulluscicidal activity against some planorbid snails, (Bekkouch et aly 2000 ; Siiva et a/.,2005). Some general studies do exist about the ultrastructure of the digestive gland of certain molluscs, the most comprehensive of which were published by Walker(1970) and Pol(1971). On the electron microscopic level, Meuleman(1972) elucidated the changes induced in the digestive gland of south African strains of B. pfefferi as a result of infection with Schistosoma mansoni. Chen et al (2004) studied the ultrastructural changes induced in the hepatopancreas of. the gastropod Haliotis diversicolor as a result of ulcerate disease. The ultrastructural effects of extracts from Tetrapleura teiraptera (Taub.) and Bayluside on the digestive gland of B. glabrata were investigated by Bode et al (1996). The hepatopancreas and reproductive system in normal Oncomelania hupensis snails and in snails after treatment with seed extracts of Camellia sinensis were ultrastructurally examined by Song et al (1997). The present work aimed to study the effect of S. elaeagnifolium on infected and uninfected B. alexandrina as indicated by its action on the fine structure of both digestive and hermaphrodite glands. MATERMLS AND METHODS Solanum elaeagnifolium (Cav.) (Family: Solanaceae) was collected from north east Sainai, near El-Arish city. 250 grams of unripe seeds of Solanum elaeagnifolium were exhaustively extracted with 95% ethanol at room ^mperature. The extract was evaporated to dryness under vacuum. From the crude extract, a stock solution was prepared. B. alexandrina snails ( noninfected and infected with S. mansoni ) were obtained from Theodor Bilharz Research Institute, Imbaba, Giza, Egypt. ULTRASTRUCTURAL STUDY ON THE EFFECT OF 3 A PLANT MOLLUSCICIDE ON B. ALEXANDRINA Experimental desigmlnfected (shedding cercaria) and uninfected B. alexandrina snails(5-6 weeks old) were treated with bubiethql concentration (lppm) of ethanol extract of Solarium elaeagnifolium, Sonx of them were exposed to molluscicidal solution for 4 days and then recovered for 1 week. Others were exposed for 10 days continuously to themoliuscicide. Snails were then dissected to obtain the digestive and hermaphrodite glands to be prepared for ultrathin section examinationsExperimental conditions: During the experiments, snails were maintained at constant conditions of temperature (26°c ± 2°c) and artificial illumination (11 hours/day). Electron microscopy: 1 mm of both the digestive gland and the hermaphrodite gland of both infected and uninfected snails were primary fixed in 2% gluteraldehyde in sodium cacodylate buffer (0.1 (j. ,PH 7.4) for two hours at 4°c and then post fixed in 1% buffer osmium tetroxide for two hours at 4°c. The samples were dehydrated in acetone and embedded for 12 hours in Epon resin (812) (Luft, 1961). Ultrathin sections were cut and then mounted on copper grids, stained with uranyle acetate and lead citrate, and examined with Jeal JEM 100 S transmission electron microscope. RESULTS The digestive gland: The digestive gland of Biomphalaria alexandrina consists of two main types of cells, the digestive and secretory cells. The digestive cells: The electron microscope examination of uninfected and infected snails has revealed the presence of long, dense microvilli along the apical areas of the plasma membranes of the digestive cells (Fig. I). It also shows scattered cilia along thetuminal surface of these cells, each containing an axial filament complex of the conventional 9+2 configuration. The microvilli, as the cilia, are bounded by apical extensions from the plasma membranes of the cells. The ground cytoplasm of such cells contains a granular endoplasmic reticulum, mitochondria and occasionally Golgi complex. Also the digestive cells possess numerous digestive vacuoles of variable size (Fig. 1). The nuclei of the digestive cells occupy the basal regions, each containing a conspicuous nucleolus. The secretory cells: 4 Abdalla M. Ibrahim et al In the secretory cells of the digestive gland, the nucleus is situated in the broad basal part of the cell. At the ultrastructural level, the nucleolus appears to consist of a dense central part, surrounded by a less compact outer zone. The chromatin occurs mainly as clumps which lie scattered through the nucleoplasm and against the nuclear envelope. Outgrowths of the outer membrane of the nuclear envelope are continuous with cisternae of granular endoplasmic reticulum (GER). The extensive GER which is composed of tubules and numerous, mainly parallel, cisternae accounts for the strong basophilia of the cytoplasm, while a well develop Golgi apparatus is located in the supranuclear region. Mitochondria often occur in close association with the secretion granules (Fig. 2). A high density of mitochondria, longer and thicker than those of the digestive cells and occasionally containing small electron-dense granules are found apical to the nucleus and over the basal cell membrane. The degeneration of the secretory cells starts with the appearance of few vacuoles containing yellow bodies in the cytoplasm. Their ultrastructural examination show that these vacuoles contain acid phosphatase and so are probably autophagosomes and recognized as degenerating cell organelles. ■ The digestive cells of infected snails displayed various signs of degeneration. The endoplasmic-reticulum became distinctly swollen and the mitochondria were reduced in number, more elongated and accumulated (Fig.3). The cytoplasmic vacuoles became abundant without any apparent inclusions, causing change in the nucleus shape, the secretory cells of digestive gland displayed numerous vacuoles also (Figs.4&5). Ultrastructural changes of digestive gland after treatment with 1 ppm ethanol extract-.of & elaeagnifolium for 4 days and recovery for 1 week, showed phosphohydrolase reactions producing an accumulation of dense stain precipitate, surrounding the nucleus which started to separate from the cytoplasm presenting .a narrow suture (Fig. 6), However, the chromatin clumps were decreased and scattered against the nuclear envelope.. This is probably an.autophagosome indicating a sign of cell orgaiielies degeneration, No more damage in the digestive gland cell content was observed after short exposure to the molluscicide except a high density of mitochondria which appeared cloudy because the density of the matrix decreased and the inner membrane became displaced and collapsed onto ULTRASTRUCTURAL STUDY ON THE EFFECT OF 5 A PLANT MOLLUSCICIDE ON B. ALEXANDRINA a limited area of the external membrane. Some of these mitochondria appeared swollen. At daughter sporocyst, the mitochondria revealed more damage ana part of them became degenerated and seemed vacuolated mitochondria (Fig. 7). The same pathogenesis was observed in the digestive gland of snails exposed to 1 ppm ethanol extract of 5. elaeagnifolium for 10 days (Fig.8). However, more degeneration occurred in the endoplasmic reticulum accompanied with neumerous vacuoles. The hermaphrodite gland: Under light microscope, the sperm of B. alexandrina appears as an extremely long thin filament with a slightly enlarged anterior tip. At higher magnifications, a small corkscrew-shaped head is discernible ; provided with an acrosome and followed by a helicoidal middle piece which ends in a short tail (Fig. 9). The helix of the middle piece appears double in most of its length but is single near the tail. The inner organization of the sperm components was better understood by the ultrathin section examination (Fig, 10). Oogonia and mature ova are relatively little and therefore rarely seen. The hermaphrodite gland appeared more affected than the digestive gland, especially in snails exposed to 1 ppm of ethanol extract of S. elaeagnifolium for 10 days. After short exposure period (4 days) and 1 week recovery to 1 ppm of the plant molluscicide, the plasma membrane of the sperms appeared as dense fibrous bands (Fig. 11). Glycogen granules in the normal sperms appear as dense granu

Ultrastructural study on the effect of a plant molluscicide (solanum elaeagnifoljum) on the digestive and hermaphrodite glands of biomphalaria alexandrina

The structure of the uninfected and infected digestive gland of the fresh-water snail, Bulinus truncatus, was studied histologically and histochemically. The digestive gland consists of two types of cells: digestive and secretory cells clearly differentiated from each other. The pathological effects of infection of the snail with cercariae of Schistosoma haematobium have resulted in considerable structural and histochemical alterations in the digestive gland. Generally, the main effects were reflected by increased cellular vacuolation and marked diminution of carbohydrates, proteins and lipids of the gland cells.

Mohamed Sh

Papers

Histological and histochemical studies on the digestive gland of Bulinus truncatus infected with Schistosoma haematobium.