Female Schistosomes Research Articles

The potential for drug resistance in schistosomiasis

Schistosomiasis is a major neglected tropical disease mainly caused by Schistosoma haematobium, S. japonicum and S. mansoni, and results in the greatest disease burden. Mass drug administration (MDA) with praziquantel (PZQ), a single drug only available for the disease, has played a vital role in schistosomiasis control. Therefore, any possibility of selection of the parasites for PZQ resistance or low sensitivity may hamper the 2030's target of global disease elimination. We had experimentally demonstrated the long-term survival and reproductive potential of single-sex (of either sex) S. japonicum infections in definitive hosts mice. What has not yet been adequately addressed is whether the long live single-sex schistosomes remain sensitive to PZQ, and what reproduction potential for those schistosomes surviving treatment may have. We therefore performed experimental mice studies to explore the treatment effectiveness of PZQ (at total doses of 200 or 400 mg/kg, corresponding to the sub-standard or standard treatment doses in humans) for single-sex S. japonicum aged three months old. The results showed that no treatment efficiency was observed on female schistosomes, whereas on male schistosomes only at PZQ 400 mg/kg a significant higher efficiency in reducing worm burdens was observed. Moreover, either schistosome males or females surviving PZQ treatment remained their reproduction potential as normal. The results indicate that long (i.e., three months) live single-sex S. japonicum can easily survive the current treatment strategy, and moreover, any schistosomes, if with PZQ resistance or low sensitivity, could be easily transmitted in nature. Therefore, in order to realize the target for the national and the global schistosomiasis elimination, there is undoubtedly a great need for refining PZQ administration and dosage, looking for alternative therapies, and/or developing vaccines against schistosome.

Degenerative Changes in the Reproductive Organs of Female Schistosomes during Maintenance In vitro

Degenerative alteration of the reproductive organs of female schistosomes in correlation with the change in egg-laying rate of schistosome pairs in vitro was studied by electron microscopy. The production of normal eggs by adult S. japonicum pairs decreased after 4 days in vitro followed by an increase of abnormal egg laying up to day 8. In S. mansoni, the yield of both normal and abnormal eggs decreased gradually from the start of maintenance in vitro in spite of a much higher pairing rate than in S. japonicum. The vitelline gland of 14-day in vitro-maintained S. japonicum stained with Fast red B, while that of S. mansoni did not. The ovary of both species exhibited regressive features after 14 days of maintenance in vitro. Ultrastructural examination showed that the vitelline cells and oocytes of S. japonicum and S. mansoni had already lost their structural integrity after 2 days in vitro and continued to exhibit signs of structural degeneration throughout the 14-day in vitro maintenance period. The regressive changes in reproductive potential of female S. mansoni maintained in vitro for 4 days could be reversed by surgically implanting the parasites into mouse mesenteric veins.

Differential adhesive properties of the surface of human blood flukesSchistosoma mansoni

While infecting a vertebrate host, blood flukes (Schistosoma mansoni) must continually resist adhesions by immune effector cells. However, the male and female schistosomes must adhere to one another in order to establish and maintain the sexual pairing process after 4 wk postinfection. Using a contact angle method, the relative adhesiveness of male and female parasites were determined. Results indicate that schistosomes restrict effector cell adhesion through developmental, sexual, and regional differences in adhesive properties.

Possible eggshell protein gene from Schistosoma mansoni

We have identified and sequenced a cDNA clone of a mRNA found only in mature female schistosomes. This mRNA is not detectably synthesized by female worms from single sex infections (unisexual females), by males or by the developing miracidia in the eggs. The clone hybridiscs to a highly abundant polyadenylated mRNA of approximately 1500 nucleotides. The nucleotide sequence of the clone predicts a polypeptide comprising two repetitive regions, a pentapeptide repeat with the consensus sequence Gly-Tyr-Asp-Lys-Tyr, and a region rich in histidine residues. Hybrid selected mRNA translated in vitro with [ 3H]tyrosine as labelled amino acid yields a polypeptide of 48 kDa (p48) that corresponds to the major [ 3H]tyrosine labelled translation product of female worm total mRNA. p48 does not label with [ 35S]methionine and is absent from the translation products of male and unisexual female mRNAs. The amino acid sequence of p48 has significant homologies to silk moth chorion proteins and we suggest that it is one of the major components of the schistosome eggshell probably accounting for the high level of [ 3H]tyrosine incorporation into the vitellaria of Schistosoma mansoni. The tyrosine content of the polypeptide suggests that it may play a role in phenol oxidase mediated cross-linking of the schistosome eggshell and in support of this we find that mushroom phenol oxidase will cause the specific cross-linking of p48 in in vitro translation products.

Schistosoma mansoni: Nitrothiazolines and the male tegument

Within 24 hr of treatment of the mouse host with BW484C, 2-[5-nitro-2-(pivaloylimino)-4-thiazoline-3-yl]diacetamide, pairs of Schistosoma mansoni exhibited “hepatic shift” and began to leave the mesenteric veins. The tegument of the males was altered, both morphologically and physiologically, while that of females was unaffected. This morphological damage to males correlated well with therapeutic efficacy against both sexes in a range of analogues of BW484C. However, parasites removed from mice after treatment but before the hepatic shift and then maintained in vitro were far from moribund as treated males could be maintained for 8 days in vitro, although this was 5 days less than males from untreated mice. Females survived as well as control worms. In contrast, male and female S. mansoni remaining in their host after therapy were invaded by host cells in the liver after 2 days. The morphological effects and reduction of the in vitro survival of males treated in the mouse and removed after 24 hr could be simulated by in vitro exposure for 24 hr to 10 -5 M BW484C. Females were not susceptible to this regime. It was concluded that worm pairs were swept to the liver as a result of drug dependent damage to the tegument of the male and that phagocytic invasion of male and female schistosomes by host cells within the liver was an important factor in the efficacy of BW484C. The biochemical events underlying the effects on the tegument of male worms remain unknown.

Characterization of a female-specific cDNA derived from a developmentally regulated mRNA in the human blood fluke Schistosoma mansoni.

We have isolated and characterized a cDNA clone that is derived from a developmentally regulated mRNA found only in mature female schistosomes. The mRNA is approximately 950 nucleotides in length and is not detectable in immature female schistosomes isolated from single-sex infections, in male worms, or in eggs. During normal bisexual infections, the mRNA species is first detected 28 days after infection (the time of worm pairing) and increases to a high level 35 days after infection, coinciding with the start of egg production. The nucleotide sequence of the cDNA shows two large open reading frames in the coding strand. Several features of the clone, including the deduced sequence of the polypeptide encoded by one of the reading frames, suggest a relationship to the silk moth chorion (egg shell) gene family. The isolation of this clone provides us with a probe for further studies of female schistosome development and is a first step toward a detailed understanding of this process at the molecular level.

Influence of mating on surface nutrient exchange in schistosomes

In schistosomes, the mating process influences male-female transfer and gender-specific exchange of nutrients. The paired male schistosome provides glucose to the female partner. Male-to-female intertegumental transfer of(14)C-labeled glucose,(14)C-labeled 3-O-methylglucose, [(14)C]2-deoxyglucose and 2-fluorodeoxyglucose has been demonstrated in schistosomes. This phenomenon has been studied extensively inSchistosoma mansoni, and confirmed inSchistosoma japonicum, as well asS. haematobium, using radioactive pulsing methods. Male schistosomes contain significantly greater quantities (nmol/mg worm water) of glucose than do females. The transfer of glucose is apparently not an energy-dependent process, but occurs along this concentration gradient. Most, if not all, of the glucose utilized by the female is transferred from the male partner via tegumentary-facilitated diffusion mechanisms, free diffusion, or some combination of these two components. Unpaired male schistosomes contain greater quantities of glycogen than do comparable paired schistosomes, indicating that the presence of a female in the gynecophoral canal depletes the reserves of the male partner; this is additional indirect evidence for male-to-female transfer of glucose. Tegumentary surface uptake of acidic amino acids has been compared in paired and separated male and female schistosomes. InS. mansoni, a saturable carrier-mediated mechanism has been defined which operates only in unpaired male and unpaired female teguments. In contrast, the uptake of aspartate and glutamate is not seen in paired worms of this species. Tegumental uptake of acidic amino acids is not observed in paired or unpaired male or femaleS. japonicum. However, inS. haematobium, significant quantities of aspartate are taken up by both paired and unpaired male schistosomes. Measurable aspartate uptake is seen in paired femaleS. haematobium, but in the separated female, there is minimal uptake of this acidic amino acid. Thus the permeability of the teguments of human schistosome species to acidic amino acids is modified in response to the paired state inS. mansoni andS. haematobium, but these characteristics are not shared byS. japonicum.

In vitro orientation of maleSchistosoma mansoni to extracts derived from female schistosomes

Chemical orientation of adult maleSchistosoma mansoni was studied during cultivation in vitro. Directional preference was assessed in culture vessels marked with compass coordinates by the application of circular statistics for determining clustering and orientation to a predicted direction. Organic solvent extracts of fresh female schistosomes and supernatant fluids from 72-hr cultures of female parasites were tested for potential chemotactic activity. Analysis showed significant (P < 0.05) directional preference and clustering of male worms towards test compounds at all time periods (24, 48, and 72 hr) with one mixture of female extracts and at one of three time periods with a second female extract. Male worms did not respond to ecdysone, cholesterol, or solvent controls by orienting in predicted direction, although clustering was observed on two of 12 occasions. These results indicate the presence of a chemoattractant compound(s) in female extracts.

Cloning of a major developmentally regulated gene expressed in mature females of Schistosoma mansoni

A cDNA library constructed from RNA isolated from adult Schistosoma mansoni has been screened by differential hybridization to identify clones corresponding to genes highly expressed by female worms. Several such cDNAs encoding the same highly abundant mRNA species were identified. Studies with one of these (pSF10) which contained a 500 base pair insert demonstrated that this gene was not expressed in immature females or eggs and encoded a polypeptide of approximately 35 000 daltons. Quantitation of the levels of RNA showed that 10% of the total RNA of female parasites was homologous to pSF10. A single gene corresponding to pSF10 was identified in Southern blotting experiments using adult worm DNA. The cloning of this gene will facilitate study of the molecular and genetic events controlling female schistosome maturation.

The mechanism and rate of glucose transfer from male to female schistosomes

Short-term in vitro pulse-labeling of the free glucose pool in pairs of Schistosoma mansoni, and measurement of the increase in [ 14C]glucose in the female partner (and the concomitant decrease in the male), has established that glucose can be transferred from male to female schistosomes. It is demonstrated that this transfer is not inhibited by ouabain in S. mansoni. Free glucose levels have been measured in Schistosoma haematobium, Schistosoma japonicum and S. mansoni. Data indicate that the transfer of glucose is not via an active mechanism, but rather the transfer of glucose occurs along a glucose concentration gradient which exists between males and females.

Interaction of Schistosoma japonicum with Salmonellae and Other Gram-Negative Bacteria

The in vitro and in vivo interaction of Schistosoma japonicum with salmonellae and other gram-negative bacteria was studied. In vitro, S. japonicum associated with salmonellae and other gram-negative bacteria, and more male than female schistosomes associated with the bacteria. By using the various strains and mutants of salmonellae, we showed that Salmonella typhimurium had a higher degree of association than did Salmonella enteritidis and that the piliated strains of S. typhimurium associated much more frequently than did the nonpiliated strains. However, in vivo studies demonstrated more frequent association of salmonellae with female than with male schistosomes and that the piliated and nonpiliated strains of salmonella did not differ in their association with the worms.

Schistosoma mansoni, S. japonicum, and S. haematobium: Permeability to acidic amino acids and effect of separated and unseparated adults

Permeability of the tegument of male and female Schistosoma mansoni was measured in vitro and a comparison was made between copulating and separated worms. In unpaired (separated) schistosomes, a carrier-mediated (selective) transport system for acidic amino acids was demonstrated. Males and females exhibited similar uptake rates for aspartate and glutamate. Half-saturation constants for aspartate (males, 0.035 ± 0.008 m M; females, 0.026 ± 0.006 m M) and glutamate (males, 0.010 ± 0.007 m M; females, 0.015 ± 0.004 m M) were determined for separated worms only. Time-course studies provided estimates of aspartate influx rates in males (7.3 pmol min −1 worm −1) and females (2.3 pmol min −1 worm −1). The most dramatic observation, however, was that, in copula, neither male nor female schistosomes took up acidic amino acids, but may have excluded these compounds. Thus, this ouabain-insensitive, mediated mechanism was operational only when the worms were unmated. In S. japonicum, no uptake of glutamate was observed in either mated or separated males and females. In S. haematobium, saturable uptake of aspartate was apparent in both mated and unmated males and females, indicating that species-specific differences in uptake of acidic amino acids existed. These studies indicate the need for cautious interpretation of data obtained from in vitro analyses of separated male and female mansonian schistosomes, and that such conditions may not reflect in vivo or in copula function.

Schistosoma mansoni: Effect of maintenance in vitro on the uptake and incorporation of leucine by adult worms

The rates of leucine uptake and incorporation into protein by adult male and female Schistosoma mansoni were not affected by maintenance in vitro for up to 10 days' duration, despite the decline in the protein content of male and female worms of approximately 35 and 55%, respectively, during this period. The effect of maintenance in vitro was obscured in paired female schistosomes by the apparent shielding of the female tegument within the gynaecophoral canal of the male. Incorporation rates were reduced by 50% in the presence of 2 mM cycloheximide whereas uptake rates were unaffected. Adult schistosomes are unable to maintain their in vivo protein levels purely by recourse to exogenous amino acids absorbed across the tegument in vitro, and the rates of uptake and incorporation of leucine appear to reflect the changing somatic requirements of the worms and are probably not correlated with the reproductive activity of adult worms in vitro. The possible role of alimentary rather than tegumental nutrition in egg production in vivo is discussed.

Protein, Glycogen, and Water Content in Schistosomes

The volume of distribution (= VD) of water was measured in Schistosoma mansoni, S. haematobium, S. japonicum, and Hymenolepis diminuta. In the rat tapeworm, H. diminuta, the volume of distribution of 3H-water was positively correlated with wet weight (r = 0.87, P less than 0.001) and this same phenomenon also was demonstrated in S. mansoni (r = 0.90, P less than 0.001). The 5-sec VDwater was constant over a range of glucose (0.01-50 mM) and phenylalanine (0.01-20 mM) concentrations in both male and female schistosomes. The VD of antipyrine, which permeates by virtue of its lipophilic properties, also was shown to correlate with protein content in S. mansoni. Protein content determined in single, isolated schistosomes was correlated with the volume of distribution of water in S. haematobium, S. japonicum, and S. mansoni males and females. Age-related variations in the protein content (of S. mansoni) and volume of distribution of water (in both S. japonicum and S. mansoni) were also defined, and the use of tritiated water content as an indicator of mass in small tissue samples was thus established. Female blood flukes recovered from mice infected for more than 90 days appeared to be characterized by a slight reduction in size with age. Schistosoma mansoni reared in the golden hamster may be slightly smaller than schistosomes of the same strain raised in outbred mice. These results provide a baseline that should be useful for future physiological and immunological studies.

Correlation between lipid partition coefficients and surface permeation in Schistosoma japonicum.

Comparison of transintegumental membrane permeability and partition coefficients of selected nonelectrolytes was attempted to correlate the parameters of lipid solubility, hydrophilicity, and membrane permeation in male and female schistosomes (parasites of the portal venous tributaries of man). Surface permeation (measured by the triple isotope technique) and octanol/water partition coefficients were determined for 17 compounds (acetamide, aminopyrine, antipyrine, benzyl alcohol, butanol, caffeine, ethanol, ethylene glycol, glycerol, inosine, mannitol, methanol, polyethylene glycol, propylene glycol, sucrose, thiourea, and urea). Linear regression analyses comparing the logarithm of the partition coefficient to transintegumental uptakes indicate a positive correlation in both sexes: R = 0.76 (P less than 0.001) for males, and R = 0.77 (P less than 0.001) for females. Similarly, linear regression analyses comparing hydrogen bond number with the logarithm of tissue uptake index demonstrate a high (negative) correlation in both males (R = 0.85, P less than 0.001) for females (R = -0.90, P less than 0.001). The male and female schistosomes showed no statistically significant differences in correlation of these parameters. Surface permeation was the same in male and female schistosomes, suggesting that male-female variations in integumental uptake rates previously observed may be restricted to metabolites which enter by way of a selective carrier system.

Schistosoma mansoni: development of primary infections in mice genetically deficient or intact in the fifth component of complement

The influence of the late components of complement (C) on the percutaneous primary infections with Schistosoma mansoni was studied in inbred mice genetically deficient or intact in C5. Worm recoveries were diminished in C5-deficient male mice as compared to C5-intact animals. Twenty-four-day-old parasites were also shorter following growth in C5-deficient animals. At 7 weeks after infection, female schistosomes, but not male parasites, were shorter and fewer eggs/schistosome pair were deposited in the livers of C5-deficient as compared to C5-intact male mice. In addition to the presence of C5, the mouse sex was found to influence the outcome of an infection. Schistosomes showed a reduced infection rate and were relatively stunted after 24 days of growth in female mice in the two strains as compared to male mice of the corresponding strains. The results suggest that C5 plays no role in defence against a primary infection in mice.

Cross Protection in Infections Due to Schistosoma mansoni Using Tegument Antigens of Fasciola hepatica

Immunity to infection due to Schistosoma mansoni was induced in CBA/J mice by using a tegument antigen from Fasciola hepatica worms. Multiple immunizations resulted in up to 83% higher worm-burden reductions than did single immunizations. The vaccine also had an adverse effect on the fecundity of female schistosome worms, which resulted in a lower number of eggs per worm pair recovered from the livers of infected mice. Mice immunized once with tegument antigens of F. hepatica survived lethal doses of S. mansoni cercariae for longer periods than did control mice. These results--lowered worm-burden recoveries, lower egg burdens per worm pairs resulting in lesser numbers of granulomatous lesions, and longer survival times--in immunized mice suggest that protection against infection due to S. mansoni is possible using soluble antigens as vaccines.

Adenylate cyclase in adults and cercariae of Schistosoma mansoni

Adenylate cyclase of adult Schistosoma mansoni is activated by serotonin (5-hydroxytryptamine). Serotonin activation is markedly enhanced by GTP. The poorly hydrolyzed analogs of GTP, guanylyl imidophosphate (Gpp(NH)p) and guanosine 5′-(3- O-thio)triphosphate activate the schistosome cyclase in the absence of serotonin. The activating effect of these nucleotides is increased in the presence of serotonin. Female adult schistosomes have only 40% as much NaF-stimulated activity as do the male parasites. In addition, the serotonin stimulation of adenylate cyclase in the females is only 20% as much as the males. Maximal activation of adenylate cyclase with NaF reveals that the adult parasites have tenfold higher activity than the cercariae. Cercarial adenylate cyclase shows very poor response to serotonin in the presence of GTP. Serotonin caused more significant activation of cercarial adenylate cyclase in the presence of guanylyl imidophosphate.

Schistosoma mansoni: in vitro uptake and incorporation of glycine by adult worms

1. 1. The uptake of [ 3H]glycine into an ethanol-extractable intracellular pool and its simultaneous incorporation into protein (TCA insoluble fraction) have been measured in adult male and female Schistosoma mansoni incubated, in vitro, in complex media. 2. 2. The patterns of uptake and incorporation were similar in male and female schistosomes incubated in media containing serum, and also in male worms incubated in the presence or absence of serum. 3. 3. The amounts of [ 3H]glycine uptake and incorporation were directly proportional to the extracellular glycine concentration and were identical in male worms (in the presence or absence of serum) and female worms. 4. 4. Emetine at a concentration producing a 95% inhibition of protein synthesis was without effect on the uptake of [ 3H]glycine into the ethanol-extractable pool in male worms.

Schistosoma mansoni: Correlation between lipid partition coefficients and the transintegumental uptake of nonelectrolytes

Comparison of transintegumental membrane permeability and partition coefficients of selected nonelectrolytes attempted to correlate the parameters of lipid solubility and membrane permeation in male and female Schistosoma mansoni. Transintegumental uptakes and octanol:water partition coefficients were determined for 10 nonelectrolytes (acetamide, antipyrine, benzyl alcohol, caffeine, ethanol, ethylene glycol, propylene glycol, sucrose, thiourea, and urea). Linear regression analyses comparing the logarithm of the partition coefficient to the transintegumental uptakes yielded values of R = 0.80 ( P < 0.001) for males, R = 0.84 ( P < 0.001) for females, and R = 0.82 ( P < 0.001) for a combined analysis of males and females. The male and female schistosomes showed no statistically significant differences in correlation of these parameters. The evidence, then, suggests that the multilaminate membrane functions in a way similar to the function of a lipid bilayer with regard to the parameters studied.