Carolyn E. Cousin

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.

Use of RAPD-PCR to differentiate genetically defined lines of an intermediate host of Schistosoma mansoni, Biomphalaria glabrata.

The genetic differentiation among several laboratory-maintained pedigree snail lines of Biomphalaria glabrata (with different susceptibility phenotypes to Schistosoma mansoni infection) was assessed with the random amplified polymorphic DNA method. Out of the 20 primers tested, 2 (OPA-01 and OPA-06) gave reproducible markers with either individual or bulked DNA samples from resistant (BS-90, 10-R2, LAC-line) and susceptible (M-line) snails. Arbitrary primer, OPA-01, amplification of BS-90 DNA identified a 180-bp strain-specific fragment and a 400-bp marker in the susceptible M-line stock. In the 10-R2 and LAC snail lines, OPA-01 specific markers of 200 bp and 550 bp were identified. Amplification with primer OPA-06 identified several major strain-specific markers in the BS-90 (150 bp, 400 bp, 800 bp) and M-line (1,100 bp) snails. The heritability of the RAPD markers was evaluated in progeny snails derived from a cross between the BS-90 and M-line stocks. Results showed that markers were inherited in a dominant or codominant fashion. The 1,100-bp M-line marker was inherited in all susceptible progeny snails analyzed.

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.

Biomphalaria glabrata peroxiredoxin: effect of schistosoma mansoni infection on differential gene regulation.

To identify gene(s) that may be associated with resistance/susceptibility in the intermediate snail host Biomphalaria glabrata to Schistosoma mansoni infection, a snail albumen gland cDNA library was differentially screened and a partial cDNA encoding an antioxidant enzyme thioredoxin peroxidase (Tpx), or peroxiredoxin (Prx), was identified. The 753bp full-length, single-copy, constitutively expressed gene now referred to as BgPrx4 was later isolated. BgPrx4 is a 2-Cys peroxiredoxin containing the conserved peroxidatic cysteine (C(P)) in the N-terminus and the resolving cysteine (C(R)) in the C-terminus. Sequence analysis of BgPrx4 from both resistant and susceptible snails revealed the presence of several (at least 7) single nucleotide polymorphisms (SNPs). Phylogenetic analysis indicated BgPrx4 to resemble a homolog of human peroxiredoxin, PRDX4. Northern analysis of hepatopancreas RNA from both resistant and susceptible snails showed that upon parasite exposure there were qualitative changes in gene expression. Quantitative real-time RT-PCR analysis showed differences in the levels of BgPrx4 transcript induction following infection, with the transcript up-regulated in resistant snails during the early phase (5h) of infection compared to susceptible snails in which it was down-regulated within the early time period. While there was an increase in transcription in susceptible snails later (48h) post-infection, this never reached the levels detected in resistant snails. A similar trend - higher, earlier up-regulation in the resistant snails but lower, slower protein expression in susceptible snails - was observed by Western blot analysis. Enzymatic analysis of the purified, recombinant BgPrx4 revealed the snail sequence to function as Prx but with an unusual ability to use both thioredoxin and glutathione as substrates.

Susceptibility of Snails to Infection with Schistosomes is influenced by Temperature and Expression of Heat Shock Proteins.

The freshwater snail, Biomphalaria glabrata is the obligate intermediate host for the transmission of the parasitic trematode, Schistosoma mansoni the causative agent of the chronic debilitating neglected tropical disease, schistosomiasis. We showed previously that in juvenile snails, early and significant induction of stress manifested by the expression of stress proteins, Hsp 70, Hsp 90 and reverse transcriptase (RT) of the non- LTR retrotransposon, nimbus, is a characteristic feature of juvenile susceptible NMRI but not resistant BS-90 snails. These latter, however, could be rendered susceptible after mild heat shock at 32°C, revealing that resistance in the BS-90 resistant snail to schistosomes is a temperature dependent trait. Here we tested the hypothesis that maintenance of BS-90 resistant snails at the permissive temperature for several generations affects the resistance phenotype displayed at the non-permissive temperature of 25°C. The progeny of BS-90 snails bred and maintained through several generations (F1 to F4) at 32°C were susceptible to the schistosome infection when returned to room temperature, shedding cercariae at four weeks post-infection. Moreover, the study of expression levels of the heat shock protein (Hsp) 70 protein by ELISA and western blot analysis, showed that this protein is also differentially expressed between susceptible and resistant snails, with susceptible snails expressing more protein than their resistant counterparts after early exposure to wild-type but not to radiation-attenuated miracidia. These data suggested that in the face of global warming, the ability to sustain a reduction in schistosomiasis by using refractory snails as a strategy to block transmission of the disease might prove challenging since non-lethal elevation in temperature, affects snail susceptibility to S. mansoni.

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: changes in the albumen gland of Biomphalaria glabrata snails selected for nonsusceptibility to the parasite.

The LAC-line strain of the snail Biomphalaria glabrata has very low susceptibility to the parasite Schistosoma mansoni and a very low reproductive potential. Upon examination of the reproductive tract of these snails, light and electron microscopy revealed obvious abnormalities in the albumen gland. Secretory cells that are normally cuboidal in susceptible NMRI (F0) snails were squamous in LAC-line snails. These LAC-line cells contained small secretory granules and negligible rough endoplasmic reticulum and Golgi, compared to large granules and an extensive array of both organelles in F0 albumen gland cells. Comparative analyses of soluble protein extracts of F0 and LAC-line albumen glands showed several qualitative differences. Among the most prominent was an 18-kDa protein in F0 snails that was remarkably reduced in the soluble protein extracts of LAC-line snails. Also, metabolic incorporation of [35S]-methionine was impaired in LAC-line albumen glands. Whether these albumen gland changes are caused by decreased susceptibility to parasitism is yet to determined.

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-