Charles H. Dorsey

Ultrastructure of the Schistosoma mansoni cercaria.

The cercaria of the schistosome parasite is a short-lived, free-swimming larval stage that is infective for the mammalian, definitive host. This atlas describes the ultrastructure of the cells that comprise the cercaria of Schistosoma mansoni, a leading causative agent of human schistosomiasis. In addition to the cells which make up the various organ systems, such as the nervous, tegumental, osmoregulatory, muscular and primordial digestive systems, also we show the ultrastructure of those cells whose organization or location are not as well defined structurally but are essential nevertheless for the success of the parasite. These latter include the various support cells, and those cells that, upon differentiation into the adult worm, serve reproductive functions. A description is also given of the cells whose sole functions are realized only at the cercarial stage, chiefly involved in the vigorous act of skin penetration. Although we include a detailed review of the ultrastructure of S. mansoni cercariae, much of the information reported has not been previously published. In summary, this paper brings together an ultrastructural description of all the cell types presently known that make up the much studied larval stage of this medically important trematode.

Schistosoma mansoni: ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules.

Abstract Against the background of cercarial fine structure, ultrastructural changes were compared in schistosomules of Schistosoma mansoni 30 min and 1 hr after their production in vivo by skin penetration and in vitro by shear pressure. The same developmental pattern was observed in schistosomules of both derivations. In vitro schistosomules, however, developed more slowly, resembled cercariae more closely, and varied less among organisms than did in vivo schistosomules. The greatest morphological changes were observed in the 1-hr in vivo schistosomules. These were as follows: (1) in tegument, formation of transient microvilli, a hepatalaminate outer membrane and accented surface invaginations, loss of glycocalyx, movement outward of cyton vesicles via bridges, accumulation of multilaminate bodies around bridge openings; (2) in the anterior organ (oral sucker), movement of head gland vesicles via the ducts into tegument followed by collapse of the gland fundus, disappearance of the circumfundal cells and two large support cells, and the appearance in these areas of membranes and parenchymal cells; (3) secretion of the acetabular gland contents, collapse of the glands and replacement by membranes and parenchymal cells; (4) peristaltic activity of the digestive tract as shown by alternate areas of lumen constriction and dilation; (5) loss of bladder and contraction of the small aboral collecting tubules; and (6) conversion of heterochromatic parenchymal cell nuclei to euchromatic. In contrast, the 1-hr in vitro shear schistosomules resembled 30-min in vivo schistosomules, retaining many cercarial features.

Schistosoma mansoni: Stimulus and transformation of cercariae into schistosomules

Schistosoma mansoni schistosomules prepared from cercariae by seven in vitro techniques had not all reached the same state of development at the end of the incubation period as scored by seven parameters: water tolerance; Cercarienhüllen Reaktion; presence of the glycocalyx; condition of the surface membrane; nuclear state; granule migration; and cryopreservability. At the end of the specific incubation period for each technique, the level of development was judged with respect to schistosomules which had developed in situ for 1 hr after penetration of the ear skin of mice. In descending order of their correspondence to in vivo schistosomules, those derived in vitro (by the procedures listed) ranked as follows: first, penetration of dried rat skin; second, centrifuging and vortexing, or incubation in serum-supplemented medium; and third, syringe passage, omnimixing, centrifuging, and incubating, or incubating alone. The only treatment common to all techniques was incubation in 37 C culture medium for 2 hr or more. This is suggested as the stimulus for the cercaria-to-schistosomule transformation.

Schistosoma mansoni: comparative development of schistosomules produced by artificial techniques.

number of steps involved by introducing the sporozoite directly into the syringe used for injection and to prevent adherence by suspending the sporozoite in bovine serum albumin were unsuccessful. In addition to the certain introduction of the sporozoite into the lumen of the cecum, initiation of infection must depend upon the viability of the organism and the probability of its coming into contact with a cell suitable for development. Although fresh cultures of oocysts were used, there was no way of knowing whether the individual sporozoites selected were infective (Shirley and Millard, 1976, loc. cit.). Eimeria tenella has been used in genetic studies, because of the availability of lines in which resistance to a number of anticoccidial drugs has been induced experimentally. Such lines, despite ste s involved by introducing the spoi tl into the syringe used for injection e t adherence by suspending the spobeing uncloned, have proved suitable for use as genetic markers in recombination experiments (Jeffers, 1974, Journal of Parasitology 60: 900904; Chapman, 1984, Zeitschrift fur Parasitenkunde 70: 437-441). Cloning would, however, be advantageous, as indicated by the instability of resistance in some lines (Chapman, 1984, Parasitology 89: 9-16). The results presented here show that clones of E. tenella may be established by inoculating single sporozoites into the cecum. They also suggest that, by a suitable choice of inoculation site, the procedures could be adapted for cloning other species of Eimeria. We would like to thank B. J. Millard for advice and Mrs. M. Shaw, P. Hesketh and B. Fisher for their assistance.

Nervous system ofSchistosoma mansoni cercaria: Organization and fine structure

As observed by transmission electron microscopy of serially sectionedSchistosoma mansoni cercaria, the nervous system is distributed throughout the three anatomic segments of the larva-i.e., the anterior organ (oral sucker), the body (midsegment), and the tail. The central ganglion, a neuropile surrounded by cell bodies, is located in the anterior area of the body segment. It tapers anteriorly into two lobes from which a pair of anterior central nerve trunks extend longitudinally. The posterior region of the central ganglion tapers into a pair of nerve trunks (posterior central nerve trunks). Twelve peripheral nerve trunks are evenly distributed around the ganglion. Six trunks course anteriad (anterior peripheral nerve trunks) and six course posteriad (posterior peripheral nerve trunks). A pair of dorsal and ventral nerve trunks, positioned opposite each other, extend the length of the tail. All nerve trunks are unsheathed. The nervous system contains three types of vesicles. Type I vesicles average 47.66±2.57 nm in diameter, vary in electron density, and have electron-lucent peripheries. Type II vesicles have a mean diameter of 18.41±2.57 nm, are electron-lucent and are concentrated mostly in the presynaptic area of the synaptic and neuromuscular junctions. The mean diameter of Type III vesicles is 57.47±16.08 nm. They are electron-dense and are concentrated mostly in the tegumental ciliated papillac and their accompanying dendrites. Two types of synaptic junctions are present. Type1 synapse has dense material incorporated in its postsynaptic membrane, while Type2 synapse has dense material of various dimensions incorporated in its presynaptic membrane and usually in its postsynaptic membrane. Synaptic and neuromuscular junctions are similar.

Schistosoma mansoni: localization of calcium-detecting reagents in electron-lucent areas of specific preacetabular gland granules.

SummaryIn an attempt to establish the exact location of calcium within the preacetabular glands of cercariae of Schistosoma mansoni, these larvae were exposed to reagents (potassium oxalate, potassium pyroantimonate, chloranilic acid, and silver nitrate) useful in the detection of calcium, and were subsequently observed with the aid of light and electron microscopes. Cercariae incubated in potassium oxalate and viewed in polarized light showed birefringence only in the preacetabular gland funduses. At the ultrastructural level, the preacetabular glands of potassium oxalate-treated cercariae had no electron-dense precipitate, but instead had translucent, irregularly shaped inclusions, similar to spaces left by volatilized calcium oxalate as described by others. Pyroantimonate treatment, on the other hand, localized the reaction in the electron-lucent areas of the light-spotted granules. The von Kossa silver nitrate procedure destroyed the secretory granules; therefore, an electron-dense precipitate was distributed throughout the gland. However, pretreatment with chloranilic acid before fixation preserved the granules, and subsequent exposure to the von Kossa silver nitrate gave a reaction identical to that obtained with the pyroantimonate alone. When viewed in polarized light, chloranilic acid-incubated cercariae showed birefringence in the fundus and duct areas.

An unsheathed biciliated sensory papilla associated with the tegument of Schistosoma mansoni.

tigate this hypothesis, pairs and ex-paired (separated) males were incubated in medium 169 with 10% human serum (Basch, 1981, Journal of Parasitology 67: 179-185) at 37 C in flowing 5% CO2. Immunofluorescence of the ex-paired males diminished after 24 hr and disappeared by the third day, whereas paired males continued to show strong reactivity. In paired females, immunofluorescence was seen only on the surface. No reactivity was seen in the underlying finger-like projections in females, which have the same cytoarchitecture as in males (Silk et al., 1969, South African Journal of Medical Sciences 34: 1-10). This observation is consistent with the presumption that the antigen is synthesized in the subtegumental cytons of the male, from which it is transferred to the surface through the finger-like projections. It is known (McLaren, 1980, Schistosoma mansoni: The parasite surface in relation to host immunity, Research Studies Press, New York, p. 14) that subtegumental cells are the site of synthesis igate this hypothesis, pairs and ex-paired (sepof tegumental inclusions, which are translocated into the tegument via microtubule-lined cytoplasmic connections. If the material immunoreactive to monoclonal 134 B2/9/4 is transferred to, but not synthesized by, the female, it should be detectable on the female surface but not in the subsequent processes or cytons. Our observations are in agreement with this hypothesis. Unisexual females, freshly perfused from mice, showed no immunofluorescence; when paired for 2 wk in vitro with either unisexual or ex-paired males, weak to moderate immunofluorescence appeared over their entire surface. Control unisexual females similarly incubated in medium 169 without males showed no immunofluores-

Schistosoma mansoni: A new parenchymal cell in cercariae

A new type of cell has been identified in cercariae of Schistosoma mansoni. The perikarya (cell bodies) of these cells were located in the body (midsegment), in an area oral to the acetabulum (ventral sucker). Cytoplasmic processes extending from the perikarya ramified throughout the parenchyma of the anterior organ (oral sucker), body, and tail segments by following the path of the nerve processes from the neuropile. The perikarya of these cells had heterochromatic nuclei and a predominance of particulate material and granules (240-360 nm) in their cytoplasm. Aggregates of granules (240-360 nm) and associated vesicles (34 nm) were scattered throughout the cytoplasmic processes of the cells and formed distinct varicosed areas. These processes often connected to the tegument in the midsegment (body) of the cercariae. The granules and associated vesicles reacted (became electron dense) with fixatives reported to be detectors of biogenic amines: The glutaraldehyde/osmium tetroxide fixation procedure rendered the granules electron dense while the glutaraldehyde/chromate/osmium tetroxide fixation procedure rendered the granules and the associated vesicles electron dense. The chromate solution of the latter procedure was responsible for the electron density of the associated vesicles. The morphology of these cells (their long ramifying cytoplasmic processes) and their reaction to chromium suggests that they are probably biogenic aminergic sensory cells.

Schistosoma mansoni: a rapid procedure for in vitro transformation of cercariae to Schistosomules.

only moderately but irreversibly affected by hycanthone and IA-4. Both drugs, however, had a more pronounced effect upon male than upon female worms. Hycanthone and IA-4 also produced a moderate inhibition on the incorporation of tritiated leucine and the effect was again more conspicuous in male than in female worms. This result is in accordance with previously reported inhibitions of protein synthesis in mammalian and bacterial systems (reviewed in Pica Mattoccia et al., loc. cit.), but it is at variance with the recently reported stimulation of protein synthesis by hycanthone in a cell-free system from S. mansoni (Lukacs et al., 1980, J. Parasitol. 66: 424-427). The reasons for this difference are unknown.