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The sterol and phospholipid composition of cercariae, schistosomula, and adult Schistosoma mansoni was analyzed by gas-liquid chromatography and high-performance liquid chromatography (HPLC). Cercariae and schistosomula contained cholesterol, desmosterol, campesterol, stigmasterol, and β-sitosterol while adults contained only cholesterol. In all stages cholesterol comprised greater than 50% of the total sterols, and in cercariae and schistosomula desmosterol comprised 38 and 21% of the total sterols, respectively. The other three sterols, campesterol, stigmasterol, and β-sitosterol, made up approximately 10% of the total. The same five sterols found in cercariae and schistosomula were present in the hepatopancreas of uninfected snails but with a much higher desmosterol concentration in the parasite, 38%, than in the snail, 2%. As in cercariae and schistosomula the three minor sterols comprised approximately 10%. Thus, the sterol composition of cercariae and schistosomula was similar but not identical to that of the snail host. Phosphatidylcholine was the major phospholipid of all three stages (50%) as determined by two HPLC procedures. The remaining phospholipids consisted of phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. In addition, in adults there were small quantities of sphingomyelin and lysophosphatidylcholine. The percentage of each phospholipid was similar among stages with the exception of a slight increase in phosphatidylserine in adults compared to cercariae and schistosomula. These results show that a characteristic lipid composition is found in cercariae, schistosomula, and adults.

Human complement activation by cercariae and schistosomula of the human parasite Schistosoma mansoni was studied in vitro. Cercariae are composed of tails which are shed after infection of the host and bodies which transform into the larvae or schistosomula after infection. After incubation in fresh normal human serum (NHS), cercarial tails bound more anti-C3 antibodies than did cercarial bodies (CB), and the tails were rapidly lysed, while the attached CB remained intact. Complement activation by cercariae was dependent on the alternative pathway but was independent of antibody, as shown by C3 deposition by hypogammaglobulinemic human sera. By transmission microscopy, the fibrillar glycocalyx on both CB and tails was stained by NHS but not by heat-inactivated serum (HI-NHS). The glycocalyx was labeled with periodate and tritiated borohydride, and parasites were incubated in NHS and HI-NHS. After solubilization, the labeled glycocalyx on organisms incubated in NHS but not HI-NHS bound anti-C3 antibodies. Of the CB incubated with eserine sulfate to prevent transformation, 78% +/- 10% were dead after culture for 24 h in NHS. In contrast, 21% +/- 12% of the CB were dead after culture in HI-NHS. Schistosomula incubated in NHS bound 37% of the amount of anti-C3 antibodies bound by cercariae but were not killed by NHS. In conclusion, the cercarial glycocalyx activated human complement, and schistosomula were less susceptible to killing than cercariae because they had less glycocalyx and activated less complement.

Human buffy coat cells adhering to schistosomula of Schistosoma mansoni that were preincubated in fluorochrome-conjugated concanavalin A (Con A), wheat germ agglutinin, lentil lectin, or purified IgG from a hyperimmunized rabbit, were examined by fluorescence and transmission electron microscopy and by freeze-fracture. All four fluorochrome-conjugated multivalent ligands were homogeneously distributed on the parasite surface after preincubation. Within 1-3 h after the addition of cells, large areas of nonfluorescence, 10-20 micrometer in diameter, were seen on the parasite surface. In addition, the fluorochromes were observed in granules within the cells. Electron microscope autoradiography of worms preincubated with 125I-Con A showed silver grains evenly distributed over the tegumental membrane. After the addition of cells, grains were seen over phagolysosomes in the cytoplasm of neutrophils adhering to the parasites. In addition, no grains were present over large areas of the tegumental membrane, which still retained its normal architecture, or over fusions between the neutrophil plasma membrane and the outer tegumental membrane. Rabbit IgG formed an electron-dense layer on the tegumental membrane which was endocytosed by neutrophils. Both neutrophils and eosinophils fused with the parasite in areas containing no electron-dense material on the surface. It is concluded that human neutrophils will endocytose a variety of multivalent ligands from the surface of schistosomula, which probably accounts for the failure of neutrophils to kill the parasite and acts to clear the parasite surface of both antigen and antibody. Presumably, the components of the parasite surface which have originally bound the ligands are also endocytosed since surface components labeled by galactose oxidase and NaB3H4 are taken into cells when examined by light microscope autoradiography. Finally, membrane fusion occurs in areas devoid of multivalent glands, which suggests that these ligands serve to bring the cells and parasites close together, but the actual fusigens probably reside in the lipids in the outer tegumental membrane.

The surfaces of miracidia of Schistosoma mansoni were examined ultrastructurally during in vitro transformation to sporocyts. Before transformation, the surface was composed of ciliated epithelial plates (EP) that were set into a reticulum of narrow syncytial ridges (SR). The EP were attached to SR by extensive pleated septate junctions that had 18–24 strands of intramembrane particles (IMP) on the protoplasmic faces and complementary pits on the ectoplasmic faces. These junctions also appeared to separate the EP plasma membrane into apical and basolateral domains with a larger number of IMPs on the latter. Transformation was induced by placing the miracidia in salt containing medium which also halted ciliary beating. In 2–5 hr, the SR expanded until they formed a syncytium covering the parasite surface, while the EP retracted and rounded up. During this time, the EP and SR were held in contact with one another by the septate junctions which became progressively convoluted. Subsequently, the EP detached from the parasite. When transforming miracidia were returned to fresh water, the cilia resumed beating and the EP detached from the parasite surface and exposed the underlying basement membrane. Those EP that remained attached were connected only by septate junctions to the expanded SR. These studies demonstrate that the formation of the syncytium occurs gradually with contact maintained between EP and SR via the septate junctions. Further, the septate junctions are similar to occluding junctions in mammalian epithelia since they segregate the plasma membrane of the EP and they have an adhesive function.

termined with accuracy. However, E. monacis and E. perforoides oocysts sporulated first with about 85% sporulating after 5 days incubation while E. os took a slightly longer time (about 6.5 days). Eimeria tuscarorensis required about 20 days for a 50% sporulation. A short sporulation time may be a factor for the relative abundance of E. monacis and E. perforoides. Depending upon the time of year, between 77% and 90% of the oocysts were E. monacis and E. perforoides. In contrast, sporulation in E. tuscarorensis required at least 4 times longer than E. monacis and E. perforoides. The relatively long sporulation period would require more time for E. tuscarorensis to achieve sufficient levels of infective oocysts in the environment to produce an increase in oocyst numbers in the host. Furthermore, E. tuscarorensis oocysts were relatively more prevalent during the warmdry summer weather months (May-August) suggesting that the thick outer oocyst wall may resist desiccation and extend viability. Eimeria os were more prevalent in the Spring and Fall, suggesting less resistance to desiccation or heat. Indeed, E. os oocyst appeared to deteriorate much sooner than other species when incubated at 28-30 C in 2.5% potassium dichromate. The deterioration usually started at the region around the micropyle. In conclusion, coccidial parasites were abundant in the woodchuck population of southern Illinois. Although E. monacis and E. perforoides were the species which predominated through the non-hibernating months of the year, E. os and E. tuscarorensis appeared to show seasonal

This study determined the amino acid and carbohydrate composition of 2 cercarial glycocalyx preparations obtained after phenol-water extraction and subsequent gel chromatography. Labeled cercariae were subjected to 85% phenol, thereby dissociating the glycocalyx into the aqueous phase, which was dialyzed and chromatographed on Sepharose CL-2B or -4B in either 4 M guanidine hydrochloride (GuHCl) or 0.1% sodium dodecyl sulfate (SDS). The label eluted primarily in the void volume and was antigenic as tested with rabbit polyclonal antibodies by immunoblotting. Approximately 6 micrograms of protein and 28 micrograms of carbohydrate were obtained from 10(5) cercariae in the antigenic void volume fraction after SDS chromatography. Threonine, serine, and glutamic acid comprised 44% of the amino acid residues of the protein. The predominant sugar was fucose. Galactosamine, glucosamine, galactose, and mannose were also detected. After GuHCl chromatography, free amino acids, predominantly glycine and serine, comprised 17% of the total protein. The carbohydrate composition was similar to that of SDS-chromatographed extracts. Phenol-water extracts eluting in the void volume of Sepharose CL-2B were compared by negative staining and scanning electron microscopy with material obtained from medium in which cercariae shed glycocalyx during transformation to schistosomula. Both the isolated material and the transformation medium contained particles 20-50 nm in diameter, with subunits of 15-20 nm. These studies show that the cercarial glycocalyx is particulate, contains mainly carbohydrate and some protein, and is solubilized by phenol-water extraction.

Metrizamide-gradient purified rat peritoneal mast cells were allowed to adhere to schistosomula of S. mansoni that had been preincubated in either minimum essential medium with 5% fetal calf serum (MEM/FCS), heat-inactivated serum from rats 8 weeks after cercarial infection (IRS) fresh normal rat serum as a source of complement (NRS), or IRS and NRS. Adherence was evaluated in sections 0.3 micrometers thick. The preincubation conditions favoring cell adherence were IRS + NRS greater than NRS greater than IRS greater than MEM/FCS. Greater adherence was seen at 60 min than at 10 min. Discharge of mast cell granulates was seen infrequently, was not greater in adherent than nonadherent cells, was not increased by any preincubation condition, and did not occur against the parasite's surface, as would be expected if antigen, antibody, and surface receptors were aggregated there. Electron microscopic examination showed that attachment to the parasite occurred through electron-dense material which was usually fibrillar in appearance. Membrane fusion, such as is seen between human neutrophils and schistosomula, did not occur.

Hatching, chemokinesis, and transformation of miracidia of Schistosoma mansoni were examined with a light microscope equipped with a video recording system. Saline, linearly and reversibly, inhibited miracidial hatching and swimming. Both hatching and swimming were inhibited at 4 C and 12 C and accelerated at 34 C relative to rates at 22 C. Hatching was an explosive event that began with ciliary beating when the egg was placed in artificial pond water (APW) and culminated in the parasite's escape from the shell in 100 to 300 msec. Broken egg shells had sharp, complementary edges. Neither miracidia nor eggs swelled prior to hatching. Accumulation of miracidia in a spot of snail conditioned water (SCW) occurred rapidly due to a 60-75% decrease in the exit rate from the spot, rather than by an increase in the entry rate. The turning rate in SCW increased tenfold and the time spent in the spot was 6 times that of controls. Eserine sulfate inhibited miracidial turning and accumulation in SCW. Parasites accumulated in a spot of serotonin by increasing their rate of turning. Miracidia transformed to sporocysts in either complex media containing serum, RPMI-1640, Hanks' salts or phosphate buffered saline, but not in amino acids or vitamins. Transformation was inhibited when miracidia were incubated with serotonin or when miracidia had not been exposed previously to APW.